Method and kit for high-sensitivity colorimetric detection of lead ions

Abstract

Sensitivity is one of important parameters for evaluating the analysis performance of the sensor. A colorimetric analysis method of a GR-5DNAzymes mediated CHA-HCR cascade amplification strategy is developed by utilizing specific binding of GR-5DNAzymes and Pb < 2 + >, so that the sensitivity of the method is improved, and the method is used for detecting Pb < 2 + >. The hemin / G quadruplex DNA enzyme is used as a signal generation element to construct a colorimetric sensing technology for high-sensitivity detection of Pb < 2 + >. According to the method, GR-5DNAzymes and Pb < 2 + > are specifically combined, a hairpin H1 is catalytically cracked, CHA circular reaction is started to form a bivalent initiation chain compound, two ends of the compound simultaneously initiate HCR reaction to form longer and more long-chain products with G quadruplex DNAzymes fragments, TMB is catalytically oxidized under the induction of H2O2 to generate blue substances, and finally the blue substances are converted into stable yellow substances under the termination action of H2SO4. According to the method, the next cascade reaction can be quickly started through a bivalent trigger chain compound formed by CHA, the detection limit is 16.71 pM, the signal amplification effect is enhanced, 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. 2+ Pb is widely distributed in the environment and has high biological toxicity. 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, it is still of great significance to develop a lead ion detection method with simple operation, low cost and strong stability. Summary of the invention

[0004] The present invention aims to provide a Pb 2+ In order to maximize the detection sensitivity, the present invention has designed a design principle based on GR-5 DNAzymes-mediated catalytic hairpin self-assembly and hybridization chain reaction cascade amplification strategy for detecting trace amounts of Pb 2+ ,like Figure 1As shown in the figure, the system consists of three parts: specific enzyme cleavage reaction of GR-5 DNAzymes, CHA, and HCR. 2+ When it does not exist, the reactants in the system are in a stable state and exist independently of each other; when the target substance Pb 2+ When present, PD and Pb 2+ It binds specifically and catalytically cleaves the rA site of H1, releasing T1 to participate in the CHA reaction, and then PD enters the next round of enzyme cleavage reaction; T1 performs base complementary pairing with the exposed sticky end region of H2, gradually opening the hairpin H2, and then the newly exposed sequence of H2 combines with H3 to form a stable H2-H3 complex, and the replaced T1 enters the next round of CHA cycle; both ends of the formed H2-H3 complex can activate the HCR reaction, because one end of H2-H3 contains a complete initiation chain sequence that can trigger HCR, and the other end is composed of two split initiation sequence fragments. After the sticky ends on both sides of the H2-H3 complex simultaneously open the hairpin H4, H5 undergoes hybridization reaction with the newly released H4 sequence, and the newly exposed H5 sticky end can destroy the H4 stem structure and then open H4. After multiple rounds of cyclic reactions, a "work-type" long-chain DNA with multiple G-rich sticky end chains is finally formed, and its G-rich sequence is in the K + Under the action of H2O2, it binds to hemin, folds to form hemin / G quadruplex DNAzymes, and catalyzes the oxidation of TMB to produce a blue substance under the induction of H2O2, which is finally converted into a stable yellow substance after termination by H2SO4. The solution system has a maximum absorption peak at 450nm when detected by UV-visible spectrophotometer. 2+ As the concentration increases, the detected colorimetric signal gradually increases. Therefore, the detection of Pb 2+ Quantitative detection of GR-5 DNAzymes and Pb 2+ It specifically binds and catalyzes the cleavage of hairpin H1, initiating the CHA cycle reaction to form a divalent initiator chain complex. Both ends of the complex simultaneously trigger HCR reactions to form longer and more long-chain products with G-quadruplex DNAzymes fragments, thereby increasing the local concentration of hemin / G-quadruplex DNAzymes and improving its catalytic activity, thereby achieving a greater signal amplification effect.

[0005] The above-mentioned PD and hairpin probes H1, H2, H3, H4, and H5 were all dissolved in 20mM Tris-HCl buffer solution (20mM tris(hydroxymethyl)aminomethane, 120mM NaCl, 20mM KCl, 10mM MgCl2, pH=7.4); the above-mentioned hemin was 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 H1, H2, H3, H4, and H5 have partially the same base sequence, and the G-rich sequence H2 chain is designed with a guanine-rich sequence; the sequences of the H1, H2, H3, H4, and H5 are the sequences shown in Table 1 SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6, and the G-rich sequence H2 chain is the sequence shown in Table 1 SEQ ID No.2.

[0007] In the above detection method, preferably, the PD concentration is 50, 75, 100, 125, or 150 nM; and further preferably, the PD concentration is 75 nM.

[0008] In the above detection method, preferably, the concentration of the hairpin probe H1 is 50, 75, 100, 125, or 150 nM; and further preferably, the concentration of the hairpin H1 is 75 nM.

[0009] In the above detection method, preferably, the concentration of the hairpin probe H2 is 50, 75, 100, 125, or 150 nM; further preferably, the concentration of the hairpin H2 is 100 nM.

[0010] In the above detection method, preferably, the concentration of the hairpin probe H3 is 50, 75, 100, 125, or 150 nM; further preferably, the concentration of the hairpin H3 is 100 nM.

[0011] In the above detection method, preferably, the concentration of the hairpin probe H4 is 50, 100, 150, 200, or 250 nM; further preferably, the concentration of the hairpin H4 is 200 nM.

[0012] In the above detection method, preferably, the concentration of the hairpin probe H5 is 50, 100, 150, 200, or 250 nM; further preferably, the concentration of the hairpin H5 is 200 nM.

[0013] In the above detection method, preferably, the hemin concentration is 100, 200, 300, 400, 500 nM; more preferably, the hemin concentration is 300 nM.

[0014] In the above detection method, preferably, the reaction time is 40, 60, 80, 100, or 120 min; and a further preferred reaction time is 80 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 25°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 Tris-HCl buffer solution to a concentration of 10 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 10 μM before use.

[0019] The above detection kit includes a third container, wherein the third container contains the hairpin probe H2, which is diluted with Tris-HCl buffer solution to a concentration of 10 μM before use.

[0020] The above detection kit comprises a fourth container, wherein the fourth container contains the hairpin probe H3, which is diluted with Tris-HCl buffer solution to a concentration of 10 μM before use.

[0021] The above detection kit comprises a fifth container, wherein the fifth container contains the hairpin probe H4, which is diluted with Tris-HCl buffer solution to a concentration of 10 μM before use.

[0022] The above detection kit comprises a sixth container, wherein the sixth container contains the hairpin probe H5, which is diluted with Tris-HCl buffer solution to a concentration of 10 μM before use.

[0023] The above detection kit comprises a seventh container, wherein the seventh 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 an eighth container, wherein the eighth container contains the color developing substrate TMB.

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

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

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

[0028] As the same technical concept, the present invention also provides a method for using a lead ion detection 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 10 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 10 μM before use. The above-mentioned detection kit includes a third container, the third 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 fourth container, the fourth 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 fifth container, the fifth container includes the hairpin probe H4, 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 H5, 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 hemin, preferably the hemin is dissolved in DMSO and diluted to a concentration of 20 μM with a HEPES buffer solution before use. The above-mentioned detection kit includes an eighth container, the eighth container contains the chromogenic substrate TMB. The above-mentioned detection kit includes a ninth container, the ninth container contains the 20mM Tris-HCl buffer solution (20mM trishydroxymethylaminomethane, 120mM NaCl, 20mM KCl, 10mM MgCl2, 1M HCl adjusts the solution pH to 7.4). The above-mentioned detection kit includes a tenth container, the tenth container contains the 2M H2SO4 solution. The above detection kit comprises an eleventh container, which contains the 25 mM HEPES buffer solution (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 200 mM NaCl, 20 mM KCl, 0.05% Triton X-100, 1% DMSO, pH=7.4).

[0030] The method of using the above detection kit is to take 1.5 μL of the solution from the first container, 1.5 μL of the solution from the second container, 2 μL of the solution from the third container, 2 μL of the solution from the fourth container, 4 μL of the solution from the fifth container each added to the solution to be tested, and 4 μL of the solution from the sixth container, and then add different concentrations of Pb 2+, mix and react at 25°C for 80 minutes; then add 3 μL of the solution in the seventh container, mix and react at 25°C for 30 minutes; add 50 μL of the solution in the eighth container, react at room temperature for 15 minutes; add 50 μL of the solution in the tenth container to terminate the reaction; add 82 μL of the solution in the eleventh container, and measure the absorbance at 450 nm with a UV-visible spectrophotometer.

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

[0032] In summary, the present invention proposes a method for RNA cleavage of Pb based on GR-5 DNAzymes. 2+ Based on the high specific recognition ability of Pb and the efficient amplification ability of CHA-HCR cascade reaction, a highly sensitive Pb 2+ Colorimetric analysis method. This method shows four advantages.

[0033] First, the cascade amplification reaction does not have any separation and washing steps, the reaction time is short, and complex design is avoided. This method can quickly start the next cascade reaction through the bivalent trigger chain complex formed by CHA, enhance the signal amplification effect, and provide some references for other multiple trigger cascade signal amplification strategies.

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

[0035] Third, compared with traditional CHA-HCR, the CHA product of this method contains two HCR initiating chain fragments, which can efficiently trigger HCR and produce more and longer long-chain complexes with G quadruplex fragments. After binding to hemin and folding, it catalyzes the TMB oxidation reaction to generate a colorimetric signal, realizing trace Pb 2+ Highly sensitive detection.

[0036] Fourth, the present invention provides a Pb 2+ The detection method and kit realize the detection of trace amounts of Pb in actual samples 2+ This method can quickly start the next cascade reaction through the bivalent trigger chain complex formed by CHA, enhance the signal amplification effect, and provide some references for other multiple trigger cascade signal amplification strategies.

[0037] Instruction Manual

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

[0039] Figure 2This is a polyacrylamide gel electrophoresis characterization diagram of the HCR strategy and CHA cycle.

[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 6 This is the concentration optimization diagram of hairpin probes H2 and H3.

[0044] Figure 7 This is the concentration optimization diagram of hairpin probes H4 and H5.

[0045] Figure 8 This is the hemin concentration optimization diagram.

[0046] Fig. 9 It is the system reaction time optimization diagram.

[0047] Fig.10 It is the temperature optimization diagram of cascade reaction.

[0048] Fig.11 Different concentrations of Pb 2+ Detect the UV absorption spectrum.

[0049] Fig.12 The wavelength of 450nm is different for different concentrations of Pb 2+ Linear relationship diagram of the determination.

[0050] Fig.13 is the specific analysis diagram, which respectively shows the blank, Ni 2+ 、Zn 2+ , Ca 2+ , Mn 2+ , Ba 2+ 、Co 2+ . DETAILED DESCRIPTION

[0052] The present invention will be further described below in conjunction with specific embodiments.

[0053] 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).

[0054] Table 1 shows the recognition probe PD, hairpin probes H1, H2, H3, H4, and H5, respectively.

[0055] Table 1: Probe sequences

[0056] Example 1: A Pb 2+ The detection method comprises the following steps: taking 1.5 μL of the solution from the first container, taking 1.5 μL of the solution from the second container, taking 2 μL of the solution from the third container and the fourth container respectively, adding 4 μL of the solution from the fifth container to the solution to be tested, and adding 4 μL of the solution from the sixth container, mixing and reacting at 25° C. for 80 min; then adding 3 μL of the solution from the seventh container, mixing and reacting at 25° C. for 30 min; adding 50 μL of the solution from the eighth container, reacting at room temperature for 15 min; adding 50 μL of the solution from the tenth container to terminate the reaction; adding 82 μL of the solution from the eleventh container, and measuring the absorbance at 450 nm by a UV-visible spectrophotometer.

[0057] The recognition probe PD, hairpin probe H1, hairpin probe H2, hairpin probe H3, hairpin probe H4 and hairpin probe H5 are all dissolved in Tris-HCl buffer; hemin is dissolved in dimethyl sulfoxide (DMSO) and diluted in HEPES buffer.

[0058] Example 2: Pb 2+ Detection feasibility analysis.

[0059] 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, lanes 1-6 are PD, H1, H2, H3, H4, and H5 respectively; lane 7 is a mixture of PD, H1, H2, and H3, and no new bands are generated, indicating that the specific cleavage reaction of GR-5 DNAzymes and the CHA cycle reaction are not active in the absence of Pb. 2+ None of them can occur when Pb is present. PD, H1, H2, and H3 coexist in the reaction system.2+ Finally, a new bright band with a higher migration position and a new band with a lower position than the H1 band were observed in lane 8. At the same time, the bands corresponding to H1, H2, and H3 disappeared. The appearance of these two new bands and the disappearance of the three old bands proved that PD and Pb 2+ After binding, H1 is cleaved and the released initiator chain T successfully activates the CHA reaction to generate a stable H2-H3 complex. Lane 9 shows that in the absence of Pb 2+ When Pb was present, all the probes in the system were mixed and reacted. No new bands appeared, proving that the reactants could maintain their original stable state and coexist in the system. Compared with lanes 7 and 9, when Pb was added, 2+ After that, the corresponding bands of H1, H2, H3, H4, and H5 in lane 10 basically disappeared, and the band of H2-H3 complex produced by CHA reaction also basically disappeared. At the same time, a bright band with a higher migration position than H2-H3 and complex was generated, indicating that Pb 2+ The specific enzyme digestion reaction of GR-5 DNAzymes and the cascade signal amplification reaction of CHA and HCR were successfully triggered, and the new band at the highest migration position was the product of the HCR reaction. The above results confirmed that the designed method is feasible in principle.

[0060] 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, in the absence of Pb 2+ The absorption spectrum response intensity of the detection system is weak, and there is almost no color change, which proves that the DNA probes in the detection system have no self-hybridization phenomenon and coexist stably and independently, so they cannot form G-quadruplex DNAzymes; adding 25nM Pb 2+ After that, the response intensity of the absorption spectrum of the detection system was significantly enhanced, and the color change was significantly deepened, indicating that PD 2+ When H1 is catalyzed to cleave, it produces initiator chain T, which in turn triggers CHA and HCR cascade amplification reactions to generate HCR products containing multiple sticky ends. The G quadruplex fragments in the ends bind to hemin and catalyze TMB to produce color changes. These results show that the designed detection method can successfully detect Pb by colorimetry. 2+ And realize signal amplification.

[0061] Example 3: PD concentration optimization.

[0062] PD is a single-stranded DNA with catalytic activity. Its concentration can affect the signal amplification effect of the entire detection system by affecting its own catalytic efficiency. When the PD concentration is too high, the catalytic cleavage efficiency of PD on H1 is low, thereby weakening the signal amplification effect of the entire system. When the concentration is too high, the possibility of opening H1 increases with the increase of its concentration, thereby increasing the background signal. PD with concentrations of 50, 75, 100, 125, and 150 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 as follows. Figure 4 As shown in the figure, when the PD concentration is 75 nM, the ratio of A / A0 is the largest. Therefore, the PD concentration of 75 nM was selected as the optimal condition.

[0063] Example 4: Optimization of hairpin probe H1 concentration.

[0064] 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 trigger a nonspecific CHA cycle amplification reaction, which then activates the cascade HCR cycle and increases the background signal in the system. Hairpin probe H1 with concentrations of 50, 75, 100, 125, and 150 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 the figure. Figure 5 As shown in the figure, when the H1 concentration is 75 nM, the ratio of A / A0 is the largest. Therefore, the H1 concentration of 30 nM is selected as the optimal condition.

[0065] Example 5: Optimization of the concentration of hairpin probes H2 and H3.

[0066] As an important part of the cascade signal amplification reaction of the system, the concentrations of H2 and H3 have a significant impact on the final signal amplification efficiency of the system. When the concentrations of H2 and H3 are too low, the signal amplification effect will be poor. When the concentrations are too high, H2 and H3 may self-hybridize to form H2-H3 complexes, thereby inducing nonspecific HCR reactions, and ultimately leading to an increase in the background signal of the system. H2 and H3 at concentrations of 50, 75, 100, 125, and 150 nM were added to the reaction system, and the other conditions were kept unchanged according to the steps of Example 1 and optimized. The results are shown in the figure. Figure 6 As shown in the figure, when the concentration of H2 and H3 is 100 nM, the ratio of A / A0 is the largest. Therefore, the concentration of H2 and H3 is 100 nM as the optimal condition.

[0067] Example 6: Optimization of the concentration of hairpin probes H4 and H5.

[0068] The G-quadruplex fragment is designed in the sequence of H4 and can be exposed through the HCR reaction. Therefore, the concentration of H4 and H5 affects the generation of the final signal of the system. Too low concentration of H4 and H5 will lead to poor signal amplification effect, and too high concentration may enhance the HCR background signal, thereby reducing the signal-to-noise ratio. Set the reaction concentration of 50, 100, 150, 200, and 250 nM, and optimize it according to the steps of Example 1 while keeping other conditions unchanged. The results are shown in Figure 7 As shown, when the concentration of H1 and H2 is 200 nM, the ratio of A / A0 is the largest, so 200 nM is selected as the optimal concentration of H1 and H2 for the next experiment.

[0069] Example 7: Optimization of hemin concentration.

[0070] The enzyme activity of G-quadruplex DNAzymes is affected by the hemin concentration, which ultimately affects the detection performance of the system. When the hemin concentration is too low, hemin reduces the production of hemin / G-quadruplex DNAzymes, resulting in a decrease in the generation rate of the final signal of the system; when the hemin concentration is too high, the background signal in the system may be enhanced due to its own catalytic effect. The hemin concentrations were set to 100, 200, 300, 400, and 500 nM respectively, and the hemin concentrations were optimized according to the steps of Example 1 while keeping other conditions unchanged. The results are shown in Figure 2. Figure 8 As shown in the figure, the ratio of A / A0 was the highest when the concentration was 300 nM. Therefore, 300 nM was selected as the optimal reaction concentration of hemin for the next step of the experiment.

[0071] Example 8: Reaction time optimization.

[0072] Reaction time is also an important factor affecting the generation of reaction signals. Before the reaction reaches saturation, increasing the reaction time will make the cascade amplification reaction of the system more complete, thereby forming more G-quadruplex DNAzymes and obtaining more colorimetric signals. Reaction times of 40, 60, 80, 100, and 120 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. 9 As shown in the figure, when the reaction time reaches 80 min, the reaction efficiency reaches the maximum and the ratio of A / A0 also reaches the maximum. Therefore, 80 min is selected as the optimal reaction time for the next step of the experiment.

[0073] Example 9: Reaction temperature optimization.

[0074] 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.10 As shown in the figure, the ratio of A / A0 reaches the maximum at 25°C. Therefore, 25°C is selected as the optimal reaction temperature.

[0075] Example 10: Different concentrations of Pb 2+ Detect UV absorption spectrum.

[0076] 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.11 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, the initiator chain T is quickly generated and the CHA reaction is activated, so that the efficiency of H2-H3 complex generation is improved, and the reaction rate of HCR is also accelerated, producing more and longer HCR products, and finally achieving the enhancement of the colorimetric signal of the system. Fig.11 The results in the illustration show that the solution color of the detection system changes with the increase of Pb 2+ It gradually darkens with increasing concentration. Fig.12 Shows Pb 2+ The relationship between concentration and absorbance at 450nm, in the range of 0.05-50nM, Pb 2+ The logarithm of the concentration and the absorbance value of this method have a good linear relationship, and the linear equation is: A = 0.3845 + 0.1381lg·C, R 2 =0.9956; According to the ratio of three times the standard deviation of the blank sample to the slope, the calculated detection limit is 10.83pM. Table 2 shows the Pb 2+ The detection performance of the detection method is better than other reported detection systems.

[0077] Example 11: Specificity analysis.

[0078] Six divalent metal ions were selected as interfering ions to selectively analyze the colorimetric detection system. 2+ 、Zn 2+ , Ca 2+ , 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; an equal volume of 20mM Tris-HCl buffer solution was used as a blank control group. Fig.13 As shown, the concentration is Pb 2+ The absorbance values ​​of other interfering metal ions with 10 times the concentration of Pb did not change significantly compared with the blank control group, and the colors of the reaction solutions were similar. 2+ The absorbance of the solution increased significantly, and the color signal of the solution deepened significantly. The results show that the colorimetric analysis method is effective for Pb 2+ The selectivity is higher than that of other divalent metal ions.

[0079] Example 12: Actual sample detection.

[0080] In order to verify the Pb established in this chapter 2+ The potential of colorimetric analysis in actual sample analysis was demonstrated by using the above analysis method to analyze Pb in drinking water and fish. 2+ The detection was carried out and the Pb content was calculated by the spike recovery method. 2+ The results are shown in Table 2. The spiked recoveries in drinking water and fish were within 98.19%–115.10%, and the RSDs fluctuated within the range of 3.44%–10.87%, indicating that the designed analytical method has good accuracy and precision and can be actually applied to Pb in water and fish. 2+ Quantitative analysis and detection. Table 2 Pb in drinking water and fish 2+ 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, H2, H3, H4, and H5.

2. Pb according to claim 1 2+ Detection system, the sequence of the PD is the sequence shown in SEQ ID No.1 in Table 1, the sequence of the hairpin probe H1 is the sequence shown in SEQ ID No.2 in Table 1, the sequence of the hairpin probe H2 is the sequence shown in SEQ ID No.3 in Table 1, the sequence of the hairpin probe H3 is the sequence shown in SEQ ID No.4 in Table 1, the sequence of the hairpin probe H4 is the sequence shown in SEQ ID No.5 in Table 1, the sequence of the hairpin probe H5 is the sequence shown in SEQ ID No.6 in Table 1, and the sequence of the hairpin probe H6 is the sequence shown in SEQ ID No.7 in Table 1.

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, H2, H3, H4, and H5 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, H2, H3, H4, and H5. 2+ , incubate at 25°C for 1 h, then add hemin (300 nM) and incubate 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 was 75 nM, the hairpin probe H1 concentration was 75 nM, the H2 and H3 concentrations were 100 nM, the H4 concentration was 200 nM, and the H5 concentration was 200 nM. The hemin concentration was 300 nM, and the 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide concentrations were both 200 μM.

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 hairpin probe H2; The kit further comprises a fourth container, wherein the fourth container contains the hairpin probe H3; The kit further comprises a fifth container, wherein the fifth container contains the hairpin probe H4; The kit further comprises a sixth container, wherein the sixth container contains the hairpin probe H5; The kit further comprises a seventh container, wherein the seventh container contains the hemin; The kit further comprises an eighth container, wherein the eighth container contains the 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide; The kit also includes a ninth container, which contains the 20 mM Tris-HCl buffer solution: 20 mM tris-hydroxymethylaminomethane, 120 mM NaCl, 20 mM KCl, 10 mM MgCl2, and 1 M HCl to adjust the solution pH to 7.

4. The kit further comprises a tenth container, wherein the tenth container contains the H2SO4 solution. The kit further includes an eleventh container, which contains the 25 mM HEPES buffer solution (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 200 mM NaCl, 20 mM KCl, 0.05% Triton X-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 1.5 μL of the solution in the first container, 1.5 μL of the solution in the second container, 2 μL of the solution in the third container, 2 μL of the solution in the fourth container, 4 μL of the solution in the fifth container, and 4 μL of the solution in the sixth container, and then add different concentrations of Pb 2 + , mixed and reacted at 25°C for 80 min; (2) Add 3 μL of the solution in the seventh container and mix and react at 25°C for 30 min; (3) adding 50 μL of the solution in the eighth container and reacting at room temperature for 15 min; (4) adding 50 μL of the solution in the tenth container to terminate the reaction; (5) Add 82 μL of the solution in the container described in item 11, and measure the absorbance at 450 nm using a UV-visible spectrophotometer.

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