Cadmium ion and lead ion detection system and detection method based on CRISPR (clustered regularly interspaced short palindromic repeats) and deoxyribozyme

Abstract

The invention discloses a cadmium ion and lead ion detection system and method based on CRISPR and deoxyribozyme, and the method comprises the following steps: forming active deoxyribozyme by using target ions and a recognition probe, cutting a redundant sequence in a substrate probe, releasing a semi-activator, and cooperatively activating the activity of CRISPR-Cas12a with the other semi-activator in the system. According to the system, the signal-to-noise ratio and the sensitivity of detection are improved by utilizing the effect of inhibiting cooperative activation by redundant fragments in semi-activators. According to the detection method, nano material modification on the substrate probe is not needed, magnetic separation is not needed, and the detection cost is reduced. The detection process only needs two steps of room temperature incubation reaction, and the two steps are carried out in one reaction tube, so that cross contamination possibly caused by multi-tube reaction is avoided, and the detection process is simplified. The method can be applied to detection of cadmium ions and lead ions in rice or serum samples, and has wide application prospects in the fields of food safety, medical detection and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a cadmium ion and lead ion detection system and method based on CRISPR and deoxyribozyme. BACKGROUND

[0002] Cadmium (Cd) and lead (Pb) are both toxic heavy metal pollutants, which can cause significant harm to human health and the environment. With their extensive application in industry and improper disposal, cadmium and lead have been enriched and polluted in soil, water and food. At present, the detection methods for heavy metal ions mainly include atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS) and atomic fluorescence spectrometry (AFS). Although these methods have good detection sensitivity, the complex sample preparation and detection process, long detection time, and expensive detection cost limit their rapid detection application for a large number of samples. Therefore, it is necessary to establish a simple, rapid and sensitive detection method for heavy metal ions to solve these challenges.

[0003] Metal-dependent deoxyribozyme (DNAzyme) refers to a nucleic acid that depends on a special metal ion to exert a specific catalytic function. Among them, a type of deoxyribozyme that can catalyze the hydrolysis of phosphate bonds is widely used in the detection of metal ions. This type of deoxyribozyme can catalyze the deprotonation of 2'-OH on ribose through metal ions to produce oxygen-containing anions, which then attack the adjacent phosphate group, resulting in the hydrolysis of the phosphodiester bond of the substrate. Based on this property, specific metal ions can be quantitatively analyzed according to the cleavage product.

[0004] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Cas proteins are widely present in bacteria and archaea, and are a unique adaptive immune defense system of prokaryotes. This system has been developed into a gene editing tool and widely used in the field of molecular biology. In addition to the cis-cutting of the target site, the CRISPR-Cas12a system has in vitro trans-cutting activity. Due to its strong signal amplification ability, this system is widely used in the construction of nucleic acid and non-nucleic acid material rapid detection biosensors.

[0005] The general strategy for constructing a heavy metal ion detection biosensor based on CRISPR and deoxyribozyme is as follows: the metal-dependent deoxyribozyme is hybridized with the substrate probe, and when the target heavy metal ion (such as Cd 2+ or Pd 2+) exists, the deoxyribozyme cuts the substrate probe and releases the activator strand. The released activator strand binds to Cas12a-crRNA and activates its trans-cleavage activity, cutting the fluorescent probe and outputting a fluorescent signal. However, the uncut activator strand on the substrate probe in this strategy can also activate CRISPR / Cas12 activity, causing high background signal or false positives. To solve this problem, some studies use magnetic separation methods to distinguish between cut and uncut substrate probes. However, magnetic separation requires modification of the substrate probe on magnetic beads or nanomatrix materials, increasing the cost of detection. At the same time, the magnetic separation step also increases the detection time and the complexity of the detection.

[0006] On the other hand, some studies have shown that "split" activator strands can also activate the trans-cleavage activity of CRISPR-Cas12a, i.e. using two "half activators" to synergistically activate CRISPR-Cas12a, and a single "half activator" cannot activate its cleavage activity. In addition, the redundant sequence in the "half activator" will greatly inhibit the efficiency of synergistic activation. Based on this, the present application uses the strategy of synergistically activating CRISPR-Cas12a with two "half activators", one of which is designed in the substrate probe and contains a redundant sequence, and the other is added to the CRISPR-Cas12a cleavage reaction solution. When there is no target ion in the detection system, the "half activator" in the substrate probe is connected to the redundant sequence and cannot synergistically activate CRISPR-Cas12a cleavage reaction with the other "half activator". When there is a target ion in the detection system, the substrate probe is cut by the deoxyribozyme, and the "half activator" is separated from the redundant sequence. At this time, the two "half activators" synergistically activate CRISPR-Cas12a and cut the fluorescent reporter probe, outputting a fluorescent signal. In the present application, when there is no target ion, the "half activator" is connected to the redundant sequence and synergistic activation cannot be performed. Without modification of the substrate probe on nanomaterials and magnetic separation, the background signal of the detection is greatly reduced, the sensitivity of the detection is improved, the complexity of the detection is reduced, and the detection process is optimized. SUMMARY

[0007] This section is intended to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification of the present application to avoid obscuring the purpose of this section, the abstract and the title of the present application, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0008] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0009] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art and provide a cadmium ion and lead ion detection system based on CRISPR and deoxyribozyme.

[0010] To solve the above technical problems, the application provides a cadmium ion and lead ion detection system based on CRISPR and deoxyribozyme, characterized in that the system comprises recognition probe Cdzyme, substrate probe PS-Cd, recognition probe Pdzyme, substrate probe PS-Pd, semi-activator SA2, LbCas12a protein, crRNA and fluorescent reporter probe FQ. 2+ Dependent deoxyribozyme; the recognition probe Pdzyme is Pd 2+ dependent deoxyribozyme.

[0011] As a preferred scheme of the cadmium ion and lead ion detection system, the sequence of the Cdzyme is shown in SEQ ID No. 1; the sequence of the PS-Cd is shown in SEQ ID No. 2; the sequence of the Pdzyme is shown in SEQ ID No. 3; the sequence of the PS-Pd is shown in SEQ ID No. 4; the sequence of the semi-activator SA2 is shown in SEQ ID No. 5; the sequence of the crRNA is shown in SEQ ID No. 6; and the sequence of the FQ is shown in SEQ ID No. 7.

[0012] As a preferred scheme of the cadmium ion and lead ion detection system, the recognition probe Cdzyme can specifically bind to Cd 2+ to form a complete deoxyribozyme, thereby shearing the substrate probe PS-Cd.

[0013] As a preferred scheme of the cadmium ion and lead ion detection system, the recognition probe Pdzyme can specifically bind to Pd 2+ to form a complete deoxyribozyme, thereby shearing the substrate probe PS-Pd.

[0014] As a preferred scheme of the cadmium ion and lead ion detection system, after the substrate probes PS-Cd and PS-Pb are sheared, the two can both synergistically activate the fluorescent probe cleavage activity of the CRISPR-Cas12a system with the semi-activator SA2.

[0015] Another object of the application is to overcome the deficiencies in the prior art and provide a detection method for a cadmium ion and lead ion system.

[0016] To solve the above technical problems, the application provides a detection method for a cadmium ion and lead ion detection system, characterized in that the detection method is as follows.

[0017] The recognition probe Cdzyme and the substrate probe PS-Cd are respectively diluted to 20 uM in buffer A, then mixed in a volume ratio of 6:5, heated at 95 DEG C for 5 minutes, and then slowly reduced to room temperature to prepare premix 1;

[0018] The recognition probe Pdzyme and the substrate probe PS-Pb are respectively diluted to 20 uM in buffer A, then mixed in a volume ratio of 6:5, heated at 95 DEG C for 5 minutes, and then slowly reduced to room temperature to prepare premix 2;

[0019] Take 4.0 uL of LbCas12a protein with a concentration of 200 nM, 8.0 uL of crRNA with a concentration of 100 nM, 2.0 uL of semi-activator SA2 with a concentration of 2 uM, 10.0 uL of buffer B, 0.5 uL of fluorescent reporter probe FQ with a concentration of 100 uM, and 56 uL of ultrapure water, mix and incubate at room temperature for 10 minutes to prepare premix 3;

[0020] Take 0.2 uL of premix 1 and 0.2 uL of premix 2 respectively, 1.0 uL of the test solution, use buffer C to make up to a final volume of 20 uL, and incubate at room temperature for 40 minutes; then add premix 3 to the above reaction tube, continue to incubate at room temperature for 20 minutes; measure the fluorescence value by fluorescence spectrophotometer, and observe the color development of the sample under ultraviolet light.

[0021] As a preferred scheme of the detection method of the application, wherein: the formula of buffer A is: 25 mM Tris-HCl and 500 mM NaCl, pH 7.4.

[0022] As a preferred scheme of the detection method of the application, wherein: the formula of buffer B is: 100 mM Tris-HCl, 500 mM NaCl, 100 mM MgCl2 and 1 mg / mL BSA, pH 7.9.

[0023] As a preferred scheme of the detection method of the application, wherein: the formula of buffer C is: 25 mM Tris-HCl and 100 mM NaCl, pH 7.4.

[0024] As a preferred scheme of the detection method of the application, wherein: when measuring the fluorescence value by fluorescence spectrophotometer, the excitation wavelength is set to 470 nm, and the fluorescence intensity is measured at an emission wavelength of 519 nm.

[0025] The application has the following beneficial effects:

[0026] The application provides a cadmium ion and lead ion detection system based on CRISPR and deoxyribonuclease, the recognition probe in the detection system can be respectively combined with Cd 2+and Pb 2+ The redundant sequence in the substrate probe is cut to form an active deoxyribozyme, and a "half-activator" nucleic acid fragment is released. The fragment cooperates with another "half-activator" fragment in the reaction system to activate the CRISPR-Cas12a transcleavage activity, cut the fluorescent reporter probe, and output the fluorescent signal. The system uses the redundant fragment in the "half-activator" to inhibit the effect of cooperative activation of CRISPR-Cas12a activity, greatly reduces the background signal of the detection, and improves the signal-to-noise ratio of the detection. The detection method does not need to modify the substrate probe with nanomaterials, and does not need magnetic separation, thereby reducing the detection cost. During the detection process, all reactions are carried out in one reaction tube, avoiding the cross contamination caused by multi-tube reactions, and simplifying the detection process. The method has high detection sensitivity, and the detection limit of Cd 2+ The highest signal-to-noise ratio can reach 20 times, and the lowest detection limit is 14.21 pM; Pd 2 + The highest signal-to-noise ratio can reach 10 times, and the lowest detection limit is 11.92 pM, and the method can be applied to the detection of Cd 2+ and Pb 2+ in rice and serum samples, and has wide application prospects in the fields of food safety and environmental monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

[0028] Figure 1 It is the detection principle diagram of the present application;

[0029] Figure 2 It is the detection flowchart of the present application;

[0030] Figure 3 It is the result of the feasibility verification of the detection of Cd 2+ in the embodiment 2 of the present application;

[0031] Figure 4 It is the result of the feasibility verification of the detection of Pd 2+ in the embodiment 3 of the present application;

[0032] Figure 5 It is the fluorescent signal response of different concentrations of Cd 2+ in the embodiment 4 of the present application, and the standard curve is drawn, wherein, Figure 5 A is the fluorescent signal response of different concentrations of Cd 2+ , and Figure 5B is the standard curve;

[0033] Figure 6 The fluorescence signal response of different concentrations of Pd in Example 5 of the present application 2+ and the standard curve is drawn, wherein, Figure 6 A is the fluorescence signal response of different concentrations of Pd 2+ , Figure 6 B is the standard curve;

[0034] Figure 7 The results of the co-detection of Cd 2+ and Pd 2+ in Example 6 of the present application;

[0035] Figure 8 The specific test results in Example 7 of the present application;

[0036] Figure 9 The results of the detection method in the present application and the inductively coupled plasma emission spectrometer for detecting Cd 2+ and Pd 2+ in Comparative Example 1 are compared, wherein, Figure 9 A is the detection of Cd 2+ in the rice sample, Figure 9 B is the detection of Pd 2+ in the serum sample. DETAILED DESCRIPTION

[0037] In order to make the above objectives, features and advantages of the present application more apparent and comprehensible, the specific embodiments of the present application will be described in detail below in conjunction with the description of the embodiments.

[0038] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the connotation of the present application, therefore the present application is not limited to the specific embodiments disclosed below.

[0039] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. In different places in this specification, "in one embodiment" does not mean the same embodiment, nor is it an independent or alternative embodiment that excludes other embodiments.

[0040] The raw materials used in the embodiments of the present application are commercially available without special instructions, and the details are shown in Table 1.

[0041] Table 1

[0042]

[0043]

[0044] LbCas12a protein was obtained by prokaryotic induction expression and purification, and the specific method is described in: Analytica Chimica Acta, 2023, 1283: 341950.

[0045] Example 1

[0046] The detection principle of the cadmium ion and lead ion detection system of the application is shown in Figure 1 .

[0047] The substrate probe (PS-Cd and PS-Pd) is composed of a semi-activator (green) and a redundant sequence (black). When Cd 2+ or Pd 2+ (or both ions coexist) exists in the detection system, the deoxyribonuclease activity is activated, the substrate probe is cut, and the semi-activator is released. Since another semi-activator (red) is pre-added in the CRISPR-Cas12a reaction solution, the two semi-activators cooperatively bind to the crRNA, activate the CRISPR-Cas12a transcleavage activity (top), cut the fluorescent reporter probe (FQ), and output the fluorescent signal. When there is no target ion in the detection system, the substrate probe remains intact. Even so, due to reasons such as strand displacement effect, the two semi-activator sequences may still be captured by the crRNA. However, due to the presence of the redundant sequence in the substrate probe, this structure inhibits the CRISPR-Cas12a transcleavage activity (bottom), and the fluorescent reporter probe (FQ) remains intact, without fluorescent signal. The oligonucleotide sequences involved in the application are shown in Table 2.

[0048] The detection process of Cd 2+ and Pb 2+ in the application is shown in Figure 2 . First, three kinds of premixes (premix 1, premix 2 and premix 3) need to be prepared. Among them, premix 1 and premix 2 are nucleic acid hybridization solutions, which can be stored at 4℃ after preparation. Premix 3 contains protein-RNA complex and needs to be prepared separately before each detection. The detection is divided into two steps of reaction, which are carried out in one reaction tube: the first step is to add premix 1, premix 2 and the liquid to be detected into the reaction tube, and incubate at room temperature for 40 minutes; the second step is to add premix 3 into the above reaction tube, and continue to incubate at room temperature for 20 minutes. After the reaction is completed, the fluorescent signal is detected.

[0049] Table 2

[0050]

[0051] Note: The underlined portion represents the CRISPR-Cas12a system recognition region. RNA sequences are shown against a gray background. Adenine ribonucleotides at cleavage sites are represented by rA. An asterisk (*) indicates a thiophosphate modification. 6-FAM is a fluorescent group, and BHQ1 is a fluorescence quencher.

[0052] The above-mentioned detection system was used to detect Cd. 2+ With Pb 2+ The method includes the following steps:

[0053] (1) Cdzyme and PS-Cd were diluted to 20 μM in buffer A (25 mM Tris-HCl, 500 mM NaCl, pH 7.4), then mixed at a volume ratio of 6:5, heated at 95 °C for 5 minutes, and then slowly cooled to room temperature to obtain premix 1 for later use.

[0054] (2)(1) Pdzyme and PS-Pd were diluted to 20 μM in buffer A (25 mM Tris-HCl, 500 mM NaCl, pH 7.4), then mixed at a volume ratio of 6:5, heated at 95 °C for 5 minutes, and then slowly cooled to room temperature to obtain premix 2 for later use.

[0055] (3) Take 4.0 μL of 200 nM LbCas12a protein, 8.0 μL of 100 nM crRNA, 2.0 μL of 2 μM SA2, 10.0 μL of buffer B (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 100 μg / mL BSA, pH 7.9), 0.5 μL of 100 μM FQ probe and 56 μL of ultrapure water, mix them and incubate at room temperature for 10 minutes to prepare premix solution 3 for later use.

[0056] (4) Take 0.2 μL of the prepared premix 1 and 0.2 μL of the prepared premix 2, and 1.0 μL of the test solution, respectively, and bring the total volume to 20 μL using buffer C. Incubate at room temperature for 40 minutes. Then add premix 3 to the above reaction tube and continue incubating at room temperature for 20 minutes. Measure the fluorescence value using a fluorescence spectrophotometer and observe the color development of the sample under ultraviolet light.

[0057] Example 2

[0058] Figure 3 For Cd 2+Results of feasibility verification: In the figure, the black line represents the presence of recognition probe (Cdzyme) and Cas12a-crRNA in the test solution; the red line represents the presence of recognition probe (Cdzyme), substrate probe (PS-Cd), and Cas12a-crRNA in the test solution; the blue line represents the presence of recognition probe (Cdzyme), substrate probe (PS-Cd), and Cd... 2+ And Cas12a-crRNA; the green line indicates that the detection solution contains recognition probe (Cdzyme), substrate probe (PS-Cd), and Cd. 2+ The purple line represents the presence of a recognition probe (Cdzyme), a substrate probe (PS-Cd), a semiactivator (SA2), and Cas12a-crRNA in the test solution; the yellow line represents the presence of a recognition probe (Cdzyme), a substrate probe (PS-Cd), and Cd2a-crRNA in the test solution. 2+ The results showed that only when Cd... 2+ A strong fluorescent signal can only be detected when Cas12a-crRNA and all DNA sequences are present simultaneously, which is consistent with the detection principle described in Example 1.

[0059] Example 3

[0060] Figure 4 For Pd 2+ Results of feasibility verification: In the figure, the black line represents the presence of recognition probe (Pdzyme) and Cas12a-crRNA in the test solution; the red line represents the presence of recognition probe (Pdzyme), substrate probe (PS-Pb), and Cas12a-crRNA in the test solution; the blue line represents the presence of recognition probe (Pdzyme), substrate probe (PS-Pb), and Cas12a-crRNA in the test solution. 2+ And Cas12a-crRNA; the green line indicates that the detection solution contains recognition probe (Pdzyme), substrate probe (PS-Pb), and Pd. 2+ The purple line represents the presence of recognition probe (Pdzyme), substrate probe (PS-Pb), semiactivator (SA2), and Cas12a-crRNA in the test solution; the yellow line represents the presence of recognition probe (Pdzyme), substrate probe (PS-Pb), and Pdzyme in the test solution. 2+ The semi-activator (SA2) and Cas12a-crRNA were observed. Experimental results showed that only when Pd... 2+ A strong fluorescent signal can only be detected when Cas12a-crRNA and all DNA sequences are present simultaneously, which is consistent with the detection principle described in Example 1.

[0061] Example 4

[0062] Figure 5 For different concentrations of Cd 2+ The fluorescence signal response was observed, and a standard curve was plotted. Cd solutions with concentrations of 0.1 nM, 2.5 nM, 7.5 nM, 12.5 nM, 17.5 nM, 25 nM, 50 nM, and 100 nM were prepared. 2+ The standard solution was tested using the detection method described in Example 1. The same sample was measured three times, and the fluorescence intensity was plotted against Cd. 2+ Concentration correlation curve. For example... Figure 5 As shown in Figure A, the fluorescence intensity increases with Cd. 2+ The concentration increases and rises, when Cd 2+ At a concentration of 25 nM, the fluorescence signal reached a saturation plateau. Imaging of the sample under UV light also showed a high concentration of Cd. 2+ The sample tube emitted a stronger fluorescence.

[0063] Select Figure 5 Cd in A 2+ Data for concentrations of 0.1 nM, 2.5 nM, 7.5 nM, 12.5 nM, 17.5 nM, and 25 nM were used, with Cd 2+ A standard curve was plotted with concentration on the x-axis and fluorescence intensity on the y-axis, as shown in the figure. Figure 5 B. The results showed that the solution fluorescence intensity was related to Cd. 2+ The concentration exhibits a linear relationship in the range of 0.1 nM to 25 nM. The linear equation is y = 1125 × [Cd] 2+ +2154, the correlation coefficient (R²) of the linear regression equation. 2 The value is 0.9938. Based on the 3σ principle (Talanta, 2014, 119:178-180), this invention addresses the issue of Cd... 2+ The limit of detection (LOD) is 14.21 pM. These results indicate that the present invention effectively detects Cd... 2+ It has good detection sensitivity.

[0064] Example 5

[0065] Figure 6 For different concentrations of Pd 2+ The fluorescence signal response was observed, and a standard curve was plotted. Pd was prepared at concentrations of 0.01 nM, 0.05 nM, 0.25 nM, 0.75 nM, 1.5 nM, 2.5 nM, 5 nM, 10 nM, and 20 nM. 2+ The standard solution was tested using the detection method described in Example 1. The same sample was measured three times, and the fluorescence intensity was plotted against Pd. 2+Concentration-dependent curve. As shown in Figure 6 The fluorescence intensity increased with the increase of Pd 2+ concentration, and reached a saturated platform when the Pd 2+ concentration was 25 nM. The sample tube also emitted stronger fluorescence under the UV light. 2+

[0066] Selecting the data of Pd 2+ concentrations of 0.01 nM, 0.05 nM, 0.25 nM, 0.75 nM, 1.5 nM and 2.5 nM in FIG. 2+ A, and taking the Pd 2+ concentration as the abscissa and the fluorescence intensity as the ordinate, a standard curve was drawn, as shown in FIG. Figure 6 B. The results showed that the fluorescence intensity of the solution was linearly related to the Pd 2+ concentration in the range of 0.01 nM to 2.5 nM. The linear equation was y = 10688x [Pd 2 ]+3811, and the correlation coefficient (R 2+ ) of the linear regression equation was 0.9910. According to the 3σ principle (Talanta, 2014, 119: 178-180), the detection limit (Limit of Detection, LOD) of Pd 2+ detection sensitivity. Figure 6

[0067] Example 6

[0068] Figure 7 The results of the Cd 2+ and Pd 2+ co-detection. Four samples to be detected were configured, which were sample 1 (blank sample, not containing Cd 2+ or Pd 2+ ), sample 2 (containing 20 nM Cd 2+ ), sample 3 (containing 20 nM Pd 2+ ), and sample 4 (containing 20 nM Cd 2+ and 20 nM Pd 2+ ). The above solutions were detected by using the detection method in Example 1, and each sample was determined three times. As shown in the figure, the fluorescence signal of sample 1 was low, and no obvious fluorescence could be observed in the sample tube. The fluorescence signals of sample 2, sample 3 and sample 4 were all high, and the three sample tubes emitted strong fluorescence under UV irradiation. The above results showed that the detection method of the present application could detect samples containing Cd 2+ , samples containing Pd 2+ , and samples containing both Cd 2+ and Pd​​2+ The sample.

[0069] Example 7

[0070] Figure 8 The sample. 2+ and Pb 2+ , Cd 2+ , Pb 2+ , Zn 2+ , Co 2+ , Ni 2+ , Mn 2+ , Ca 2+ , Ba 2+ , Mg 2+ . The above solutions were detected using the detection method in Example 1, and three determinations were repeated for the same sample. The results showed that the first three samples detected strong fluorescence signals, and the three sample tubes emitted strong fluorescence under ultraviolet irradiation. The fluorescence signal intensity of the remaining samples was close to that of the blank sample, and the sample tubes had no fluorescence under ultraviolet irradiation. The results showed that the detection method of the application had good detection specificity.

[0071] Example 8

[0072] In order to evaluate the detection performance of the Cd 2+ and Pd 2+ detection method of the application in food or biological samples, rice samples containing Cd 2+ and serum samples containing Pd 2+ were prepared. The preparation method of the rice sample containing Cd 2+ was as follows: the ground rice powder was dissolved with concentrated hydrochloric acid and incubated at 100℃ for 3h, then sodium hydroxide was added to adjust the pH to neutral, and then different concentrations of cadmium ions were added to the rice liquid sample to prepare the sample to be detected. The preparation method of the serum sample containing Pd 2+ was as follows: different concentrations of Pd 2+ were added to fetal bovine serum.

[0073] The rice sample and the serum sample were detected using the detection method in Example 1, and three determinations were repeated for the same sample. The detection results were compared with the amount of Cd 2+ or Pd 2+ added, and the recovery rate and relative standard deviation were calculated, as shown in Tables 3 and 4. When detecting Cd 2+ in the rice sample, the average recovery rate calculated by the detection of the application was between 98.06% and 102.5%, and the detection relative standard deviation was between 1.383% and 4.842%. When detecting Pd 2+The average recovery rate of the detection calculation is between 94.22% and 110.0%, and the relative standard deviation is between 2.405% and 6.062%. The above results show that the detection method of the present application has good accuracy and repeatability.

[0074] Table 3

[0075]

[0076] Table 4

[0077]

[0078] Comparative Example 1

[0079] Figure 9 The detection method in the present application and the results of inductively coupled plasma emission spectrometer detection of Cd 2+ and Pd 2+ are compared. According to the method in Example 8, rice samples containing Cd 2+ and serum samples containing Pd 2+ are prepared. The detection method in Example 1 is used to detect the rice samples and serum samples, respectively. For the same batch of samples, the concentration of Cd 2+ and Pd 2+ is detected by inductively coupled plasma emission spectrometer, and the detection results are compared with the detection results obtained by the detection method of the present application. The results show that there is no significant difference between the detection results obtained by the detection method of the present application and the results obtained by inductively coupled plasma emission spectrometer, indicating that the detection results obtained by the detection method of the present application have high accuracy.

[0080] Comparative Example 2

[0081] This comparative example is a research paper published in 2023, "An ultrasensitive Cd 2+ detection biosensor based on DNAzyme and CRISPR / Cas12a coupled with hybridization chain reaction" (Analytica Chimica Acta, 2023, 1283:34195). In Comparative Example 2, Cd 2+The dependent deoxyribozyme cuts the substrate probe to release single-stranded nucleic acid, triggers the hybridization chain reaction (HCR), amplifies the CRISPR-Cas12a activation sequence, and then activates the CRISPR-Cas12a transcleavage activity to cut the fluorescent reporter probe and output the fluorescent signal. Compared with Comparative Example 2, the present application has the following improvements in detection effect: (1) The signal-to-noise ratio of the present application is greatly improved. In Example 4 of the present application, the maximum signal-to-noise ratio of Cd 2+ detection is more than 20 times, while in Comparative Example 2, the maximum signal-to-noise ratio of Cd 2+ detection is only 1.8; (2) The detection process of the present application is simple. In Example 1 of the present application, the detection reaction only needs 2 steps, and the whole process can be incubated at room temperature. All detection reactions are carried out in 1 reaction tube, while in Comparative Example 2, the detection reaction needs to be divided into 3 steps. The first 2 steps are room temperature incubation reactions, and the last step needs to be incubated at 37°C. The 3 steps need to be carried out in 3 different reaction tubes, which increases the risk of cross contamination and the complexity of operation. (3) The detection time of the present application is shorter. In Example 1 of the present application, the 2-step detection reaction can be completed within 60 minutes, while the 3-step reaction in Comparative Example 2 takes more than 80 minutes.

[0082] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application. They should be included in the scope of the present application.

Claims

1. A CRISPR and deoxyribozyme-based cadmium ion and lead ion detection system, characterized in that, The application relates to a method for detecting a target nucleic acid sequence, comprising the following steps: The recognition probe Cdzyme, the substrate probe PS-Cd, the recognition probe Pdzyme, the substrate probe PS-Pd, the semi-activator SA2, the LbCas12a protein, the crRNA and the fluorescent reporter probe FQ are used. wherein the recognition probe Cdzyme is Cd 2+ Dependent deoxyribozyme; recognition probe Pdzyme is Pd 2+ Dependent deoxyribozyme.

2. The system for detecting cadmium and lead ions as claimed in claim 1, wherein: The sequence of the recognition probe Cdzyme is shown in SEQ ID No. 1; the sequence of the substrate probe PS-Cd is shown in SEQ ID No. 2; the sequence of the recognition probe Pdzyme is shown in SEQ ID No. 3; the sequence of the substrate probe PS-Pd is shown in SEQ ID No. 4; the sequence of the semi-activator SA2 is shown in SEQ ID No. 5; the sequence of the crRNA is shown in SEQ ID No. 6; and the sequence of the FQ is shown in SEQ ID No.

7.

3. The system for detecting cadmium and lead ions as claimed in claim 1, wherein: The recognition probe Cdzyme can specifically bind to Cd 2+ The combination forms a complete deoxyribozyme, thereby cleaving the substrate probe PS-Cd.

4. The system for detecting cadmium and lead ions as claimed in claim 1, wherein: The recognition probe Pdzyme is capable of specifically binding to Pd 2+ The binding forms a complete deoxyribozyme, which cleaves the substrate probe PS-Pd.

5. The system for detecting cadmium and lead ions as claimed in claim 1, wherein: After the substrate probes PS-Cd and PS-Pb are cleaved, the semi-activator SA2 can be used to cooperatively activate the fluorescent probe cleavage activity of the CRISPR-Cas12a system.

6. A method for detecting cadmium ion and lead ion based on CRISPR and deoxyribozyme, characterized in that, The detection method is as follows: The recognition probe Cdzyme and the substrate probe PS-Cd are respectively diluted to 20 muM in buffer A, then mixed in a volume ratio of 6:5, heated at 95 DEG C for 5 minutes, and then slowly cooled to room temperature to prepare a premix 1; The recognition probe Pdzyme and the substrate probe PS-Pb are respectively diluted to 20 muM in buffer A, then mixed in a volume ratio of 6:5, heated at 95 DEG C for 5 minutes, and then slowly cooled to room temperature to prepare a premix 2; 4.0 muL of LbCas12a protein with a concentration of 200 nM, 8.0 muL of crRNA with a concentration of 100 nM, 2.0 muL of semi-activator SA2 with a concentration of 2 muM, 10.0 muL of buffer B, 0.5 muL of fluorescent reporter probe FQ with a concentration of 100 muM and 56 muL of ultrapure water are mixed and incubated at room temperature for 10 minutes to prepare a premix 3; 0.2 muL of the prepared premix 1 and 0.2 muL of the prepared premix 2 are taken, 1.0 muL of a to-be-detected solution is taken, buffer C is used to make up to a final volume of 20 muL, and incubation is carried out at room temperature for 40 minutes; then the premix 3 is added to the above reaction tube, and incubation is continued at room temperature for 20 minutes; the fluorescence value is measured by using a fluorescence spectrophotometer, and the sample coloration is observed under an ultraviolet lamp.

7. The method of claim 6, wherein: The buffer A is formulated as follows: 25 mM Tris-HCl and 500 mM NaCl, pH 7.

4.

8. The method of claim 6, wherein: The buffer B is formulated as follows: 100 mM Tris-HCl, 500 mM NaCl, 100 mM MgCl2 and 1 mg / mL BSA, pH 7.

9.

9. The method of claim 6, wherein: The buffer C is formulated as follows: 25 mM Tris-HCl and 100 mM NaCl, pH 7.

4.

10. The method of claim 6, wherein: When the fluorescence value is measured by using the fluorescence spectrophotometer, the excitation wavelength is set to 470 nm, and the fluorescence intensity is measured at an emission wavelength of 519 nm.

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