Lead ion detection method based on nucleic acid amplification and kit

Abstract

Sensitivity is one of important parameters for evaluating the analysis performance of the sensor. A bivalent GR-5DNAzymes mediated colorimetric sensing analysis method is developed by utilizing specific binding of GR-5DNAzymes and Pb < 2 + >, and the method is combined with a self-assembly cascade amplification reaction of EDC and a Y-type probe. GR-5DNAzymes are automatically assembled through a palindromic sequence of a terminal extension chain to form bivalent DNAzymes, under the action of Pb < 2 + >, two hairpin probes containing rA sites are cracked at the same time, an initiation chain is released, self-assembly of EDC and a Y-type probe is started in sequence, a system finally forms a large amount of trivalent G quadruplex DNAzymes, an oxidation reaction of single-component TMB is formed, a reaction solution is turned into blue from colorless, and the reaction is completed. After the reaction is terminated by H2SO4, the color of the solution is finally in a yellow state. According to the method, EDC and a Y-type probe are used for self-assembly cascade connection, trivalent G quadruplex DNAzymes are generated, and the detection limit is 3.06 pM. The output of the detection signal is further enhanced, the detection sensitivity is improved, and some references can be provided for other multi-trigger cascade signal amplification strategies.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety detection, and particularly relates to a lead ion (Pb 2+ )Detection methods and kits. Background Art

[0002] Lead is a common heavy metal element. Lead ion (Pb 2+ ) is widely distributed in the environment and has high biological toxicity. Even trace amounts of Pb 2+ It will also eventually cause serious health problems to the human body due to the bioaccumulation effect. Lead ions in food usually come from a variety of channels, including soil, water sources, industrial pollution, pesticides, pollution during food processing, and lead content in food packaging materials. Many countries have enacted relevant laws to regulate Pb in water and food. 2+ The content of lead in water and food has serious effects on human health. Long-term excessive intake of lead may cause a variety of health problems, including nervous system damage, intellectual development disorders, anemia, kidney damage and reproductive system problems. Especially for children and pregnant women, lead intake may cause long-term effects on intellectual and behavioral development. Therefore, the use of Pb 2+ Screening of contaminated water and food is of great significance for ensuring food safety and human health and safeguarding the rights and interests of consumers, which depends on the development of effective and reliable detection methods.

[0003] At present, atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma atomic emission spectrometry (ICP-AES), dithizone spectrophotometry, etc. are widely used for the determination of Pb 2+ These traditional detection methods have the advantages of being able to detect multiple different elements at the same time and having a short analysis time. However, due to the defects of expensive equipment and instruments, high operating costs, complex pre-treatment of the substances to be tested and the need for professionally trained technicians, the application of traditional detection methods in on-site detection is limited and it is difficult to use them for on-site detection of Pb in water and food. 2+ Therefore, the development of Pb 2+ Detection methods are still of great importance. Summary of the invention

[0004] The present invention aims to provide a Pb 2+ In order to maximize the detection sensitivity, the present invention designs a colorimetric sensing analysis method based on bivalent GR-5 DNAzymes, combined with EDC and Y-type probe self-assembly cascade amplification reaction, to achieve Pb 2+ Highly sensitive detection. Figure 1As shown in the figure, the sequence of PD consists of a GR-5 DNA zymes region and a palindromic sequence region; the palindromic sequence can spontaneously form a double-stranded structure through base complementary pairing, so that PD automatically generates a bivalent GR-5 DNA zymes (2PD) after annealing. 2+ , 2PD cannot cleave H1, making the subsequent cascade amplification reaction impossible. 2+ When 2PD is combined with Pb 2+ It is activated by the interaction, and then catalyzes the cleavage of the phosphodiester bond at the rA site of H1, while releasing two initiator chains T, further starting the EDC cycle; T first binds to the toe region of the three-stranded complex E1-E2-E3, triggering a chain displacement reaction, gradually replacing E2, and generating a new three-stranded complex E1-T-E3; subsequently, E4 complementarily pairs with the newly exposed toe region of the E1-T-E3 complex, further displacing E1 and T chains, and generating a new double-stranded complex F-E3; the replaced T continues to initiate the next round of EDC reaction, and E1 and E2 respectively initiate the next-level YSA reaction. Both E1 and E2 contain complete initiator chain fragments that can trigger the YSA reaction, and can hybridize with the stem of H2 to open H2; subsequently, H3 and H4 are also opened one after another, eventually forming a Y-shaped probe, and the replaced E1 and E2 enter the next round of self-assembly reaction respectively; since the designed H2, H3, and H4 all contain G4 sequences, the formed Y-shaped probe has three G4 sequence sticky ends, at hemin and K + Under the action of Pb, the trivalent hemin / G quadruplex DNAzymes are folded and catalyzed to form the oxidation reaction of single-component TMB, so that the reaction solution changes from colorless to blue. After the reaction is terminated by H2SO4, the solution color finally turns yellow. The colorimetric signal response intensity of the solution system at 450nm is detected by UV-visible spectrophotometer. 2+ The concentration increases and becomes stronger, so it can achieve Pb 2+ Trace detection of Pb 2+ With the increase of concentration, the rate of PD-catalyzed cleavage of H1 increases, and the initiator chain T is quickly generated, which in turn activates the subsequent EDC cycle and Y-type probe self-assembly cycle reaction, produces a large number of Y-type G quadruplex DNAzymes, and enhances its catalytic activity, thereby achieving a greater signal amplification effect and ultimately realizing the enhancement of the system colorimetric signal.

[0005] The above-mentioned PD, hairpin probe H1, three-stranded complex E1-E2-E3, F, hairpin probes H2, H3, H4 are all dissolved in 20mM Tris-HCl buffer solution (20mM tris-hydroxymethylaminomethane, 120mM NaCl, 20mM KCl, 10mM MgCl2, pH value of the solution is adjusted to 7.4); the above-mentioned hemin is dissolved in 25mM HEPES buffer solution (25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 200mM NaCl, 20mM KCl, 0.05% Triton X-100, 1% DMSO, pH = 7.4).

[0006] The above-mentioned E1, E2, and E3 have partially the same base sequences, H2, H3, and H4 have partially the same base sequences, and the G-rich sequence H2 chain is designed with a guanine-rich sequence; the sequences of the PD, E1, E2, E3, F, H2, H3, and H4 are the sequences shown in Table 1 SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQID No.7, and SEQ ID No.8, and the G-rich sequence H2 chain is the sequence shown in Table 1 SEQ ID No.6.

[0007] In the above detection method, preferably, the PD concentration is 20, 40, 80, 100 nM; further preferably, the PD concentration is 40 nM.

[0008] In the above detection method, preferably, the concentration of the hairpin probe H1 is 20, 40, 60, 80, or 100 nM; and more preferably, the concentration of H1 is 60 nM.

[0009] In the above detection method, preferably, the concentration of the three-stranded complex E1-E2-E3 is 30, 40, 50, 60, or 70 nM; further preferably, the concentration of the three-stranded complex E1-E2-E3 is 50 nM.

[0010] In the above detection method, preferably, the probe F concentration is 30, 40, 50, 60, or 70 nM; further preferably, the F concentration is 100 nM.

[0010] In the above detection method, preferably, the concentration of the hairpin probe H2 is 50, 100, 150, 200, or 250 nM; further preferably, the H2 concentration is 150 nM.

[0011] In the above detection method, preferably, the concentration of the hairpin probe H3 is 50, 100, 150, 200, or 250 nM; and further preferably, the H3 concentration is 150 nM.

[0012] In the above detection method, preferably, the concentration of the hairpin probe H4 is 50, 100, 150, 200, or 250 nM; and further preferably, the H4 concentration is 150 nM.

[0013] In the above detection method, preferably, the hemin concentration is 200, 400, 600, 1000 nM; more preferably, the hemin concentration is 600 nM.

[0014] In the above detection method, preferably, the reaction time is 20, 40, 60, 80, or 100 min; and further preferably, the reaction time is 60 min.

[0015] In the above detection method, preferably, the reaction temperature is 4, 25, 37, or 45°C; and a further preferred reaction temperature is 37°C.

[0016] As the same technical concept, the present invention also provides a Pb 2+ Detection kit: The kit comprises the nucleic acid sequence and reagents described in the above detection method.

[0017] The above detection kit comprises a first container, wherein the first container contains the PD, which is diluted with a Tris-HCl buffer solution to a concentration of 2 μM before use.

[0018] The above detection kit comprises a second container, wherein the second container contains the hairpin probe H1, which is diluted with Tris-HCl buffer solution to a concentration of 2 μM before use.

[0019] The above detection kit comprises a third container, wherein the third container contains the three-chain complex E1-E2-E3, which is diluted with Tris-HCl buffer solution to a concentration of 10 μM before use.

[0020] The above detection kit includes a fourth container, which contains the hairpin probe F, which is diluted with Tris-HCl buffer solution to a concentration of 10 μM before use.

[0020] The above detection kit comprises a fifth container, wherein the fifth container contains the hairpin probe H2, which is diluted with Tris-HCl buffer solution to a concentration of 10 μM before use.

[0021] The above detection kit includes a sixth container, which contains the hairpin probe H3, which is diluted with Tris-HCl buffer solution to a concentration of 10 μM before use.

[0022] The above detection kit includes a seventh container, wherein the seventh container contains the hairpin probe H4, which is diluted with Tris-HCl buffer solution to a concentration of 10 μM before use.

[0023] The above detection kit comprises an eighth container, wherein the eighth container contains the hemin. Preferably, the hemin is dissolved in DMSO and diluted with a HEPES buffer solution to a concentration of 20 μM before use.

[0024] The above detection kit comprises a ninth container, and the ninth container contains the color developing substrate TMB.

[0025] The above-mentioned detection kit includes a tenth container, and the tenth container contains the 20mM Tris-HCl buffer solution (20mM tris-hydroxymethylaminomethane, 120mM NaCl, 20mM KCl, 10mM MgCl2, 1M HCl adjustment solution, pH=7.4).

[0026] The above detection kit comprises an eleventh container, and the eleventh container contains the 2M H2SO4 solution.

[0027] The above detection kit includes a twelfth container, which contains the 25mM HEPES buffer solution (25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 200mM NaCl, 20mM KCl, 0.05% Triton X-100, 1% DMSO, pH=7.4).

[0028] As the same technical concept, the present invention also provides a Pb 2+ Instructions for use of the test kit.

[0029] The above-mentioned detection kit uses a method, the above-mentioned detection kit includes a first container, the first container includes the PD, and is diluted with a Tris-HCl buffer solution to a concentration of 2 μM before use. The above-mentioned detection kit includes a second container, the second container includes the hairpin probe H1, and is diluted with a Tris-HCl buffer solution to a concentration of 2 μM before use. The above-mentioned detection kit includes a third container, the third container includes the three-chain complex E1-E2-E3, and is diluted with a Tris-HCl buffer solution to a concentration of 10 μM before use. The above-mentioned detection kit includes a fourth container, the fourth container includes the hairpin probe F, and is diluted with a Tris-HCl buffer solution to a concentration of 10 μM before use. The above-mentioned detection kit includes a fifth container, the fifth container includes the hairpin probe H2, and is diluted with a Tris-HCl buffer solution to a concentration of 10 μM before use. The above-mentioned detection kit includes a sixth container, the sixth container includes the hairpin probe H3, and is diluted with a Tris-HCl buffer solution to a concentration of 10 μM before use. The above-mentioned detection kit includes a seventh container, the seventh container contains the hairpin probe H4, and is diluted to a concentration of 10 μM with a Tris-HCl buffer solution before use. The above-mentioned detection kit includes an eighth container, the eighth container contains the hemin, preferably the hemin is dissolved in DMSO, and is diluted to a concentration of 20 μM with a HEPES buffer solution before use. The above-mentioned detection kit includes a ninth container, the ninth container contains the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). The above-mentioned detection kit includes a tenth container, the tenth container contains the 20mM Tris-HCl buffer solution (20mM trishydroxymethylaminomethane, 120mM NaCl, 20mM KCl, 10mM MgCl2, 1M HCl to adjust the solution pH to 7.4). The above-mentioned detection kit includes an eleventh container, the eleventh container contains the 2M H2SO4 solution. The above detection kit includes a twelfth container, which contains the 25mM HEPES buffer solution (25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 200mM NaCl, 20mM KCl, 0.05% Triton X-100, 1% DMSO, pH=7.4).

[0030] The method of using the above detection kit is to take 4 μL of solution from the first container, 6 μL of solution from the second container, 1 μL of solution from the third container, 2 μL of solution from the fourth container, 3 μL of solution from the fifth container, 3 μL of solution from the sixth container, and 3 μL of solution from the seventh container, and then add different concentrations of Pb 2+, mixed and reacted at 37°C for 60 minutes, then 6 μL of the solution in the eighth container was taken to continue the reaction for 30 minutes, 50 μL of the solution in the ninth container was added to react at room temperature for 15 minutes; 50 μL of the solution in the eleventh container was added to terminate the reaction; finally, 72 μL of the solution in the twelfth container was added, and the UV-visible spectrophotometer was used to measure the wavelength at 450 nm The absorbance at .

[0031] The present invention has outstanding substantive features and remarkable technical progress, which are as follows:

[0032] In summary, the present invention proposes a colorimetric sensing analysis method based on bivalent GR-5 DNAzymes, combined with EDC and Y-type probe self-assembly cascade amplification reaction. To achieve Pb 2+ This method shows four advantages.

[0033] First, the self-assembled cascade reaction does not have any separation and washing steps, the reaction time is short, complex design is avoided, and the operation is simple.

[0034] Second, the entire detection system that does not require the addition of any protease is conducive to reducing detection costs, while avoiding the decrease in detection stability caused by protease inactivation.

[0035] Third, the use of bivalent GR-5 DNAzymes effectively improved the signal amplification rate.

[0036] Fourth, the self-assembly cascade of EDC and Y-type probes was used to generate trivalent G-quadruplex DNAzymes, which further enhanced the output of the detection signal and improved the detection sensitivity.

[0037] Instruction Manual

[0038] Figure 1 It is a Pb 2+ Detection schematic diagram.

[0039] Figure 2 Polyacrylamide gel electrophoresis characterization of the entropy-driven catalytic reaction and Y-cascade amplification strategy mediated by type probe self-assembly bivalent GR-5 DNAzymes.

[0040] Figure 3 It is the feasibility diagram of the sensing platform detection.

[0041] Figure 4 It is the concentration optimization diagram of PD.

[0042] Figure 5 This is the concentration optimization diagram of the hairpin probe H1.

[0043] Figure 6It is the concentration optimization diagram of E1-E2-E3 mixture.

[0044] Figure 7 This is the concentration optimization diagram of hairpin probe F.

[0045] Figure 8 This is the concentration optimization diagram of hairpin probes H2, H3, and H4.

[0046] Fig. 9 This is the hemin concentration optimization diagram.

[0047] Fig.10 It is the system reaction time optimization diagram.

[0048] Fig.11 It is the temperature optimization diagram of cascade reaction.

[0049] Fig.12 Figure 2 is a kinetic colorimetric analysis of the cascade amplification strategy of monovalent and bivalent GR-5 DNAzymes.

[0050] Fig.13 Different concentrations of Pb 2+ Detect the UV absorption spectrum.

[0051] Fig.14 The wavelength of 450nm is different for different concentrations of Pb 2+ Linear relationship diagram of the determination.

[0052] Fig.15 is the specific analysis diagram, which respectively shows the blank, Ni 2+ 、Zn 2+ , Ca 2+ , Mn 2+ , Ba 2+ 、Co 2+ . DETAILED DESCRIPTION

[0054] The present invention will be further described below in conjunction with specific embodiments.

[0055] Tris, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), hemin, TritonX-100, and dimethyl sulfoxide (DMSO) were purchased from Shanghai Shenggong Biotechnology Co., Ltd.; sodium chloride (NaCl), potassium chloride (KCl), and magnesium chloride (MgCl2) were purchased from Xilong Science. Ultrapure water (18.2MQ·cm) was used in this experiment, and the DNA used in the experiment was synthesized by Shanghai Shenggong Biotechnology Co., Ltd. and purified by high performance liquid chromatography (HPLC).

[0056] Table 1 shows the sequences of PD, hairpin probes H1 and H2, H3, H4, E1, E2, E3, F, PD-0, and YT, respectively.

[0057] Table 1: Probe sequences

[0058] A Pb 2+ The detection method is as follows: 4 μL of solution is taken from the first container, 6 μL of solution is taken from the second container, 1 μL of solution is taken from the third container, 2 μL of solution is taken from the fourth container, 3 μL of solution is taken from the fifth container, 3 μL of solution is taken from the sixth container, and 3 μL of solution is taken from the seventh container, and then different concentrations of Pb are added. 2+ , mix and react at 37°C for 60 minutes, then take 6 μL of the solution from the eighth container and continue to react for 30 minutes, add 50 μL of the solution from the ninth container and react at room temperature for 15 minutes; add 50 μL of the solution from the eleventh container to terminate the reaction; finally take 72 μL of the solution from the twelfth container and measure the absorbance at 450 nm using a UV-visible spectrophotometer.

[0059] The recognition probe PD, hairpin probe H1, hairpin probe H2, hairpin probe H3, hairpin probe H4, hairpin probe F, E1, E2, and E3 are all dissolved in Tris-HCl buffer; hemin is dissolved in dimethyl sulfoxide (DMSO) and diluted in HEPES buffer.

[0060] Example 2: Pb 2+ Detection feasibility analysis

[0061] In order to verify the feasibility of the above cascade amplification strategy design principle, PAGE with a concentration of 10% was used to characterize it. Figure 2 As shown, lane 1 contains only PD, and only bright bands with higher migration positions appear, indicating that the designed PD can stably form bivalent GR-5DNAzymes, namely 2PD, after annealing treatment; lanes 2 to 6 are H1, E1, E2, E3, and F, respectively; lane 7 is a single F-E3 complex formed after annealing treatment; lane 8 is a single E1-E2-E3 complex; lane 9 is a sample after the mixed reaction of 2PD, H1, E1-E2-E3 complex, and F, and no new bands appear, indicating that they are in the absence of Pb 2+ When Pb is added to the reaction system, it remains stable and can coexist in the same system. 2+ After that, the bands of H1, E1-E2-E3, and F in lane 10 almost completely disappeared, and four new bands appeared. The migration positions of three of the new bands corresponded to those of the F-E3 complex, E1, and E2, respectively, and the migration position of the other new band was slightly lower than that of H1, proving that in the presence of Pb2+ When present, 2PD can specifically recognize and bind Pb 2+ , thus activating the catalytic activity, catalyzing the cleavage of H1 to produce the initiator chain T, resulting in the disappearance of the H1 band and the appearance of a new band with a migration rate slightly faster than that of the H1 band; the generated T successfully initiates the EDC cycle, generates the F-E3 complex and replaces E1 and E2. Therefore, Figure 2 The electrophoresis results confirmed that the bivalent GR-5 DNAzymes cyclic cleavage reaction and EDC cyclic reaction were 2+ The electrophoresis results of the Y-type probe self-assembly are shown in Figure 2 Lanes a to d are E1, H2, H3, and H4, respectively; Lane e contains a mixture of H2, H3, and H4, and no new bands appear, indicating that H2, H3, and H4 can stably coexist in the same solution system when EDC circulation product E1 does not appear, and there is no self-hybridization reaction; Lane f is the product of the mixed reaction of E1 and H2, and the new band that appears is caused by the E1-H2 complex generated after E1 opens H2; When E1, H2, and H3 react in a mixed reaction, Lane g shows a similar migration position. For the brighter band of E1-H2 complex, the band corresponding to E1-H2 complex disappears at the same time, proving that the reaction generates E1-H2-H3 complex; when H4 is added to the reaction, a bright band with a very slow migration rate appears in lane h, and the corresponding bands of H2, H3, and H4 also disappear, indicating that H1-H2-H3 complex is successfully generated; the electrophoresis results of lanes f–h prove that H2, H3, and H4 gradually assemble to form a Y-shaped probe under the action of E1, namely, H2-H3-H4 complex. The above two electrophoresis results prove that the entropy-driven catalytic reaction based on bivalent GR-5 DNAzymes and the cascade amplification strategy of Y-shaped probe self-assembly are used to detect Pb 2+ It is feasible.

[0062] The changes in the absorption spectra and colors of the solutions under different conditions were further verified by UV-visible spectrophotometer and smartphone camera. Figure 3 As shown, no Pb 2+ The absorbance of the existing detection system at 450nm is very weak and there is no obvious color change, which proves that the DNA probe in the detection system does not undergo hybridization reaction and maintains the original stable structure, so the background signal generated is very low; adding 25nM Pb 2+ After that, the absorbance of the detection system increased significantly and the color of the solution became darker, indicating that 2PD 2+ In the case of catalytic H1 cleavage to produce initiator chain T, T starts the EDC cycle and releases E1 and E2, which in turn triggers the self-assembly of the Y-shaped probe to form a Y-shaped G quadruplex DNAzymes, catalyzing the single-component TMB to produce a color change. These results show that the design of colorimetric analysis can realize Pb2+ Quantitative detection of .

[0063] Example 3: PD concentration optimization.

[0064] PD concentration has an important influence on the signal amplification efficiency and background signal of the system. When the PD concentration is too low, the catalytic cracking efficiency of PD at a lower concentration on H1 is low, thereby weakening the signal amplification effect of the entire system. When the concentration is too high, the possibility of PD opening H1 increases with the continued increase in PD concentration, resulting in an increase in background signal. PD with concentrations of 20, 40, 60, 80, and 100 nM was added to the reaction system, and the other conditions were kept unchanged according to the steps of Example 1 to optimize it. The results are shown in Figure 1. Figure 4 As shown in the figure, when the PD concentration is 40 nM, the ratio of A / A0 is the largest. Therefore, the PD concentration of 40 nM was selected as the optimal condition.

[0065] Example 4: Optimization of hairpin probe H1 concentration.

[0066] The concentration of H1 has an important influence on the signal amplification efficiency and background signal of the system. When the concentration of H1 is too low, the signal amplification effect is poor. When the concentration is too high, H1 may initiate a nonspecific EDC cycle amplification reaction, thereby triggering the self-assembly of the Y-type probe to form a Y-type G quadruplex DNAzymes, which increases the background signal in the system. H1 with concentrations of 20, 40, 60, 80, and 100 nM was added to the reaction system, and the system was optimized according to the steps of Example 1 while keeping other conditions unchanged. The results are shown in FIG. Figure 5 As shown in the figure, when the H1 concentration is 60 nM, the ratio of A / A0 is the largest. Therefore, the H1 concentration of 60 nM is selected as the optimal condition.

[0067] Example 5: Optimization of E1-E2-E3 mixture concentration.

[0068] The concentration of E1-E2-E3 has an important influence on the signal amplification efficiency and background signal of the system. If the concentration of E1-E2-E3 is too low, the release amount of E1 and E2 may be reduced, resulting in an increased possibility of subsequent incomplete reactions, thereby making the signal amplification effect of the system worse. If the concentration is too high, it may cause DNA chain waste and produce higher background signals, reducing the signal-to-noise ratio. E1-E2-E3 with concentrations of 30, 40, 50, 60, and 70 nM was added to the reaction system, and the other conditions were kept unchanged according to the steps of Example 1 to optimize it. The results are shown in Figure 2. Figure 6 As shown in the figure, when the concentration of E1-E2-E3 is 50 nM, the ratio of A / A0 is the largest. Therefore, 50 nM of E1-E2-E3 concentration is selected as the optimal condition.

[0069] Example 6: Optimization of hairpin probe F concentration.

[0070] The concentration of F has an important impact on the background of the system. If the concentration is too low, the release of E1 and E2 may be reduced, resulting in an increased possibility of incomplete reactions, thereby worsening the signal amplification effect of the system. If the concentration is too high, it may cause DNA chain waste and generate higher background signals, reducing the signal-to-noise ratio. Set the reaction concentrations to 30, 40, 50, 60, and 70 nM, and optimize them according to the steps of Example 1 while keeping other conditions unchanged. The results are shown in the figure. Figure 7 As shown, when the F concentration is 50 nM, the ratio of A / A0 is the largest, so 50 nM is selected as the optimal concentration of F for the next experiment.

[0071] Example 7: Optimization of the concentration of hairpin probes H2, H3, and H4.

[0072] The Y-shaped G quadruplex is formed by the self-assembly of H2, H3, and H4 through the Y-shaped probe, and its concentration affects the generation of the system signal. When the concentration of H2, H3, and H4 is too low, the signal amplification effect is poor. When the concentration is too high, the background signal of the system may be enhanced, thereby reducing the ratio of A / A0. H2, H3, and H4 with concentrations of 50, 100, 150, 200, and 250 nM were added to the reaction system, and the other conditions were kept unchanged according to the steps of Example 1 to optimize it. The results are shown in Figure 2. Figure 8 As shown in the figure, when the concentration of H2, H3, H4 is 150 nM, the ratio of A / A0 is the largest. Therefore, the concentration of H2, H3, H4 is 150 nM as the optimal condition.

[0073] Example 8: Optimization of hemin concentration.

[0074] The hemin concentration affects the catalytic activity and thus the generation of the reaction system signal. When the hemin concentration is too low, the catalytic activity is low, and the production of hemin / G quadruplex DNAzymes is reduced, resulting in a decrease in the generation rate of the final signal of the system; when the hemin concentration is too high, the nonspecific amplification reaction in the system is enhanced, thereby increasing the possibility of background signal enhancement. The hemin concentrations were set to 200, 400, 600, 800, and 1000 nM, respectively, and the hemin concentrations were optimized according to the steps of Example 1 while keeping other conditions unchanged. The results are as follows: Fig. 9 As shown in the figure, the ratio of A / A0 was the highest when the concentration was 600 nM. Therefore, 600 nM was selected as the optimal reaction concentration of hemin for the next step of the experiment.

[0075] Example 9: Reaction time optimization.

[0076] The detection performance of the system is also related to the cascade reaction time. When the reaction has not reached equilibrium, appropriately extending the reaction time is conducive to generating more trivalent G-quadruplex DNAzymes, thereby obtaining a stronger colorimetric signal. Reaction times of 20, 40, 60, 80, and 100 min were selected, and the reaction time was optimized according to the steps of Example 1 while keeping other conditions unchanged. The results are shown in FIG. Fig.10 As shown in the figure, when the reaction time reaches 60 min, the reaction efficiency reaches the maximum and the ratio of A / A0 also reaches the maximum. Therefore, 60 min is selected as the optimal reaction time for the next step of the experiment.

[0077] Example 10: Reaction temperature optimization.

[0078] The reaction temperature has an important influence on the signal generation of the reaction system. When the temperature is too low, the reaction rate of the entire system is low; when the temperature is too high, it will cause nonspecific binding between hairpins, making the background signal higher. The reaction temperature was set to 4, 25, 37, and 45°C respectively. According to the steps of Example 1, keeping other conditions unchanged, the reaction temperature of the system was optimized. The results are shown in Fig.11 As shown in the figure, the ratio of A / A0 reaches the maximum at 37°C. Therefore, 37°C is selected as the optimal reaction temperature.

[0079] Example 11: Reaction temperature optimization.

[0080] Example 12: Validation of the dynamic colorimetric analysis results between the monovalent and bivalent GR-5 DNAzymes cascade amplification strategies.

[0081] The PD involved in the monovalent GR-5 DNAzymes cascade amplification strategy does not have a palindromic sequence, while the PD in the bivalent GR-5 DNAzymes cascade amplification strategy self-assembles to form bivalent DNAzymes (2PD) due to the palindromic sequence designed to promote dimer formation. 2+ In the presence of , both PD and 2PD can catalyze the cleavage of hairpin H1, further initiate the cascade signal amplification reaction to generate Y-shaped G quadruplex, catalyze TMB oxidation reaction, and then generate colorimetric signal. Fig.12 As shown, in the absence of Pb 2+ When Pb 2+When the presence of GR-5 DNAzymes was present, the colorimetric signal of the monovalent GR-5 DNAzymes cascade amplification strategy became stronger with the increase of reaction time, and basically reached equilibrium at around 90 minutes; while the colorimetric signal of the bivalent GR-5 DNAzymes cascade amplification strategy increased sharply and reached saturation at 60 minutes, which was 30 minutes shorter than the monovalent cascade reaction time. Within the same reaction time, the absorbance of the bivalent reaction system was higher than that of the monovalent reaction system. The above results prove that within the same reaction time, the bivalent GR-5 DNAzymes cascade amplification system has a higher signal amplification efficiency and is more conducive to achieving Pb 2+ Trace detection.

[0082] Example 13: Different concentrations of Pb 2+ Detect UV absorption spectrum.

[0083] In order to evaluate the detection performance of the designed detection method, after obtaining the optimal conditions, we used UV-visible spectrophotometer to measure a series of different concentrations of Pb 2+ The colorimetric signal responsiveness intensity in the wavelength range of 420-500nm. Fig.13 As shown in the figure, the colorimetric signal response intensity at 450 nm increases with the increase of Pb 2+ The concentration increases, which is because as Pb 2+ With the increase of concentration, the rate of PD-catalyzed cleavage of H1 increases, and the initiator chain T is quickly generated, which in turn activates the subsequent EDC cycle and Y-type probe self-assembly cycle reaction, produces a large number of Y-type G-quadruplex DNAzymes, and finally achieves the enhancement of the colorimetric signal of the system. Fig.13 The results in the illustration show that the solution color of the detection system changes with the increase of Pb 2+ It gradually deepens with the increase of concentration. Fig.14 Pb 2+ The relationship between concentration and absorbance at 450nm is within the range of 5pM–25nM. 2+ The logarithm of the concentration and the absorbance value of this method have a good linear relationship, and the linear equation is: A = 0.5178 + 0.1463lg·C, R 2 =0.9978; the detection limit calculated based on the blank sample concentration plus three times the standard deviation principle is 3.06pM. Table 2 proves that compared with some other reported methods, the detection limit of the analytical method established in this chapter is lower, and its detection performance is better than other reported detection systems.

[0084] Example 14: Specificity analysis.

[0085] Six divalent metal ions were selected as interfering ions to selectively analyze the colorimetric detection system. 2+ 、Zn 2+ , Ca2+ , Mn 2+ , Ba 2+ 、Co 2+ and 10 nM Pb 2+ They were added to the reaction system for reaction, and then the colorimetric signal intensity at 450nm was measured; the blank control group was replaced with an equal volume of 20mM Tris-HCl buffer solution instead of the measured metal ion solution. Fig.15 As shown, compared with the blank control group, the concentration of Pb 2+ The absorbance values ​​of other interfering metal ions at 10 times did not change significantly, and the colors of the reaction solutions were similar; while only adding 10 nM Pb 2+ The absorbance of the solution increased significantly and the color of the solution deepened significantly. The results show that the colorimetric analysis method is effective for Pb 2+ Has good selectivity.

[0086] Example 15: Actual sample detection.

[0087] In order to prove that this analytical method can be used to detect Pb in real samples 2+ In this study, Pb in drinking water and fish 2 + The spike recovery method was used to detect Pb 2+ The spike recovery was calculated. The results are shown in Table 2. The spike recovery in drinking water and fish ranged from 99.47% to 111.09%, and the relative standard deviation fluctuated from 4.99% to 8.63%. The results show that the analytical method designed in this chapter is effective in the determination of Pb in drinking water and fish. 2+ The actual detection effect is good. Table 2 Pb in drinking water and fish samples 2+ Detection of spike recovery experiment Note: “ND” means not detected.

Claims

1. A lead ion (Pb 2+ ) detection system, comprising hemin, a chromogenic substrate and a reaction termination solution, characterized in that The components also include PD, hairpin probes H1, E1, E2, E3, H2, H3, and H4.

2. Pb according to claim 1 2+ Detection system, the sequence of the PD is the sequence shown in Table 1 SEQ ID No.1, the sequence of the PD-0 is the sequence shown in Table 1 SEQ ID No.2, the sequence of the hairpin probe H1 is the sequence shown in Table 1 SEQ ID No.3, the sequence of the hairpin probe E1 is the sequence shown in Table 1 SEQ ID No.4, the sequence of the hairpin probe E2 is the sequence shown in Table 1 SEQ ID No.5, the sequence of the hairpin probe E3 is the sequence shown in Table 1 SEQ ID No.6, the sequence of the hairpin probe F is the sequence shown in Table 1 SEQ ID No.7, the sequence of the hairpin probe YT is the sequence shown in Table 1 SEQ ID No.8, the sequence of the hairpin probe H2 is the sequence shown in Table 1 SEQ ID No.9, the sequence of the hairpin probe H3 is the sequence shown in Table 1 SEQ ID No.10, and the sequence of the hairpin probe H4 is the sequence shown in Table 1 SEQ ID No.

11.

3. Pb according to claim 1 2+ The detection system is characterized in that The chromogenic substrate is TMB.

4. Pb according to claim 1 2+ The detection system is characterized in that The reaction termination solution is H2SO4 solution.

5. A method for preparing the Pb as claimed in claim 1 2+ The method of the detection system is characterized in that The steps include: (1) the PD and hairpin probes H1, E1, E2, E3, H2, H3, and H4 were annealed at 95° C. for 5 min respectively; (2) After the treatment in step (1), different concentrations of Pb were added to PD, hairpin probes H1, E1-E2-E3, F, H2, H3, and H4. 2 + , mixed and reacted at 37°C for 60 min, then hemin (600 nM) was added and the reaction continued for 30 min; (3) After the treatment of steps (1) and (2), 50 μL of single-component TMB colorimetric solution was added to the reaction solution, and the reaction was continued at room temperature for 15 min; (4) Terminate the reaction using 50 μL of 2M H2SO4 solution; (5) Add 20 mM Tris-HCl buffer solution to make the final volume 200 μL, and measure the absorbance at 450 nm using a UV-visible spectrophotometer.

6. Pb according to claim 4 2+ The detection method is characterized in that The annealing treatment is carried out in a 95° C. water bath for 5 minutes, followed by slow cooling to room temperature.

7. Pb according to claims 1 to 5 2+ The detection method is characterized in that The PD concentration is 40nM, the hairpin probe H1 concentration is 60nM, the E1-E2-E3 concentration is 50nM, the F concentration is 50nM, the hairpin probe H2 concentration is 150nM, the H3 concentration is 150nM, the hairpin probe H4 concentration is 150nM, the hemin concentration is 300nM, and the 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide concentrations are both 200μM.

8. A method for preparing the Pb as claimed in claim 1 2+ A detection kit for the detection method, characterized in that The kit further comprises a first container, wherein the first container contains the PD; The kit further comprises a second container, wherein the second container contains the hairpin probe H1; The kit further comprises a third container, wherein the third container contains the E1-E2-E3 complex; The kit further comprises a fourth container, wherein the fourth container contains the hairpin probe F; The kit further comprises a fifth container, wherein the fifth container contains the hairpin probe H2; The kit further comprises a sixth container, wherein the sixth container contains the hairpin probe H3; The kit further comprises a seventh container, wherein the seventh container contains the hairpin probe H4; The kit further comprises an eighth container, wherein the eighth container contains the hemin; The kit further comprises a ninth container, wherein the ninth container contains the 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide; The kit further comprises a tenth container, wherein the tenth container comprises the 20 mM Tris-HCl buffer solution: 20 mM tris(hydroxymethyl)aminomethane, 120 mM NaCl, 20 mM KCl, 10 mM MgCl2, 1 M HCl adjusting solution, pH=7.

4. The kit further comprises an eleventh container, wherein the eleventh container contains the H2SO4 solution. The kit further comprises a twelfth container, which comprises the 25 mM HEPES buffer solution (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 200 mM NaCl, 20 mM KCl, 0.05% TritonX-100, 1% DMSO, pH=7.4).

9. A Pb 2+ The method for using the detection kit is characterized by comprising the Pb 2+ The detection kit further comprises the following steps: (1) Take 4 μL of the solution in the first container, 6 μL of the solution in the second container, 1 μL of the solution in the third container, 2 μL of the solution in the fourth container, 3 μL of the solution in the fifth container, 3 μL of the solution in the sixth container, and 3 μL of the solution in the seventh container, then add different concentrations of Pb 2+ , mixed and reacted at 37°C for 60 min; (2) adding 6 μL of the solution in the eighth container and continuing the reaction for 30 min; (3) adding 50 μL of the solution in the ninth container and reacting at room temperature for 15 min; (4) adding 50 μL of the solution in the eleventh container to terminate the reaction; (5) Add 72 μL of the solution in the container described in step 12, and measure the absorbance at 450 nm using a UV-visible spectrophotometer.

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