CN114015694A

CN114015694A - Nucleic acid aptamer for detecting copper ions and application thereof

Info

Publication number: CN114015694A
Application number: CN202110361808.2A
Authority: CN
Inventors: 白卫滨; 倪新雨; 蒋鑫炜; 孙建霞; 李旭艳
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2022-02-08

Abstract

The invention discloses a nucleic acid aptamer for detecting copper ions and application thereof, wherein the nucleic acid aptamer is single-stranded DNA with the length of 42nt, and the nucleic acid sequence comprises GGCAGGGTGAGGTTGAGGTCCG basic groups. The copper ion aptamer has high affinity, good specificity, low synthesis cost, stable property and easy modification; obtained by magnetic bead-SELEX screening method, and Cu²⁺The binding force is high, and the specificity is good; the established aptamer colloidal gold colorimetric detection method for copper ions uses an enzyme-labeling instrument for characterization detection, has the advantages of wide detection range, high sensitivity, low detection limit of 1.99ng/mL, rapid detection and high scene matching degree of on-site real-time rapid detection; the established method for detecting copper ions by aptamer colloidal gold is convenient to operateThe method has the advantages of simple steps, no need of marking, low cost and portability, and can provide a novel copper ion analysis and detection method for the field of on-site real-time rapid detection including food safety.

Description

Nucleic acid aptamer for detecting copper ions and application thereof

Technical Field

The invention relates to the technical field of detection of copper ions, in particular to a nucleic acid aptamer for detecting copper ions and application thereof.

Background

Copper is one of the most important metals for industrial applications due to its ductility, low corrosion, alloying capacity, high thermal and electrical conductivity properties. Besides, copper is also a trace mineral element necessary for human body, and is an important component of protein and enzyme in organism, and many important enzymes need to participate in copper for starting and activating. In an adult, the copper content is about 100-150 mg, which is essential for maintaining normal operation and body health of organs, but excessive copper intake can cause corresponding organ injury of a human body. Disruption of the metabolic balance of copper ions in the body can lead to a range of diseases such as anemia, vitiligo, chronic liver disease, alzheimer's disease, wilson's disease, and the like. Therefore, analyzing and detecting the copper content in the environment and food are of great significance to environmental protection and human health.

Copper in the environment and organisms is mostly Copper ions (Cu)²⁺) And the forms of the compounds exist, the detection methods of copper ions are various, and the traditional detection methods comprise atomic absorption spectrometry, inductively coupled plasma emission spectrometry, inductively coupled plasma mass spectrometry and the like. In recent years, researchers are dedicated to developing rapid detection methods for copper aiming at the defects that the traditional detection method is expensive, high in detection cost, incapable of being applied to field detection and the like, and common rapid detection methods comprise a fluorescence method, a colorimetric method, an electrochemical method and the like. Compared with the traditional detection method, the rapid detection method can detect the target substance in a short time, has the characteristics of high detection sensitivity, low detection cost, no need of large-scale instruments and the like, and plays an important role in the scenes needing rapid detection, such as farmer, customs and the like.

In all types of biosensors, the aptamer is used as a target substance recognition molecule, and a colloidal gold colorimetric method is used as a detection means, so that the biosensor not only has good specificity, high sensitivity, low detection limit and high analysis speed, but also is simple and convenient to operate, and is easy to miniaturize and carry.

However, no method for detecting copper ions by using a specific aptamer-based colloidal gold colorimetric method has been reported so far.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a nucleic acid aptamer for detecting copper ions and application thereof.

The first object of the present invention is to provide an aptamer for detecting copper ions.

The second purpose of the invention is to provide a kit for detecting copper ions by a nucleic acid aptamer-based colloidal gold colorimetric method.

The third object of the invention provides the application of any one of the copper ion aptamers in detecting copper ions.

The fourth purpose of the invention is to provide the application of any one of the copper ion nucleic acid aptamers in preparing a kit for detecting copper ions.

The fifth object of the invention provides a method for colorimetric detection of copper ions based on colloidal gold of a copper ion aptamer.

In order to achieve the purpose, the invention is realized by the following scheme:

the invention claims a nucleic acid aptamer for detecting copper ions, wherein the nucleic acid aptamer is single-stranded DNA (ssDNA) with the length of 42nt, and the nucleic acid sequence comprises GGCAGGGTGAGGTTGAGGTCCG bases.

Preferably, the aptamer sequence forms two stem-loop structures, the structure of which is shown in FIG. 2, Cu²⁺More concentrated on the first loop. The aptamer equilibrium dissociation constant K_DThe value was 48.3X 10^-6±26.7×10^-6mol/L。

The invention also provides a kit for detecting copper ions by a colloidal gold colorimetric method based on the aptamer, and the kit contains any one of the copper ion aptamers.

Preferably, the colloidal gold in the kit is prepared by reducing chloroauric acid with trisodium citrate.

Preferably, the colloidal gold in the kit is gold nanoparticles with the diameter of 13 nm.

The application of any copper ion aptamer in detecting copper ions also belongs to the protection scope of the invention.

The application of any copper ion aptamer in preparing a kit for detecting copper ions also belongs to the protection scope of the invention.

The invention also claims a method for detecting copper ions by colloidal gold colorimetry based on copper ion aptamers, which comprises the steps of fully reacting any one of the aptamers with a colloidal gold solution, fully reacting with a sample to be detected, fully reacting with NaCl, detecting absorbance values under the conditions of 650nm and 520nm, and calculating the copper ion concentration of the sample to be detected according to a standard curve.

Preferably, 40 μ L of 0.3 μ M aptamer solution and 100 μ L of 13nm colloidal gold solution are fully reacted for 15min, 40 μ L of sample solution to be tested is fully reacted for 10min, and then 20 μ L of 0.8M NaCl solution is fully reacted for 5 min.

Preferably, the established linear working equation takes the concentration logarithm value of the copper ion solution as an abscissa, and the corresponding A_650nm/A_520nmThe variation values are ordinate.

Preferably, the colloidal gold is gold nanoparticles with the diameter of 13nm, and is prepared by reducing chloroauric acid by trisodium citrate.

Compared with the prior art, the invention has the following beneficial effects:

(1) the copper ion specific aptamer is used for identifying and detecting the copper ion specific aptamer, has the advantages of high affinity, good specificity, low synthesis cost, stable property, easiness in modification and the like, and is more suitable for detecting heavy metal ions;

(2) the copper ion specific aptamer used in the invention is obtained by a magnetic bead-SELEX screening method, has high binding force with divalent copper ions and good specificity.

(3) The nucleic acid aptamer colloidal gold colorimetric detection method established in the invention uses an enzyme-labeling instrument for characterization detection, has the advantages of wide detection range, high sensitivity, low detection limit of 1.99ng/mL, rapid detection and high scene matching degree of on-site real-time rapid detection;

(4) the method for detecting the copper ions by the aptamer colloidal gold established in the invention has the advantages of convenient operation, simple steps and no need of marking, and in addition, the aptamer and the colloidal gold solution used in the method have low cost and are convenient to carry, so that a novel copper ion analysis and detection method can be provided for the field of on-site real-time rapid detection including food safety.

Drawings

FIG. 1 is a diagram of qPCR monitoring for each round of screening of copper ion aptamers according to the invention.

FIG. 2 is a schematic diagram of the sequence information of the copper ion aptamer of the invention, (a) simulation of the secondary structure of the copper ion aptamer MFold, (b) isothermal titration microcalorimetry (ITC) of the copper ion aptamer.

FIG. 3 is a linear working equation for detecting copper ions based on a copper ion aptamer colloidal gold colorimetric method, wherein the copper ion gradient concentration is 5ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL and 2500 ng/mL.

FIG. 4 is the result of the evaluation of the specificity of each ion after colorimetric detection based on the aptamer colloidal gold according to the present invention; wherein the specific ion solution comprises nickel ions (Ni)²⁺) Aluminum ion (Al)³⁺) Cobalt ion (Co)²⁺) Cadmium ion (Cd)²⁺) Calcium ion (Ca)²⁺) Lead ion (Pb)²⁺) And barium ion (Ba)²⁺) And a mixed solution of each ion.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 screening of copper ion aptamers based on the magnetic bead-SELEX technique

First, experiment method

Fixing the library and incubating the magnetic beads with the library: the library is partially paired with a biotin modified complementary strand, streptavidin magnetic beads are connected with biotin on a partial complementary strand to achieve the purpose of fixing the magnetic beads and the library, the library sequence information is shown in table 1, and the library sequence is obtained by a large number of experiments designed in the early stage.

1. The specific steps of library immobilization are:

(1) the library and primers were centrifuged at 12000rpm for 10 min. Under a fume hood, 260. mu.L of ultrapure water is added into the library to dissolve the library to 5. mu.M; 50 μ L of ultrapure water was added to bring the volume to 100 μ M. And (3) uniformly mixing the diluted library and the primer by vortex, fully dissolving and then centrifuging. Wherein the library and primer sequence information is shown in Table 1;

table 1 screening library sequence information:

the above dry powder of ssDNA oligonucleotides is provided by Onputummai Biotech, Inc., Anhui province

(2) Adding the dissolved 29 mu L of primer into the library, uniformly mixing, and centrifuging after the final concentration of the primer is 10 mu M;

(3) library and primer complementary pairing: subpackaging the uniformly mixed solution into PCR octaplex tubes, and slowly renaturing by using a PCR instrument under the following conditions: keeping at 95 deg.C for 10min, slowly cooling to 60 deg.C, keeping for 1min, then slowly cooling to 25 deg.C at a speed of 0.1 deg.C/S, and keeping at 25 deg.C for 30 min;

2. the incubation of the magnetic beads with the library comprises the following specific steps:

(1) taking 50 mu L of solution with good renaturation for ultraviolet detection, and recording A_260nmA value of C₁；

(2) 1mL of magnetic beads are sucked in the first round of screening, 400 mu L of screening buffer solution is used for washing the magnetic beads each time, the washing is carried out for 6 times, the using amount of each round of screening magnetic beads is 70 mu L, and 200 mu L of screening buffer solution is used for washing the magnetic beads each time;

(3) adding the solution with good renaturation into the activated magnetic beads, mixing uniformly, incubating for 1h at room temperature by a rotary shaking table, taking 50 mu L of supernatant for ultraviolet detection, and recording A_260nmA value of C₂. According to C₂/C₁The concentration of nucleic acid not immobilized on the magnetic beads in the supernatant was judged to judge the immobilization efficiency of the library:

fixed efficiency ═ C₁-C₂)/C₁×100％

(II) washing of the library

Removing the supernatant, adding 400 mu L of screening buffer solution into the magnetic beads, sucking the supernatant (named wash1, sample reservation qPCR detection) after the magnetic beads are resuspended, repeating the operation for 6 times to obtain wash2-wash6, adding the prepared target in time for elution and incubation, and preventing the magnetic beads from being dried in the middle.

(III) target elution: separating sequences which are not combined with the target or have weak combination force from sequences combined with the target, and the method comprises the following specific steps:

(1) in the first round of screening, Cu (NO) is added₃)₂The mixture was diluted to 500. mu.M with the selection buffer, 400. mu.L was added to the washed magnetic beads, and the shaker was rotated to incubate at room temperature for 45 min. Adding the reverse screening substance Co (NO) from the second round₃)₂. The fourth and fifth screening change of the positive screen material is CuSO₄Is to reject NO₃ ^-And SO₄ ^2-The effect of (3) excluding specific adsorption.

(2) Fixing the magnetic beads by using a strong magnet, and sucking the supernatant of the positive sieve into a new centrifugal tube, and recording as Elution. The latter 6 library washes were subjected to qPCR assays.

(IV) emulsion PCR amplification

Adopting emulsion PCR amplification to prevent preferential amplification, and the specific steps are as follows:

(1) setting an emulsion PCR amplification system and parameters; taking out 2mL of PCR mix, dissolving, centrifuging, adding into a 50mL centrifuge tube, adding all Elutions, and fully and uniformly mixing by swirling the centrifuge tube. Adding 8mL of ePCR micro-droplet generated oil, carrying out vortex for more than 2min by a vortex instrument, standing for 1-3 min, and evenly subpackaging the vortexed emulsion into 12 rows of eight-connected pipes after a layered transparent liquid is found out. The PCR amplification solution system and the reaction parameter settings of the emulsion are shown in tables 2 and 3;

TABLE 2 emulsion PCR mix solution System

Wherein, lib18S 1-FAM: 5 '-FAM-ATTGGCACTCCACGCATAGG-3'

lib20A2-ployA：5’-AAAAAAAAAAAAAAAAAAASpacer18TTCACGGTAGCACGCATAGG-3’

TABLE 3 emulsion PCR reaction parameters

(2) And (3) recovering amplification products: averagely subpackaging the emulsion in the eight-connected tubes into 2 15mL centrifuge tubes;

(3) and (3) concentrating the PCR product by using n-butyl alcohol, namely filling two 15mL centrifuge tubes filled with the PCR product with n-butyl alcohol in a fume hood, shaking up and down to shake the centrifuge tubes sufficiently, and centrifuging the centrifuge tubes. After centrifugation, the solution was clear and separated. The upper clear liquid and the middle oily emulsion are aspirated, the lower PCR amplification product is transferred to a corresponding 1.5mL centrifuge tube according to volume, and the upper solution n-butanol is removed by centrifugation until the bottom PCR product is concentrated to 100. mu.L.

(V) preparation of Single-stranded DNA

The single chain obtained by adopting a long-chain method and a short-chain method of denaturing PAGE electrophoresis is convenient for the next round of screening, and the specific steps are as follows:

(1) adding the concentrated product into 100 μ L2 × TBE urea loading buffer solution, heating at 95 deg.C for 10min with PCR instrument, centrifuging instantaneously, and immediately loading for electrophoresis.

(2) After preparing the polyacrylamide gel electrophoresis plate, 1 XTBE electrophoresis buffer is added into the inner electrophoresis tank and the outer electrophoresis tank. Electrophoresis was run using a constant temperature voltage of 300V.

(3) After electrophoresis was complete, the correct strip was selected with a blade under a hand-held uv lamp. When cutting out the target band, care is taken not to cut the blank glue at the upper edge beyond the width of 2mm, otherwise the lengthened reaction strand of polyA is easily cut.

(4) Putting the target strip into a crumb rubber centrifuge tube, and centrifuging. After the target band is completely broken, 1.5mL of screening buffer is added, and the mixture is boiled in boiling water for 15 min. After centrifugation, the supernatant liquid was transferred to a 15mL centrifuge tube and repeated once, and the boiled single-stranded solution was transferred to the same 15mL centrifuge tube.

(5) The ssDNA was concentrated in n-butanol. Adding n-butanol into the gel boiling liquid in a 15mL centrifuge tube, and uniformly observing the state of the uniformly mixed liquid by turning upside down. When the solution appeared slightly turbid, the solution was centrifuged to prevent the concentrated product from drying out. The PCR concentrate was placed in a 1.5mL centrifuge tube, centrifuged transiently, the upper n-butanol layer removed, and the concentrate was aspirated.

(6) A microdialysis container and a fixing ring are added to a clean PE glove. The concentrated single-stranded library is added to a dialysis container. Pressing the container opening by using a symmetrical cover of the dialysis membrane to ensure whether the dialysis membrane is complete, adding 40mL of screening buffer solution into a 50mL centrifuge tube, putting a microdialysis device into the centrifuge tube to ensure that the lower surface of the dialysis membrane is perpendicular to the liquid level below the liquid level as much as possible, inverting the centrifuge tube, dialyzing overnight at 4 ℃, wherein each round of dialysis needs to be replaced by new screening buffer solution. The next day, the dialysis membrane was punctured to aspirate the ssDNA solution, and the concentration of ssDNA was calculated.

(VI) qPCR detection

And (3) according to qPCR result analysis, judging the screening condition of each round, and specifically comprising the following steps:

(1) the qPCR mix was first dissolved at low temperature and centrifuged at 5000rpm for 30 s.

(2) And (3) taking a qPCR eight-link tube, adding 30 mu L of qPCR mix, Elution and wash1-6 samples into each hole, respectively adding 2 mu L of the qPCR mix into the qPCR mix, reserving 1 tube of blank qPCR mix, adding 2 mu L of screening buffer solution as negative control, covering a centrifugal tube, and then instantly centrifuging and uniformly mixing the samples to start qPCR detection. The qPCR detection system and reaction parameters are shown in tables 4 and 5;

TABLE 4qPCR mix solution System

Wherein, libV1-S1: 5'-ATTGGCACTCCACGCATAGG-3'

libV1-A2:5’-TTCACGGTAGCACGCATAGG-3’

TABLE 5 qPCR mix solution System

(3) And (5) according to the qPCR result analysis, judging the screening condition of the round.

Wherein the screening buffer is 20mM Tris-HCl, 150mM NaCl, 5mM KCl, 1mM MgCl2, 1mM CaCl2 pH 7.4.

Second, experimental results

Screening Cu by using magnetic bead-SELEX screening technology²⁺Aptamers were screened in a total of six rounds. The monitoring of qPCR for each round of screening is shown in FIG. 1, wherein the green curve from the first round to the fourth round of screening represents washing of magnetic beads 1-6 times (wash1-6), and the sixth round of screening represents washing of magnetic beads 1-5 times; in the fifth round and the sixth round of screening, the orange curve shows that the time for rinsing the magnetic beads for the sixth time is increased to 30min, and the time for cleaning the magnetic beads for the last time in the last round of screening is increased to better eliminate specific adsorption; in the first to sixth round of screening, the red curve is a positive screening curve, wherein the first, second, third and sixth round positive screens use Cu (NO)₃)₂The fourth round and the fifth round of screening materials are replaced by CuSO₄The positive sieve material being replaced to exclude NO₃ ^-Interference of (2); the blue curve in the second to sixth screening is the reverse screening curve, and the reverse screening material uses Co (NO)₃)₂Wherein the first round of screening does not add a back-screening material; in the first to sixth screening rounds, no material was added to the black curve, i.e., negative control. From the second wheelStarting with the counter sieve, the Cq value (also called "Ct value", i.e. the cycle threshold, referring to the number of cycles that the fluorescence signal of the reaction system has undergone when it reaches the set threshold) of the copper ions in the positive sieve is 13.58, which is greater than the Cq value of the cobalt ions in the counter sieve by 11.54, and the Cq value of the copper ions in the positive sieve is 6.03, which is significantly smaller than the Cq value of the cobalt ions by 18.55, although the copper ions in the positive sieve are replaced by CuSO ions in the fourth sieve₄The Cq value of the copper ions in the fourth screening is 8.32 and is slightly larger than that of the cobalt ions by 8.24, but the copper ions in the fifth screening are still CuSO₄When the Cq value of copper ion is 6.6 and the Cq value of cobalt ion is 18.86, NO is excluded₃ ^-Impact on screening. Clearly indicating that the nucleic acid sequences recognized and bound to copper ions were enriched in each round and could be stopped after the sixth round of screening was completed.

Obtaining the original sequence and K thereof by screening_DThe values are shown in Table 6. K_DThe value represents the degree of dissociation into free aptamer and free target, K_DHigh values indicate weak binding of the aptamer to the target, K_DA low value indicates a strong binding of the aptamer-target complex. Equilibrium dissociation constant K_DThe smaller the value, the more the aptamer and the target Cu are represented²⁺The greater the binding force.

Table 6: partial screening sequence information and K for copper ion aptamer_DValue of

The original sequence obtained by screening is longer, so that the synthesis cost is increased, and the uncertainty of the three-dimensional conformation of the sequence is introduced. The reasonable length of the truncated sequence after SELEX screening is a key step for obtaining an effective sequence. Then, modification work such as length clipping and base substitution is carried out on the basis of the respective original sequences, and then the modified sequences are subjected to affinity verification using ITC to find Cu²⁺The highest affinity sequence. Namely, a single-stranded DNA of 42nt in length, comprising GGCAGGGTGAGGTTGAGGTCCG bases as a copper ion aptamer, as shown in FIG. 2. The result shows that the copper ion aptamer chain can form two loop ringsStructure of equilibrium dissociation constant K_DThe value was 48.3X 10^-6±26.7×10^-6mol/L, its affinity is obviously high with the original sequence, and ITC verification shows that Cu is involved²⁺More on the first loop.

Example 2 establishment of method for detecting copper ions by aptamer colloidal gold colorimetry

First, experiment principle

The colloidal gold solution can be aggregated under the high-salt environment, and macroscopically, the colloidal gold solution is changed from wine red to bluish purple or even gray; when the aptamer is added into the colloidal gold solution, the aptamer chain is adsorbed to the AuNPs surface through physical adsorption, so that the colloidal gold is protected from interference of high-salt ions, but when Cu exists in the solution²⁺In the presence of (A), the aptamer forms a specific conformation to recognize Cu²⁺And dissociated from the AuNPs surface, at which time the colloidal gold is not protected by the aptamer, and then the aggregation color changes from wine red to blue-violet under the high-salt environment.

Second, Experimental methods

The method for detecting copper ions by using the aptamer established by the colloidal gold colorimetric method shown in the table 6 comprises the following specific steps:

s1, preparing colloidal gold: the flask and rotor used for the synthesis were taken out from a 10% nitric acid solution, thoroughly washed with ultrapure water, and dried for use. Firstly, 50mL of ultrapure water is added into a triangular flask, and the uniform rotating speed is set to be 500 r/min. 1mL of 1% chloroauric acid is uniformly added into water, and 2mL of 1.5% trisodium citrate solution is rapidly added when the water is about to boil to prepare 13nm colloidal gold solution. Changing the color of the solution in the triangular flask from light yellow to dark purple to black and finally changing the solution into wine red, timing for 10min, stopping heating, cooling to room temperature, supplementing to 50mL with ultrapure water, and storing at 4 ℃ for later use;

s2, optimizing NaCl concentration: adding 80 mu L of ultrapure water into 100 mu L of 13nm colloidal gold solution, standing for 15min, then adding 20 mu L of NaCl solutions (0M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M and 1.1M) with different concentrations to ensure that the total volume of the system is 200 mu L, completely mixing, balancing for 5min, and scanning at 400-800 nm ultraviolet-visible spectrum. Each NaCl concentration corresponds to 3 replicates. The NaCl concentration of 0.8M is the best when the color of the colloidal gold solution begins to change.

Optimization of aptamer concentration: adding 40 mu L of aptamer solutions (0 mu M, 0.1 mu M, 0.2 mu M, 0.3 mu M, 0.4 mu M, 0.5 mu M, 0.6 mu M, 0.7 mu M and 0.8 mu M) with different concentrations into 100 mu L of 13nm colloidal gold solution for reaction for 15min, then adding 40 mu L of ultrapure water, then adding 20 mu L of 0.8M NaCl solution to ensure that the total volume of the system is 200 mu L, fully mixing the mixture, then balancing the mixture for 5min, and scanning the mixture at 400-800 nm ultraviolet-visible spectrum. Each aptamer concentration corresponds to 3 replicates. The optimal concentration of the aptamer is 0.3 mu M when the colloidal gold solution system is not discolored and is stable.

③ optimizing the pH value: firstly, 0.1mol/L HCl or 0.25mol/L K is used₂CO₃The pH values of the colloidal gold are adjusted to 5.0, 6.0, 7.0, 8.0 and 9.0. Wherein the pH of 6.0 is the environmental condition of the colloidal gold without any treatment. mu.L of 0.3. mu.M aptamer solution was added to 100. mu.L of 13nm colloidal gold solution for reaction for 15min, and then 40. mu.L of 100ng/mL Cu was added²⁺The solutions were reacted for 10min, respectively. And then, adding 20 mu L of 0.8M NaCl solution to ensure that the total volume of the system is 200 mu L, completely mixing uniformly, and then balancing for 5min to scan at 400-800 nm ultraviolet-visible spectrum. Each pH corresponds to 3 replicates. To explore the detection method of Cu in different pH environments²⁺The best detection level is determined under the environment of pH 6.0, and the pH 6.0 is the optimal pH.

Construction of Cu²⁺Aptamer detection system: at about 100. mu.L (extinction coefficient of 2.01X 10 according to beer's law 13nm AuNPs)⁸M^-1·cm^-1AuNPs concentration of about 14nM) was added to a solution of colloidal gold at 13nM pH 6.0 for reaction for 15min, 40. mu.L of 0.3. mu.M aptamer solution was added; then adding 40 mu L of different solutions of Cu respectively²⁺Reacting for 10 min; adding 20 μ L of 0.8M NaCl solution to make the total volume of the system 200 μ L, mixing completely, balancing for 5min, and passing through enzyme labeling instrument to obtain A_650nm/A_520nmThe value can be calculated by Cu²⁺And (4) concentration.

S3, sequentially detecting the gradient concentrations of 5ng/mL, 50ng/mL, 100ng/mL and 500ng/mL, 1000ng/mL and 2500ng/mL Cu²⁺Solution, linear working equation established by using the logarithmic value of the concentration of the copper ion solution as the abscissa, and corresponding A_650nm/A_520nmThe variation values are ordinate.

S4, pre-treating the sample, then using a standard labeling method to label the sample (namely adding a copper ion solution prepared by copper nitrate), using the copper ion aptamer colloidal gold to detect the copper ions in the sample in a colorimetric manner after labeling, and calculating the corresponding actual detection concentration of the copper ions through the established linear working equation to finish the quantitative detection of the copper ions in the labeled sample.

Wherein the pretreatment comprises the following steps: making the sample to be tested into sample to be tested by passing through 0.22 μm microporous filter membrane, and detecting whether the solution contains Cu by ICP-MS²⁺. Because the ICP-MS is used for detecting the condition that the content of copper ions in a sample is extremely low or even none before the detection is carried out by using the method, an experimental method of adding standard and recycling can be adopted.

Second, experimental results

The result is shown in fig. 3, where the working equation is specifically y 0.1878 logcpcu²⁺(ng/mL) +0.4247, wherein cCu²⁺Represents Cu²⁺Concentration, y represents A_650nm/A_520nmValue, linear relation good, R²0.9731, the method detection limit reaches 1.99ng/mL, completing the establishment of the detection method.

Example 3 aptamer colloidal gold colorimetric specificity evaluation

First, experiment method

Selecting nickel ion (Ni)²⁺) Aluminum ion (Al)³⁺) Cobalt ion (Co)²⁺) Cadmium ion (Cd)²⁺) Calcium ion (Ca)²⁺) Lead ion (Pb)²⁺) And barium ion (Ba)²⁺) A total of 7 ions were used as interfering ions to construct Cu in example 2²⁺And respectively carrying out incubation detection on the interfering ion solution with the concentration of 10 mu g/mL and the mixed ion solution thereof by using a colloidal gold colorimetric method under an aptamer detection system. Recording A after incubation of different interfering ions by the detection method_650nm/A_520nmValue, and A at a copper ion concentration of 100ng/mL_650nm/A_520nmThe values were compared.

Second, experimental results

The results are shown in FIG. 4. From these results, it was found that A was observed after 7 kinds of interfering ion solutions and their composition were mixed solutions having a concentration of 10. mu.g/mL_650nm/A_520nmAll values are Cu²⁺About 1/2, and addition of 100ng/mL Cu was also observed from the color²⁺The colloidal gold solution is obviously changed into purple, and the color of the colloidal gold is basically not changed after other interfering ions and the mixed solution are added. The comparison shows that: the colorimetric method does not generate specific phenomenon to other heavy metal ions, and the method has good construction specificity and high selectivity.

EXAMPLE 4 quantitative determination of copper ions in actual spiked samples

First, experiment method

Pretreatment: 3 samples of tap water, commercially available purified water and airless soda water are selected for detection. Wherein the sample pretreatment modes of tap water and commercially available purified water are as follows: collecting water sample, filtering with 0.22 μm microporous filter membrane to obtain sample to be tested, and detecting whether the solution contains Cu by ICP-MS²⁺The pretreatment mode of the airless soda water is to mix the sample with ultrapure water in a volume ratio of 1:1 and then treat the two samples.

After sample preparation is finished, a standard adding method is adopted to respectively add copper ion solution into commercial drinking water so that the standard adding concentration of the copper ions is 5ng/mL, and the standard adding concentration of the copper ions is 50ng/mL after the standard adding is carried out on an airless soda water sample. The detection method is used for detecting copper ions in 3 samples, corresponding actual detection concentration, detection recovery rate and relative standard deviation of the copper ions are calculated through the linear working equation established in the embodiment 2, quantitative detection of the copper ions in the 2 standard-added samples is completed, and ICP-MS is used for carrying out comparative detection on the samples.

Second, experimental results

The results show that the detection results of the 3 samples are consistent with the detection results of the ICP-MS. The results are shown in Table 7.

TABLE 7 aptamer colloidal gold colorimetry actual sample plus standard testTest Cu²⁺Results

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An aptamer for detecting copper ions, wherein the aptamer is a single-stranded DNA with the length of 42nt, and the nucleic acid sequence comprises GGCAGGGTGAGGTTGAGGTCCG bases.

2. The aptamer according to claim 1, wherein the aptamer sequence forms two stem-loop structures.

3. A kit for detecting copper ions by a nucleic acid aptamer-based colloidal gold colorimetric method, wherein the kit contains the copper ion nucleic acid aptamer according to any one of claims 1 or 2.

4. The kit according to claim 3, wherein the colloidal gold in the kit is prepared by reducing chloroauric acid with trisodium citrate.

5. The kit according to claim 3, wherein the colloidal gold in the kit is gold nanoparticles with a diameter of 13 nm.

6. Use of the copper ion aptamer according to any one of claims 1 or 2 for detecting copper ions.

7. Use of the copper ion aptamer of any one of claims 1 or 2 in the preparation of a kit for detecting copper ions.

8. A method for colorimetric detection of copper ions by using colloidal gold based on a copper ion aptamer, which is characterized in that the aptamer of claim 1 is fully reacted with a colloidal gold solution, fully reacted with a sample to be detected, fully reacted with NaCl, detected in absorbance values at 650nm and 520nm, and the concentration of the copper ions in the sample to be detected is calculated according to a standard curve.

9. The colorimetric detection method of copper ions from colloidal gold according to claim 8, wherein the colloidal gold is gold nanoparticles with a diameter of 13nm, and is prepared by reducing chloroauric acid with trisodium citrate.