Nucleic acid aptamer capable of specifically recognizing hexavalent chromium form, nucleic acid aptamer derivative and application of nucleic acid aptamer derivative

Abstract

The invention discloses a nucleic acid aptamer capable of specifically recognizing hexavalent chromium forms, a nucleic acid aptamer derivative and application of the nucleic acid aptamer and the nucleic acid aptamer derivative. The surface of a working electrode of an electrochemical sensing chip is modified with a nucleic acid aptamer functional sequence which can specifically recognize different forms of hexavalent chromium and has an electrochemical activity label; the electrochemical sensor is assembled, and the concentration of the hexavalent chromium in two specific forms can be quantitatively analyzed by utilizing the difference of indication signals of electrochemical active markers. The electrochemical sensor with ultrahigh sensitivity is prepared, and rapid sensing detection of different hexavalent chromium forms is realized by using electrochemical response signals of the electrochemical sensor. The method provided by the invention has good specificity, sensitivity and stability, can realize in-situ long-time accurate monitoring of different hexavalent chromium forms in various complex samples, can effectively avoid matrix interference, and has good practical application prospects in the technical fields of food, biology and environment.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid aptamers, and in particular to a nucleic acid aptamer capable of specifically recognizing hexavalent chromium forms, a nucleic acid aptamer derivative and applications thereof. Background Art

[0002] As a strong carcinogenic pollutant, the toxicity and environmental behavior of hexavalent chromium are highly dependent on its chemical form (CrO4 2- / Cr2O7 2- / HCrO4 - ), the bioavailability, migration and transformation patterns and genetic toxicity of different forms vary significantly. Although existing detection technologies (such as spectrophotometry and chromatography) can determine the total chromium content, it is difficult to achieve form resolution, and there are problems such as expensive equipment (IC-ICP-MS), cumbersome steps (need ion separation), and weak anti-interference (Fe 3+ / Hg 2+ Interference with color development) and other defects. In addition, the traditional method cannot analyze the dynamic changes of morphology in real time, resulting in a lack of accurate data support for environmental risk assessment and governance decision-making, which seriously restricts the improvement of the effectiveness of heavy metal pollution prevention and control. The current sensing analysis technology based on nucleic acid aptamers as recognition molecules has shown some application potential in heavy metal ion detection. Among the currently known technologies, the document with Chinese invention application number CN202311289907.X discloses a nucleic acid aptamer that specifically recognizes hexavalent chromium ions and its application. The invention uses sodium chromate as the screening object, and obtains a nucleic acid aptamer with a length of 80bp by screening chromate using the Capture-SELEX technology. The binding force K between the two is determined by affinity. d The specificity of this sequence for chromate was 24 nM, and the binding between other ions was examined. Chinese invention patent document CN202010565393.6 discloses a hexavalent chromium aptamer consisting of a 62-base DNA sequence with an affinity of 6.1 μM. This shows that while existing aptamer technology can target specific binding to the total amount of hexavalent chromium and chromate, it still suffers from limitations such as poor specificity due to long sequences and high synthesis costs.

[0003] However, the specific recognition of hexavalent chromium forms faces three bottlenecks: ① The difference in charge density and spatial configuration between anionic forms is small (CrO4 2- With Cr2O7 2- The size difference is only 0.3nm). The existing nucleic acid aptamers are sensitive to CrO4 2- With Cr2O7 2- The recognition difference between them is insufficient (Kd differs by a hundred times); ② pH fluctuations in the environmental medium trigger spontaneous morphological transformation, resulting in distortion of the recognition signal; ③ Coexistence of anions in complex matrices (SO4 2- / NO3- ) competitive binding, resulting in a decrease in detection sensitivity by 1-2 orders of magnitude. There is an urgent need to develop new aptamer sensing methods that combine morphological selectivity, environmental stability, and anti-interference properties to overcome the limitations of existing technologies for hexavalent chromium toxicity assessment and risk management. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art. The purpose of the present invention is to provide a nucleic acid aptamer, a nucleic acid aptamer derivative and applications thereof that specifically recognize hexavalent chromium forms.

[0005] To achieve the above objectives, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a nucleic acid aptamer that specifically recognizes a hexavalent chromium form, wherein the hexavalent chromium form includes dichromate and chromate. The nucleotide sequence of the dichromate nucleic acid aptamer is shown in SEQ ID No. 1, and the nucleotide sequence of the chromate nucleic acid aptamer is shown in SEQ ID No. 2.

[0007] Furthermore, a certain position on the nucleotide sequence of the dichromate and chromate nucleic acid aptamers is phosphorylated, oxygenated, methylated, aminated, sulfhydrated, fluorinated or isotopized.

[0008] In a second aspect, the present invention provides a nucleic acid aptamer derivative that specifically recognizes hexavalent chromium forms, wherein the nucleic acid aptamer derivative is derived from the nucleotide sequence of the nucleic acid aptamer, and the nucleotide sequence of the derived nucleic acid aptamer includes any one of the following three sequences:

[0009] The homology with the nucleotide sequence of the nucleic acid aptamer is greater than 60%;

[0010] a sequence that hybridizes with the nucleotide sequence of the nucleic acid aptamer;

[0011] The nucleotide sequence of the nucleic acid aptamer is transcribed into an RNA sequence.

[0012] In a third aspect, the present invention provides the use of a nucleic acid aptamer or a nucleic acid aptamer derivative in distinguishing between dichromate and chromate.

[0013] Further, the following steps are included:

[0014] (1) Immobilization and modification of nucleic acid aptamer or its derivative probe on the working electrode

[0015] The nucleotide sequence of the nucleic acid aptamer or its derivative is modified by sulfhydryl, amino and carboxyl groups, and fixed to the surface of a reactive working electrode (such as a functional material covered with nanogold, graphene, etc.) by a covalent coupling reaction; it is captured to the working electrode surface by a non-covalent method based on base hybridization pairing, biotin, digoxigenin, enzyme or folic acid;

[0016] (2) Selection of electrochemically active probe system

[0017] Electrochemical signal response groups are introduced by modifying the nucleic acid aptamer or its complementary sequence with electrochemically active groups (methylene blue-MB, ferrocene, nanomaterials, quantum dots, etc.). The selection of electrochemically active groups for the nucleotide sequence modification of the nucleic acid aptamer or its derivatives follows the principle that the oxidation / reduction potential of the electrochemically active groups has obvious differences.

[0018] (3) Electrochemical analysis test

[0019] Electrochemical testing and analysis were performed using a three-electrode system, with a Pt electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and a gold electrode or glassy carbon electrode modified with electroactive dichromate and chromate nucleic acid aptamers as the working electrode. The test and analysis were performed in a buffer solution, and the electrochemical signals were recorded.

[0020] (4) Electrochemical signal analysis

[0021] The constructed test electrode was used to measure the electrochemical signals of a series of chromate and dichromate standard solutions with different concentrations, and the generated signals were analyzed. Finally, a standard curve was drawn. By detecting and analyzing dichromate and chromate in the samples to be tested under different conditions, the morphological distribution state and toxicity of hexavalent chromium in different environmental media were quantitatively judged and explored based on the difference and ratio between the two signals.

[0022] Furthermore, a sensing platform is composed of a glassy carbon electrode, gold nanoparticles modified on the glassy carbon electrode, and a signal probe; the signal probe is composed of a first signal probe and a second signal probe, the first signal probe is a ferrocene-labeled hairpin structure, and the second signal probe is a methylene blue-labeled hairpin structure; the first signal probe is used to identify dichromate, and the second signal probe is used to identify chromate; when the first probe binds to the dichromate in the sample to be tested, the nucleic acid aptamer forms a hairpin structure, and the ferrocene is closer to the electrode surface; when the second probe binds to the dichromate in the sample to be tested, the nucleic acid aptamer forms a hairpin structure, and the methylene blue is closer to the electrode surface.

[0023] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0024] (1) The present invention proposes a dual-aptamer collaborative recognition sensor strategy, which constructs a 40-bp sequence aptamer electrode recognition interface by precise screening and using CrO4 2- 、Cr2O7 2- The recognition between the nucleic acid and its signal sequence induces conformational switching of the nucleic acid, realizing signal amplification (detection limit as low as 0.01 μg / L), and can accurately distinguish different forms of hexavalent chromium with high affinity and high specificity. It has the advantages of good stability, non-toxicity, and easy modification. The present invention can be applied to intelligent quality control of industrial wastewater and precise monitoring of human exposure, reducing the cost of hexavalent chromium detection by 70% (compared with chromatography) and increasing the detection time by 10 times, providing a new tool for dynamic management and control of environmental health risks.

[0025] (2) The nucleic acid aptamer of the present invention has the property of being easily modified. By modifying the signal substance, the recognition event output between different hexavalent chromium forms and their nucleic acid aptamers can be converted into other signals (such as electrical signals, etc.), thereby achieving high-sensitivity detection and differentiation of different hexavalent chromium forms. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0027] Figure 1 In the embodiment of the present invention, two nucleic acid aptamer probes are respectively combined with CrO4 2- With Cr2O7 2- The affinity determination results of

[0028] Figure 2 CrO4 in the embodiment of the present invention 2- With Cr2O7 2- Figure 2 shows the specificity test results of the nucleic acid aptamer probes;

[0029] Figure 3 The nucleic acid aptamer electrochemical sensor constructed in the embodiment of the present invention is sensitive to different concentrations of CrO4 2- With Cr2O7 2- Test results;

[0030] Figure 4 CrO4 in the present invention 2- With Cr2O7 2- Figure 2 shows the results of the nucleic acid aptamer sensor specificity test for target ions;

[0031] Figure 5 CrO4 in the present invention 2- With Cr2O7 2- Figure 2 shows the results of the target ion sensitivity test of the nucleic acid aptamer sensor. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0033] The object of the present invention is achieved through the following technical solutions:

[0034] A hexavalent chromium (Cr 6+ )Specific form of dichromate (Cr2O7 2- ) and chromate (CrO4 2- ) nucleic acid aptamer, the nucleotide sequence of which includes the DNA fragment shown in the following sequence, which can accurately identify and distinguish the two important forms of hexavalent chromium (CrO4 2- With Cr2O7 2- ), the nucleotide sequence of the nucleic acid aptamer is as follows:

[0035] SEQ ID NO: 1 (dichromate aptamer):

[0036] 5'-GACTCGTCAAGGATACAGCCTTGTTTCCGCTCGGCGTGAC-3';

[0037] SEQ ID NO: 2 (chromate aptamer):

[0038] 5'-ATGGCACAAGCCATAACATATGTGATAGTGGCGTTGTCGT-3';

[0039] The nucleotide sequence of the aforementioned nucleic acid aptamer is selected from naturally occurring or artificially synthesized sequences, or the same sequence from any other source.

[0040] A nucleic acid aptamer sequence of different forms of hexavalent chromium (dichromate and chromate), wherein the aptamer sequence comprises a sequence identical to all nucleotides of the characteristic sequence.

[0041] A certain position on the nucleotide sequence of the nucleic acid aptamer can be phosphorylated, oxygenated, methylated, fluorinated, aminated, sulfhydrylated or isotopized.

[0042] The nucleotide sequence of the nucleic acid aptamer can be combined with biotin, digoxigenin, fluorescent substances, nanomaterials, polyethylene glycol, peptides, proteins, enzymes or folic acid labels (even connected to radioactive substances, etc.).

[0043] The above-mentioned nucleic acid aptamers can be derived into other nucleic acid aptamers, and the nucleotide sequence of the derived nucleic acid aptamers can be any one of the following three sequences:

[0044] (1) The homology with the nucleotide sequence of the nucleic acid aptamer listed in this embodiment is greater than 60% (for example, the nucleic acid aptamer sequence can be deleted or partially complementary nucleotides can be added);

[0045] (2) a sequence that hybridizes with the nucleotide sequence of the nucleic acid aptamer listed in this example;

[0046] (3) RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer listed in the embodiment above.

[0047] The backbone of the nucleotide sequence of the nucleic acid aptamer listed in the above embodiment can also be derived into a phosphorothioate backbone, and the above nucleic acid aptamer can also be transformed into a corresponding locked nucleic acid or peptide nucleic acid.

[0048] The potassium dichromate and potassium chromate nucleic acid aptamers of the above-mentioned embodiment have the following uses: ① Use in the detection of environmental, food and biological harmful factors; ② Use in separation and purification; ③ Use in drug design and development; ④ Use in the preparation of hexavalent chromium morphological analysis detection probes.

[0049] The nucleic acid aptamers of the above embodiment are mainly screened using the magnetic bead capture method, and the specific screening process includes the following steps:

[0050] (a) Optimizing the Nucleic Acid Library: The DNA library structure consists of three parts. The center is a 40-base random sequence. Flanking the random sequence are two DNA arms, which are necessary for PCR amplification of the 20 fixed bases. The fixed sequence at the 5' end hybridizes with an immobilized probe (BDNA). The immobilized probe is a DNA strand complementary to the fixed sequence in the library, labeled with biotin at one end, and serves to immobilize the library.

[0051] Random library Lib:

[0052] 5'-ATTGGCACTCCACGCATAGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCTATGCGTGCTACCGTGAA-3' (Note: N represents any base among A, T, C, and G);

[0053] Forward primer: 5′-ATTGGCACTCCACGCATAGG-3′;

[0054] Reverse primer: 5′-TTCACGGTAGCACGCATAGG-3′;

[0055] Fluorescent forward primer: 5′-FAM-ATTGGCACTCCACGCATAGG-3′;

[0056] Reverse primer with spacer: 5'-AAAAAAAAAAAAAAAAAAAAAAAA-spacer 18-TTCACGGTAGCACGCATAGG-3'

[0057] BDNA immobilization probe: 5'-CCTATGCGTGGAGTGCCAAT-biotin-3'

[0058] (b) Primary screening: Library pretreatment method: 1.3 nmol of library (about 10 14 DNA chains), 2.9nmol BDNA was dissolved in 289μL DPBS buffer (0.9mmol / L CaCl2, 2.7mmol / L KCl, 1.5mmol / L KH2PO4, 0.5mmol / LMgCl2, 136.9mmol / L NaCl, 8.1mmol / L Na2HPO4), pre-denatured at 95℃ for 10min, then cooled to 60℃ at a rate of 0.1℃ per second and maintained for 1min, and then cooled to 25℃ at 0.1℃ per second for slow denaturation. The hybridization library was added to 1mL of streptavidin magnetic beads that had been washed 5 times and incubated at room temperature in a shaker for half an hour. The magnetic beads were then adsorbed using a magnet, the supernatant was removed, and the magnetic beads were suspended and washed 6 times with 400μL DPBS buffer. The dichromate (or chromate) target solution was then added and placed in a shaker and incubated at room temperature for 40min. The magnetic beads are adsorbed by a magnet, and the supernatant solution is recovered to obtain a primary screening nucleic acid library containing a complex of DNA and dichromate (or chromate).

[0059] (c) Purification: The primary screening nucleic acid library obtained in step (b) was amplified by PCR using the primers listed in step (a) with a fluorescent primer at the 5' end and a spacer at the 3' end. The amplified products were separated by single-stranded prep using SDS denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The secondary nucleic acid library was then prepared by boiling the gel, concentrating it with n-butanol, and dialysis. The PCR reaction conditions were as follows: denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds for N (optimal number of cycles); and extension at 72°C for 7 minutes. Gradient PCR was used, using the initial nucleic acid library as a template, to optimize the annealing temperature, ultimately achieving an annealing temperature of 60°C. The optimal number of amplification cycles for each screening round was determined through round optimization. After round optimization, the remaining libraries were amplified under the same conditions. The post-PCR library was prepared for single-stranded DNA sequencing for the next round of screening. The single-stranded DNA sequencing method was as follows: PCR product was mixed with n-butanol at a volume ratio of 1:5, vortexed for 30 seconds, and centrifuged at 6000 rpm for 15 seconds. The upper n-butanol layer was removed, and the lower layer, reduced to approximately 50 μL, was retained. An equal volume of 2× TBE was added to the solution and heated at 95°C for 10 minutes. While still hot, the sample was loaded onto a denaturing polyacrylamide gel and run at 300 V for 20 minutes. Fluorescent bands were excised and collected under UV illumination, and the gel was shredded. 1.2 mL of DPBS buffer was added to the shredded gel, and the gel was boiled at 95°C for 15 minutes. The supernatant was collected and repeated one to two times. A 1:5 volume ratio of n-butanol was added to the collected solution, vortexed for 30 seconds, and centrifuged at 6000 rpm for 3 minutes. The upper n-butanol layer was removed. Repeat 2-3 times to make the final sample volume about 100 μL. Finally, dialyze the sample in DPBS solution in a 4°C refrigerator overnight.

[0060] (d) Cycling: The starting nucleic acid library is replaced with the secondary nucleic acid library obtained above and steps (b) to (c) are repeated, using new streptavidin-labeled magnetic beads in each cycle, until a nucleic acid library containing nucleic acid aptamers that bind to hexavalent chromium with high affinity and high specificity is obtained.

[0061] (e) Evaluation of screening efficiency: qPCR is used to determine the screening efficiency, that is, the enrichment of DNA during the screening process. The detection signal is the Cq value of the DNA library during the qPCR process, which is converted into the retention rate of the library by dichromate (or chromate). First, a series of nucleic acid library solutions with different concentrations are prepared and measured using qPCR to obtain a linear equation related to the concentration and Cq value. Then, the concentration of the DNA library that is competed off by dichromate (or chromate) is determined, and the library retention rate is obtained based on the ratio of the measured amount to the input amount. The higher the screening efficiency, the higher the retention rate. When the retention rate reaches a plateau, the library is cloned and sequenced.

[0062] Investigation 1: The affinity between dichromate and chromate and their nucleic acid aptamers in the above-mentioned embodiment was mainly determined by qPCR method. The specific determination process includes the following steps:

[0063] (1) Standard curve between nucleic acid concentration and qPCR Cq value

[0064] The linear relationship between the aptamer probe concentration and the Cq value determined by qPCR was determined: nucleic acid library Lib solutions of different concentrations (1000, 100, 10, 1, and 0.1 pM) were prepared using DPBS as the solvent; 10 μL of the prepared solution was pipetted and 2 μL of the pre-prepared mixture solution (containing dNTPs, evagreen enzyme, forward and backward primers, and buffer) was added, and the mixture was shaken and mixed. The obtained Cq value was linearly fitted with the logarithm of the concentration.

[0065] (2) qPCR determination of the affinity of the probe to dichromate (or chromate):

[0066] First, 200 μL of streptavidin-coated magnetic microsphere suspension was transferred, and then the magnetic beads were adsorbed using a magnet, the supernatant was removed, and the beads were washed four times with 200 μL of DPBS buffer;

[0067] Prepare 2 μL of 100 μM immobilized probe in DPBS, incubate and hybridize with the aptamer probe first, then incubate with the washed streptavidin magnetic microspheres for 35 min. Then, use a magnet to adsorb the magnetic beads and remove the supernatant. Then, wash twice with 200 μL of DPBS buffer, resuspend in 200 μL of DPBS, and divide into five equal parts.

[0068] Add equal volumes of CrO4 at different concentrations (1, 4, 16, 64, 256 μM) to the magnetic bead suspension containing the nucleic acid aptamer probe. 2- (or Cr2O7 2- ) solution, react at room temperature for 15 minutes, then use a magnet to adsorb the magnetic beads and remove the upper solution, which are labeled 1, 2, 3, 4, and 5 respectively. Pipette 10 μL of the above reaction labeled solution into the corresponding numbered qPCR tubes, and add 2 μL of pre-configured mixture solution (including dNTP, evagreen enzyme, forward and backward primers, and buffer) to each tube, and then shake and mix them. Use qPCR to quantitatively analyze the nucleic acid in the solution, and bring the Cq value obtained by the measurement into the above established linear equation to obtain the number of aptamer molecules that compete under different target concentrations, and use Y=B max *X / (K d +X)(Y is the retention rate of nucleic acid molecules, X is the CrO4 used 2-(or Cr2O7 2- ) corresponds to the concentration, B is a constant, and max means the maximum value) formula is used to fit and calculate the K of the binding between the nucleic acid aptamer and the target. d Value. Figure 1 As shown in the figure, the final fitted dichromate, chromate and its nucleic acid aptamer K d The values were 1.2 μmol / L and 0.75 μmol / L respectively.

[0069] Investigation 2: The specificity between dichromate, chromate and their nucleic acid aptamers in the above-mentioned embodiment was mainly determined by fluorescence recovery method. The specific determination process includes the following steps:

[0070] (1) Probe design

[0071] Dichromate aptamer fluorescent probe:

[0072] 5'-GACTCGTCAAGGATACAGCCTTGTTTCCGCTCGGCGTGAC-FAM-3';

[0073] Chromate aptamer fluorescent probe:

[0074] 5'-ATGGCACAAGCCATAACATATGTGATAGTGGCGTTGTCGT-FAM-3';

[0075] The 5' end of the aptamer complementary sequence is labeled with a BHQ-2 fluorescence quencher group (quenching probe):

[0076] Dichromate quenching probe:

[0077] 5'-BHQ-2-GTCACGCCGAGCGGAAACAAGGCTGTATCCTTGACGAGTC-3';

[0078] Chromate quenching probe:

[0079] 5'-BHQ-2-GTCACGCCGAGCGGAAACAAGGCTGTATCCTTGACGAGTC-3';

[0080] Dichromate aptamer electrochemical sensing probe:

[0081] 5'-SH-AAAAGACTCGTCAAGGATACAGCCTTGTTTCCGCTCGGCGTGAC-Fc-3';

[0082] Chromate aptamer electrochemical sensing probe:

[0083] 5'-SH-AAAAATGGCACAAGCCATAACATATGTGATAGTGGCGTTGTCGT-MB-3';

[0084] (2) Determination of morphological selectivity: Using DPBS as solvent, prepare solutions of aptamer fluorescent probe and quencher probe at a concentration of 2 μM, pipette 75 μL of each solution, mix them, shake and mix, and incubate at room temperature for 30 min. The mixed solution is divided into three equal parts (50 μL each), and then 50 μL of 2 μM (CrO4 2- or Cr2O7 2- ) solution, CrO4 2- With Cr2O7 2- Each of the three reaction solutions was used as a negative control and reacted at room temperature for 10 min. The solution was then transferred to a 96-well plate and a multifunctional scanner was used to perform fluorescence emission spectrum scanning to obtain the fluorescence emission spectrum curves of the three reaction solutions. Figure 2 It can be seen that the two nucleic acid aptamer probes obtained are respectively effective for CrO4 2- 、Cr2O7 2- It has high reaction selectivity, proving that the nucleic acid aptamer provided by the present invention can react with CrO4 2- and Cr2O7 2- It has high morphological selectivity.

[0085] Investigation 3: The electrochemical method was used to determine dichromate and chromate. The specific determination process included the following steps:

[0086] CrO4 modified with thiol and electroactive groups 2- With Cr2O7 2- The concentration of both nucleic acid aptamer sequences was set to 2 μM, and the mixture was pretreated with TCEP for half an hour. The mixed solution was then dropped onto the working electrode surface containing gold material by drop coating and allowed to stand overnight at 4°C. CrO4 was added at final concentrations of 10 nM, 20 nM, 40 nM, 80 nM, 160 nM, 320 nM, 400 nM, and 500 nM, respectively. 2- With Cr2O7 2- 50 μL of the solution was reacted for 5 to 25 minutes and then measured in phosphate buffer. Figure 3 As shown in the electrochemical spectrum, it can be seen that as CrO4 2- With Cr2O7 2- As the concentration of Cr increases, the electrochemical signal gradually increases, and the electrochemical signal is consistent with the Cr 6+The concentrations showed a good linear relationship. The detection method also exhibited a good linear range from 20nM to 320nM. This demonstrates that the nucleic acid aptamer electrochemical detection method provided by this patent can quickly and effectively sense and analyze hexavalent chromium forms.

[0087] Investigation 4: Electrochemical sensor for CrO4 2- With Cr2O7 2- Specificity analysis

[0088] CrO4 at a concentration of 350nM 2- With Cr2O7 2- and 2 μM Mg 2+ 、Fe 3+ 、Zn 2+ 、Tl + 、Al 3+ 、Cu 2+ , Ca 2 + , Pb 2+ 、Cd 2+ 、SO4 2- 、NO3 - 、AsO3 3- Add it dropwise to the constructed nucleic acid aptamer sensor electrode, react at room temperature for 10 minutes, and use square wave voltammetry to measure and obtain its electrochemical response signal. And record the highest peak response signal, such as Figure 4 As shown in the results, the constructed nucleic acid aptamer electrochemical sensor can resist the interference of various metal cations and anions. Therefore, the modified electrode constructed using the two obtained nucleic acid aptamers is a kind of electrochemical sensor that can resist the interference of CrO4 2- With Cr2O7 2- Specific binding sensor.

[0089] Investigation 5: Electrochemical sensor for CrO4 2- With Cr2O7 2- Sensitivity analysis

[0090] The concentration of CrO4 was 350nM 2- With Cr2O7 2- and 2 μM Mg 2+ 、Fe 3+ 、Zn 2+ 、Tl + 、Al 3+ 、Cu 2+ , Ca 2+ , Pb 2+ 、Cd 2+ 、SO4 2- 、NO3 - 、AsO3 3-The mixed solution was added dropwise to the constructed nucleic acid aptamer sensing electrode, and reacted at room temperature for 10 minutes. The electrochemical response signal was obtained by square wave voltammetry. The response signal was recorded as follows: Figure 5 The results showed that the constructed nucleic acid aptamer electrochemical sensor can highly selectively recognize CrO4 under the coexistence of various metal cations and anions. 2- With Cr2O7 2- ions. The results showed that the modified electrode constructed using the two obtained nucleic acid aptamers is a kind of electrode that can 2- With Cr2O7 2- Sensitive sensor.

[0091] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A nucleic acid aptamer that specifically recognizes hexavalent chromium forms, characterized in that: The hexavalent chromium form includes dichromate and chromate. The nucleotide sequence of the dichromate nucleic acid aptamer is shown in SEQ ID No. 1, and the nucleotide sequence of the chromate nucleic acid aptamer is shown in SEQ ID No.

2.

2. The nucleic acid aptamer that specifically recognizes hexavalent chromium forms according to claim 1, characterized in that A certain position on the nucleotide sequence of the dichromate and chromate nucleic acid aptamers is phosphorylated, oxygenated, methylated, aminated, sulfhydrated, fluorinated or isotopized.

3. A nucleic acid aptamer derivative that specifically recognizes hexavalent chromium forms, characterized in that: The nucleic acid aptamer derivative is derived from the nucleotide sequence of the nucleic acid aptamer according to claim 1 or 2, and the nucleotide sequence of the derived nucleic acid aptamer includes any one of the following three sequences: The homology with the nucleotide sequence of the nucleic acid aptamer is greater than 60%; a sequence that hybridizes with the nucleotide sequence of the nucleic acid aptamer; The nucleotide sequence of the nucleic acid aptamer is transcribed into an RNA sequence.

4. Use of the nucleic acid aptamer according to claim 1 or 2 or the nucleic acid aptamer derivative according to claim 3 in distinguishing between dichromate and chromate.

5. The use according to claim 4, characterized in that The following steps are involved: (1) Immobilization and modification of nucleic acid aptamer or its derivative probe on the working electrode The nucleotide sequence of the nucleic acid aptamer or its derivative is modified by sulfhydryl, amino and carboxyl groups and fixed on the reactive working electrode surface by covalent coupling reaction; it is captured on the working electrode surface by non-covalent means based on base hybridization pairing, biotin, digoxigenin, enzyme or folic acid; (2) Selection of electrochemically active probe system Electrochemical signal response groups are introduced by modifying the aptamer or complementary sequence with electrochemically active groups. The selection of electrochemically active groups modified with the nucleotide sequence of the aptamer or its derivatives follows the principle that the oxidation / reduction potential of the electrochemically active groups has obvious differences. (3) Electrochemical analysis test Electrochemical testing and analysis were performed using a three-electrode system, with a Pt electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and a gold electrode or glassy carbon electrode modified with electroactive dichromate and chromate nucleic acid aptamers as the working electrode. The test and analysis were performed in a buffer solution, and the electrochemical signals were recorded. (4) Electrochemical signal analysis The constructed test electrode was used to measure the electrochemical signals of a series of chromate and dichromate standard solutions with different concentrations, and the generated signals were analyzed. Finally, a standard curve was drawn. By detecting and analyzing dichromate and chromate in the samples to be tested under different conditions, the morphological distribution state and toxicity of hexavalent chromium in different environmental media were quantitatively judged and explored based on the difference and ratio between the two signals.

6. The use according to claim 5, characterized in that The sensing platform is composed of a glassy carbon electrode, gold nanoparticles modified on the glassy carbon electrode, and a signal probe; the signal probe is composed of a first signal probe and a second signal probe, the first signal probe is a ferrocene-labeled hairpin structure, and the second signal probe is a methylene blue-labeled hairpin structure; the first signal probe is used to identify dichromate, and the second signal probe is used to identify chromate; when the first probe binds to the dichromate in the sample to be tested, the nucleic acid aptamer forms a hairpin structure, and the ferrocene is closer to the electrode surface; when the second probe binds to the dichromate in the sample to be tested, the nucleic acid aptamer forms a hairpin structure, and the methylene blue is closer to the electrode surface.

Images