A nucleic acid aptamer specifically recognizing hexavalent chromium form, a nucleic acid aptamer derivative and application thereof

Abstract

The application discloses a nucleic acid aptamer specifically recognizing hexavalent chromium forms, a nucleic acid aptamer derivative and application thereof, and relates to an electrochemical sensor prepared by modifying a nucleic acid aptamer functional sequence with specific recognition for different forms of hexavalent chromium and with an electrochemically active label on the surface of a working electrode of an electrochemical sensing chip, and utilizing the difference of the indicator signal of the electrochemically active label to quantitatively analyze the concentrations of two specific forms of hexavalent chromium respectively. The application prepares an electrochemical sensor with ultra-high sensitivity, and realizes rapid sensing and detection of different forms of hexavalent chromium by using the electrochemical response signal. The method provided by the application has good specificity, sensitivity and stability, can realize in-situ and long-time accurate monitoring of different forms of hexavalent chromium in various complex samples, can effectively avoid matrix interference, and has good practical application prospect in the fields of food, biology and environment technology.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid aptamer technology, specifically to a nucleic acid aptamer that specifically recognizes the hexavalent chromium form, nucleic acid aptamer derivatives, and their applications. Background Technology

[0002] Hexavalent chromium is a potent carcinogen, and its toxicity and environmental behavior are highly dependent on its chemical form (CrO4). 2- / Cr2O7 2- / HCrO4 - Different forms of chromium exhibit significant differences in bioavailability, migration and transformation patterns, and genotoxicity. While existing detection techniques (such as spectrophotometry and chromatography-coupled methods) can determine total chromium content, they struggle to achieve speciation and suffer from drawbacks such as expensive equipment (IC-ICP-MS), cumbersome procedures (requiring ion separation), and weak resistance to interference (Fe). 3+ / Hg 2+ Traditional methods suffer from drawbacks such as interference with color development. Furthermore, they cannot analyze dynamic morphological changes in real time, leading to a lack of accurate data support for environmental risk assessment and governance decisions, severely hindering the improvement of heavy metal pollution control effectiveness. Currently, sensing and analysis technologies based on nucleic acid aptamers as recognition molecules have shown some application potential in heavy metal ion detection. Among known technologies, Chinese invention application number CN202311289907.X discloses a nucleic acid aptamer that specifically recognizes hexavalent chromium ions and its application. This invention uses sodium chromate as a screening target, and obtains an 80bp nucleic acid aptamer by using Capture-SELEX technology to screen chromate ions. The binding force K between the two is determined by affinity measurement. d The specificity of this sequence for chromate was investigated by examining its binding to other ions, with a binding affinity of 24 nM. Chinese invention patent document CN202010565393.6 discloses a nucleic acid aptamer for hexavalent chromium, which is a 62-base DNA sequence with an affinity of 6.1 μM. Therefore, while existing nucleic acid aptamer technology can bind to specific forms of hexavalent chromium in terms of both total amount and chromate content, it still suffers from drawbacks such as poor specificity due to excessively long sequences and high synthesis costs.

[0003] However, the specific identification of hexavalent chromium species faces three bottlenecks: ① The differences in charge density and spatial configuration among anionic species are small (CrO4) 2- With Cr2O7 2- With a size difference of only 0.3 nm, existing nucleic acid aptamers for CrO4 2- With Cr2O7 2- The differences in recognition between them are insufficient (Kd differs by a factor of 100); ② pH fluctuations in the environmental medium cause spontaneous transformation of the speciation, leading to distortion of the recognition signal; ③ Coexistence of anions (SO4) in complex matrices. 2- / NO3- Competition and combination of factors reduce 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 interference resistance to overcome the limitations of existing technologies in assessing the toxicity and managing the risks of hexavalent chromium. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art. The purpose of the present invention is to provide a nucleic acid aptamer that specifically recognizes the hexavalent chromium form, nucleic acid aptamer derivatives and their applications.

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

[0006] In a first aspect, the present invention provides a nucleic acid aptamer that specifically recognizes hexavalent chromium forms, wherein the hexavalent chromium forms include 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, oxymethylated, methylated, aminated, thiolated, fluorinated, or isotopized.

[0008] Secondly, the present invention provides a nucleic acid aptamer derivative that specifically recognizes the hexavalent chromium form, 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 of the nucleotide sequence with the nucleic acid aptamer is greater than 60%;

[0010] The 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] Thirdly, the present invention provides the application of nucleic acid aptamers or nucleic acid aptamer derivatives in distinguishing dichromate and chromate.

[0013] Furthermore, it includes the following steps:

[0014] (1) Immobilization and modification of nucleic acid aptamers or their derivative probes on the working electrode

[0015] Nucleic acid aptamers or their derivatives are modified with thiol, amino, and carboxyl groups and then covalently coupled to immobilize them on the surface of reactive working electrodes (such as those covered with functional materials like gold nanoparticles or graphene); or they are captured onto the working electrode surface non-covalently using base hybridization, biotin, digoxigenin, enzymes, or folic acid.

[0016] (2) Selecting an electrochemically reactive probe system

[0017] Electrochemical signal response groups are introduced by modifying nucleic acid aptamers or complementary sequences with electrochemically active groups (methylene blue-MB, ferrocene, nanomaterials, quantum dots, etc.). The selection of electrochemically active groups for nucleotide sequence modification of nucleic acid aptamers or their derivatives should follow the principle that the oxidation / reduction potentials of the electrochemically active groups are significantly different.

[0018] (3) Electrochemical analysis and testing

[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 testing and analysis were carried out in a buffer solution, and the electrochemical signals were recorded.

[0020] (4) Electrochemical signal analysis

[0021] Electrochemical signals of a series of standard solutions of chromate and dichromate at different concentrations were measured using the constructed test electrode, and the generated signals were analyzed. Finally, the standard curve was plotted. By detecting and analyzing dichromate and chromate in the test samples under different conditions, the distribution of hexavalent chromium in different environmental media and its toxicity were quantitatively determined and explored based on the differences and ratios between the two signals.

[0022] Furthermore, a sensing platform is constructed from a glassy carbon electrode, gold nanoparticles modified on the glassy carbon electrode, and a signal probe. The signal probe consists 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 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 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) This invention proposes a dual-aptamer synergistic recognition sensor strategy, which constructs a recognition interface for sequence 1 and sequence 2 nucleic acid aptamers with a base length of 40bp through precise screening, and utilizes CrO4 2- Cr2O7 2- The recognition between its signal sequences induces conformational switching of nucleic acids, achieving signal amplification (detection limit as low as 0.01 μg / L). It 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. This invention can be applied to intelligent classification and treatment of industrial wastewater and precise monitoring of human exposure, reducing the cost of hexavalent chromium detection by 70% (compared to chromatography) and improving detection time by 10 times, providing a new tool for dynamic management of environmental health risks.

[0025] (2) The nucleic acid aptamer of the present invention has the characteristic of easy modification. 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), thereby achieving high-sensitivity detection and differentiation of different hexavalent chromium forms. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0027] Figure 1 In this embodiment of the invention, two nucleic acid aptamer probes are respectively reacted with CrO4 2- With Cr2O7 2- Affinity test results;

[0028] Figure 2 CrO4 in the embodiments of the present invention 2- With Cr2O7 2- The results of specificity tests on nucleic acid aptamer probes are shown in the figure.

[0029] Figure 3 The nucleic acid aptamer electrochemical sensor constructed in this embodiment of the invention reacts with different concentrations of CrO4. 2- With Cr2O7 2- Test results;

[0030] Figure 4 CrO4 in the example of this invention 2- With Cr2O7 2- The results of the nucleic acid aptamer sensor's specificity test for target ions are shown in the figure.

[0031] Figure 5 CrO4 in the example of this invention 2- With Cr2O7 2- The results of the nucleic acid aptamer sensor's sensitivity test to target ions are shown in the figure. Detailed Implementation

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

[0033] The objective of this invention is achieved through the following technical solution:

[0034] A hexavalent chromium (Cr) of the present invention 6+ ) Specific forms of dichromate (Cr2O7) 2- ) and chromate (CrO4) 2- The nucleic acid aptamer of chromium, whose nucleotide sequence includes the DNA fragment shown below, is capable of accurately identifying and distinguishing between two important forms of hexavalent chromium (CrO4). 2- With Cr2O7 2- The nucleic acid aptamer of ), the nucleotide sequence of which is shown below:

[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 sequences of the above-mentioned nucleic acid aptamers are selected from naturally occurring or artificially synthesized sequences, or the same sequences from any other source.

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

[0041] A specific position on the nucleotide sequence of the aforementioned nucleic acid aptamer can be phosphorylated, oxymethylated, methylated, fluorinated, aminated, thiolated, or isotopized.

[0042] The nucleotide sequences of the aforementioned nucleic acid aptamers can be bound to biotin, digoxigenin, fluorescent substances, nanomaterials, polyethylene glycol, peptides, proteins, enzymes, or folic acid labels (or even radioactive substances).

[0043] The above-mentioned nucleic acid aptamers can be used to derive other nucleic acid aptamers. The nucleotide sequences of the derived nucleic acid aptamers can be any one of the following three sequences:

[0044] (1) The nucleotide sequence of the nucleic acid aptamer listed in this embodiment has more than 60% homology (for example, some complementary nucleotides can be deleted or added to the above nucleic acid aptamer sequence);

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

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

[0047] The nucleotide sequence backbone of the nucleic acid aptamers listed in the above embodiments can also be derived into a thiophosphate backbone, and the above nucleic acid aptamers can also be modified into corresponding locked nucleic acids or peptide nucleic acids.

[0048] The potassium dichromate and potassium chromate nucleic acid aptamers described in this 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 speciation analysis and detection probes.

[0049] The nucleic acid aptamers in this embodiment are mainly screened using the magnetic bead capture method. The specific screening process includes the following steps:

[0050] (a) Optimization of the nucleic acid library: The DNA library structure is divided into three parts. The middle part is a 40-base random sequence; on either side of the random sequence are two DNA arms, which are the sequences required for PCR amplification of 20 fixed bases. Simultaneously, the 5' fixed sequence hybridizes with the fixed probe (BDNA). The fixed probe is a DNA strand with one end labeled with biotin, complementary to the library's fixed sequence, used for library fixation.

[0051] Random Library Lib:

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

[0053] Positive phase primer: 5'-ATTGGCACTCCACGCATAGG-3';

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

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

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

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

[0058] (b) Initial screening: Library pretreatment method: 1.3 nmol of library (approximately 10 14 2.9 nmol of B DNA was dissolved in 289 μL of DPBS buffer (0.9 mmol / L CaCl2, 2.7 mmol / L KCl, 1.5 mmol / L KH2PO4, 0.5 mmol / L MgCl2, 136.9 mmol / L NaCl, 8.1 mmol / L Na2HPO4), pre-denatured at 95 °C for 10 min, then cooled to 60 °C at a rate of 0.1 °C / s and held for 1 min, and then slowly annealed to 25 °C at a rate of 0.1 °C / s. The hybridization library was added to 1 mL of streptavidin-modified magnetic beads that had been washed 5 times, and incubated on a shaker at room temperature for 30 minutes. The magnetic beads were then adsorbed using a magnet, and the supernatant was removed. The beads were then resuspended in 400 μL of DPBS buffer and washed 6 times. Then, dichromate (or chromate) target solution was added, and the mixture was incubated on a shaker at room temperature for 40 min. Magnetic beads are adsorbed by a magnet, and the supernatant solution is recovered to obtain a preliminary nucleic acid library containing a complex of DNA and dichromate (or chromate).

[0059] (c) Purification: The initial screening nucleic acid library obtained in step (b) was subjected to PCR amplification using the primers listed in (a) with fluorescent primers at the 5' end and primers with spacers at the 3' end. The amplified products were separated into single strands using SDS-denaturing PAGE, and then further processed by gel boiling, n-butanol concentration, and dialysis to form a secondary nucleic acid library. The PCR amplification conditions were: 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for N (optimal number of rounds) of amplification cycles; extension at 72℃ for 7 min. Gradient PCR was used, with the initial nucleic acid library as a template to optimize the annealing temperature, ultimately achieving an annealing temperature of 60℃. The optimal number of amplification rounds for each round of screening products was obtained through round optimization. After round optimization, the remaining libraries were amplified under the same conditions. The PCR-generated library was prepared into single strands for the next round of screening. The preparation 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 solution separated into layers; the upper n-butanol layer was removed, and the lower layer was retained, with a volume of approximately 50 μL. An equal volume of 2×TBE solution was added to the lower layer, and the mixture was heated at 95°C for 10 min. While still hot, the sample was loaded onto a denaturing gel PAGE and run at 300V for 20 min. Fluorescent bands were excised and collected under UV light, and the gel was fragmented. 1.2 mL of DPBS buffer was added to the fragmented gel, and the mixture was boiled at 95°C for 15 min. After centrifugation, the supernatant was collected, and this process was repeated 1–2 times. A 1:5 volume ratio of n-butanol solution was added to the collected solution, vortexed for 30 seconds, and centrifuged at 6000 rpm for 3 min. The upper n-butanol layer was removed. Repeat 2-3 times to achieve a final sample volume of approximately 100 μL. Finally, dialyze the sample overnight in DPBS solution at 4°C.

[0060] (d) Cycling: Replace the starting nucleic acid library with the secondary nucleic acid library obtained above and repeat steps (b) to (c) above, using new streptavidinized 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) Screening Efficiency Evaluation: Screening efficiency, i.e., the enrichment of DNA during the screening process, was determined using qPCR. The detection signal was the Cq value of the DNA library during the qPCR process, which was converted into the retention rate of the library by dichromate (or chromate). First, a series of nucleic acid library solutions of different concentrations were prepared and measured using qPCR to obtain a linear equation relating concentration to Cq value. Then, the concentration of DNA library acquired by dichromate (or chromate) competition was measured, and the library retention rate was obtained based on the ratio of measured amount to input amount. Higher screening efficiency resulted in a higher retention rate. When the retention rate reached a plateau, the library was cloned and sequenced.

[0062] Examination 1: The affinity between dichromate, chromate and their nucleic acid aptamers in this embodiment was mainly determined by qPCR. The specific determination process includes the following steps:

[0063] (1) Determine the standard curve between nucleic acid concentration and Cq value determined by qPCR.

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

[0065] (2) Determination of the affinity of the probe for dichromate (or chromate) by qPCR:

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

[0067] Prepare 2 μL of 100 μM fixed probe with DPBS, incubate with aptamer probe for hybridization, 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 DPBS buffer, resuspend in 200 μL DPBS, and divide into 5 equal parts.

[0068] Equal volumes of CrO4 at different concentrations (1, 4, 16, 64, 256 μM) were added to the magnetic bead suspensions containing the nucleic acid aptamer probes. 2- (or Cr2O7) 2- The solution was reacted at room temperature for 15 min. Then, magnetic beads were adsorbed using a magnet, and the supernatant solution was transferred and labeled 1, 2, 3, 4, and 5. 10 μL of the labeled solution was transferred to the corresponding numbered qPCR tubes, and 2 μL of pre-prepared mixture solution (containing dNTPs, Evagreen enzyme, forward and reverse primers, and buffer) was added to each tube. The mixture was then vortexed and qPCR was used to quantify the nucleic acids in the solution. The obtained Cq value was substituted into the established linear equation to obtain the number of aptamer molecules that competed at different target concentrations, and Y = B... max *X / (K d +X)(Y is the retention rate of nucleic acid molecules, and X is the CrO4 used. 2-(or Cr2O7) 2- (For the corresponding concentration, B is a constant, and max means the maximum value.) The formula is used to fit and calculate K, the binding force between the nucleic acid aptamer and the target. d Value. For example... Figure 1 As shown, the final fitted dichromate, chromate, and their nucleic acid aptamer K were obtained. d The values ​​were 1.2 μmol / L and 0.75 μmol / L, respectively.

[0069] Examination 2: The specificity among dichromate, chromate, and their nucleic acid aptamers in this embodiment was mainly determined using the fluorescence recovery method. The specific determination process includes the following steps:

[0070] (1) Probe design

[0071] Dichromate nucleic acid aptamer fluorescent probe:

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

[0073] Chromate nucleic acid aptamer fluorescent probe:

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

[0075] The 5' end of the aptamer's complementary sequence is labeled with a BHQ-2 fluorescent 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 nucleic acid aptamer electrochemical sensing probe:

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

[0082] Chromate-containing nucleic acid aptamer electrochemical sensing probe:

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

[0084] (2) Determination of morphological selectivity: Using DPBS as solvent, solutions of aptamer fluorescent probe and quencher probe at a concentration of 2 μM were prepared separately. 75 μL of each solution was transferred, mixed, and incubated at room temperature for 30 min. The mixed solution was then divided into three equal portions (50 μL each). 50 μL of 2 μM (CrO4) solution was added to each of the three portions. 2- or Cr2O7 2- ) solution, CrO4 2- With Cr2O7 2- Each solution served as a negative control, and the reaction was carried out at room temperature for 10 min. The solutions were then transferred to 96-well plates, and fluorescence emission spectra were obtained using a multifunction scanner. The fluorescence emission spectra of the three reaction solutions were then obtained. Figure 2 As can be seen from the data, the two obtained nucleic acid aptamer probes are respectively effective against CrO4. 2- Cr2O7 2- The high selectivity demonstrates that the nucleic acid aptamer provided by this invention can react with CrO4. 2- and Cr2O7 2- It has high morphological selectivity recognition.

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

[0086] CrO4 modified with thiol and electroactive groups respectively was used 2- With Cr2O7 2- Nucleic acid aptamer sequences were prepared, and both were pretreated with TCEP for half an hour. The mixed solution was then drop-cast onto the surface of a gold-containing working electrode and incubated overnight at 4°C. CrO4 was then 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- React 50 μL of solution for 5–25 minutes, and then measure in phosphate buffer. Results are as follows: Figure 3 As shown in the electrochemical spectral results, with the increase of CrO4 2- With Cr2O7 2- As the concentration of Cr increases, the electrochemical signal gradually increases, and the electrochemical signal is related to Cr. 6+A good linear relationship was observed between the concentrations. Furthermore, the detection method exhibited good linearity within the range of 20 nM to 320 nM. This demonstrates that the nucleic acid aptamer electrochemical detection method provided in this patent can rapidly and effectively perform sensing analysis of hexavalent chromium speciation.

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

[0088] CrO4 with a concentration of 350 nM was respectively 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 sample was dropped onto the constructed nucleic acid aptamer sensing electrode and reacted at room temperature for 10 min. The electrochemical response signal was then measured using square wave voltammetry. The peak response signal was recorded. Figure 4 As shown, the results indicate that the constructed nucleic acid aptamer electrochemical sensor is resistant to interference from various metal cations and anions. Therefore, the modified electrode constructed using the two obtained nucleic acid aptamers is a type of electrode capable of reacting with CrO4. 2- With Cr2O7 2- Sensors that bind specifically.

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

[0090] CrO4 with a concentration of 350 nM 2- With Cr2O7 2- and each mixed with 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 the reaction was carried out at room temperature for 10 min. The electrochemical response signal was then measured using square wave voltammetry. The response signal was recorded, such as... Figure 5 As shown, the results indicate that the constructed nucleic acid aptamer electrochemical sensor can selectively recognize CrO4 under conditions where multiple metal cations and anions coexist. 2- With Cr2O7 2- Ions. The results show that the modified electrode constructed using the two obtained nucleic acid aptamers is capable of reacting with CrO4. 2- With Cr2O7 2- Sensitive sensor.

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

Claims

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

2.

2. The aptamer of claim 1, wherein the aptamer specifically recognizes the hexavalent chromium species. A certain position on the nucleotide sequence of the dichromate and chromate aptamer is phosphorylated, oxymethylated, methylated, aminated, sulfhydrylated, fluorinated or isotopically labeled.

3. Use of the aptamer of claim 1 or 2 in the preparation of a sensor for distinguishing dichromate and chromate.

4. Use according to claim 3, characterized in that, The method comprises the following steps: (1) immobilization modification of the aptamer probe on the working electrode The nucleotide sequence of the aptamer is modified by thiol, amino and carboxyl groups to be covalently coupled to the surface of the working electrode with reactivity; and is captured to the surface of the working electrode in a non-covalent manner based on base hybridization pairing, biotin, digoxin, enzyme or folate; (2) selection of an electrochemical reaction active probe system The nucleotide sequence of the aptamer is modified by introducing an electrochemical signal response group with an electrochemically active group, and the selection of the electrochemically active group of the nucleotide sequence of the aptamer is required to follow the principle that the oxidation / reduction potential of the electrochemically active group has a significant difference; (3) electrochemical analysis test The three-electrode system is used for electrochemical test analysis, the Pt electrode is used as the counter electrode, the Ag / AgCl electrode is used as the reference electrode, the gold electrode or glassy carbon modified with the electroactive dichromate and chromate aptamer is used as the working electrode, the test analysis is performed in a buffer solution, and the electrochemical signal is recorded; (4) electrochemical signal analysis A series of different concentrations of chromate and dichromate standard solutions are determined by using the constructed test electrode, the generated signals are analyzed, and finally the standard curve is drawn; the dichromate and chromate in the samples under different conditions are detected and analyzed, the difference and proportion between the signals are determined, and the distribution state of the hexavalent chromium form and the toxicity thereof in different environmental media are quantitatively judged and explored.

5. Use according to claim 4, characterized in that, The sensing platform is composed of a glassy carbon electrode, nano-gold modified on the glassy carbon electrode and signal probes; the signal probes are 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 for identifying dichromate, and the second signal probe is used for identifying chromate; when the first probe combines with dichromate in the sample to be tested, the aptamer forms a hairpin structure, and ferrocene is closer to the electrode surface; when the second probe combines with dichromate in the sample to be tested, the aptamer forms a hairpin structure, and methylene blue is closer to the electrode surface.

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