Cadmium ion analysis method based on feedback type HCR-Exo reaction

Abstract

The invention discloses a cadmium ion analysis method based on a feedback type HCR-Exo reaction, and belongs to the field of food safety detection. According to the method, the trigger chain T triggers the synergistic effect of HCR and Exo III, so that two nucleic acid amplification technologies are alternately carried out, and signal exponential amplification is realized. As the initiation chain T can be efficiently regenerated and recycled in the reaction process, the system can obviously enhance an electrochemical luminescence signal, thereby improving the detection sensitivity. Experimental results show that the detection limit of the system to cadmium ions is as low as 0.13 pM, and the system has ultra-sensitive detection performance. Besides, the system has the characteristic of high modularization, can be flexibly adapted to different recognition elements by replacing the recognition sequence of the hairpin probe, constructs a universal biosensing platform, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of food safety testing, specifically to a cadmium ion analysis method based on a feedback-type HCR-Exo reaction. Background Technology

[0002] Cadmium ions (Cd) 2+ Heavy metals, a common toxic contaminant in food, can accumulate in the human body through the food chain. Long-term excessive intake can lead to serious health problems such as vascular damage, cancer, and impaired immune system.

[0003] Cd detection 2+ Traditional methods for detecting Cd2 include inductively coupled plasma atomic emission spectrometry (ICP-AES) and ion-selective electrode (ISE), which offer good selectivity and sensitivity. However, their reliance on expensive, large-scale instruments and time-consuming analytical procedures makes them unsuitable for rapid on-site detection. In contrast, novel detection methods such as colorimetry, photoelectrochemical methods, and ion imprinting have attracted significant attention due to their low cost and ease of operation. Colorimetry allows for visual assessment but suffers from lower quantitative accuracy. Photoelectrochemical methods offer real-time monitoring and high sensitivity, but their stability is susceptible to variations in electrolyte pH, properties, and electrode fabrication processes. Ion imprinting exhibits high affinity for the target analyte, but its results are easily affected by environmental factors. Therefore, developing a stable and accurate method for detecting Cd2 is crucial. 2+ The detection method has important practical significance.

[0004] Isothermal nucleic acid amplification (HCR) technology, as an emerging signal amplification strategy, has demonstrated significant application value in the detection of heavy metals, fungal toxins, and pathogens due to its advantages such as isothermal operation, rapid response, and high sensitivity. Hybridization chain reaction (HCR) in isothermal nucleic acid amplification technology possesses excellent anti-interference capabilities and low background signal; it only requires the priming strand to trigger the cascade assembly of DNA hairpin structures, ultimately forming double-stranded DNA nanowires with repeating units; and it is easily functionalized, making it a research hotspot in recent years. However, traditional monolayer HCR systems can only achieve linear signal amplification (1:N), and their detection sensitivity still has room for improvement. Therefore, a strategy of combining multiple DNA amplification technologies is proposed, such as coupling HCR with other amplification technologies, to construct a detection system with exponential signal amplification capabilities. This multi-stage combination technique can effectively overcome the sensitivity limitations of single-stage amplification and shows significant advantages in the detection of heavy metals, proteins, and viruses. In enzyme-assisted amplification technology, exonuclease III (Exo-III) stands out due to its unique catalytic properties: simple probe design and no labeling required; high efficiency of 3'-5' exonuclease activity; specific recognition and cleavage of blunt or concave 3'-OH ends of double-stranded DNA; high catalytic efficiency and rapid generation of a large number of detectable signal molecules.

[0005] In summary, the innovative combination of HCR and Exo-III to establish a novel cascaded signal amplification system for detecting trace heavy metals in food is of great significance in the field of food safety. Summary of the Invention

[0006] To address the problems existing in the prior art, this application proposes a cadmium ion analysis method based on a feedback-type HCR-Exo reaction.

[0007] According to a first aspect of the present invention, a feedback-based HCR-Exo reaction system is provided, the reaction system comprising target DNA (T), hairpin probe H1, hairpin probe H2, S* / IT complex, and exonuclease Exo-III; the target DNA (T) is composed of a*-b*; the hairpin probe H1 comprises b*, c, b, and a; the hairpin probe H2 comprises c*, b, c, and d; the S* / IT complex comprises an S*-Ru chain and an IT chain, wherein the S*-Ru chain comprises a, d*, and c*, and the IT chain contains a base sequence completely identical to that of the target DNA (T).

[0008] Further, in the hairpin probe H1, b* and b form a complementary double chain as the stem of the hairpin probe H1, c is the loop portion of the hairpin structure, and a is the 3' sticky end extending from the stem of the hairpin structure; in the hairpin probe H2, c* and c form a complementary double chain as the stem of the hairpin probe H2, b is the loop portion of the hairpin structure, and d is the 3' sticky end extending from the stem of the hairpin structure; the 5' end of the S*-Ru chain is bounded by Ru(bpy)3 2+ Modification.

[0009] Further, the sequence of the hairpin probe H1 is shown in SEQ ID NO.1; the sequence of the hairpin probe H2 is shown in SEQ ID NO.2; the sequence of S* is shown in SEQ ID NO.3; the sequence of the IT strand is shown in SEQ ID NO.4; and the sequence of the target DNA (T) is shown in SEQ ID NO.5.

[0010] Furthermore, the reaction system also includes a recognition unit AT and a blocking chain B; the recognition unit AT is initiated by a T(a*-b*) sequence and Cd. 2+ The aptamer sequence is composed of: the blocking chain B partially hybridizes with the recognition unit AT to prevent the release of the trigger chain T from causing signal leakage; the sequence of the recognition unit AT is shown in SEQ ID NO.6; the sequence of the blocking chain B is shown in SEQ ID NO.7.

[0011] According to a second aspect of the present invention, a nucleic acid detection method for a feedback-type HCR-Exo reaction system as described above is provided, comprising the following steps:

[0012] A1. The S*-Ru chain and the IT chain were reacted under Tris-HCl buffer conditions to obtain the S* / IT complex;

[0013] A2. Subsequently, the hairpin probe H1, the hairpin probe H2, the exonuclease Exo-III, and different concentrations of the target DNA (T) were added to the S* / IT complex for reaction.

[0014] A3. Subsequently, ECL detection was performed in the TPrA solution with a detection voltage of 0.6V to 1.6V and a scan rate of 100mV / s.

[0015] Further, the reaction conditions in step A1 are 35℃ for 30 min, and the reaction conditions in step A2 are 35℃ for 60 min; the concentration of the S*-Ru chain is 1 μM, the concentration of the IT chain is 800 nM, the concentration of the hairpin probe H1 is 200 nM, the concentration of the hairpin probe H2 is 200 nM, and the concentration of the exonuclease Exo-III is 0.4 U / μL.

[0016] According to a third aspect of the present invention, a Cd reaction system for a feedback-type HCR-Exo as described above is proposed. 2+ The analytical method includes the following steps:

[0017] B1. The S*-Ru chain and the IT chain are reacted under Tris-HCl buffer conditions to obtain the S* / IT complex;

[0018] B2. React the recognition unit AT and the blocking chain B to obtain the AT / B complex;

[0019] B3. Mix the S* / IT complex and the AT / B complex, then add the hairpin probe H1, the hairpin probe H2, the exonuclease Exo-III, and different concentrations of Cd. 2+ To carry out the reaction;

[0020] B4. Subsequently, ECL detection was performed in the TPrA solution with a detection voltage of 0.6V to 1.6V and a scan rate of 100mV / s.

[0021] Further, the reaction conditions in steps B1 and B2 are both 35℃ for 30 min, and the reaction conditions in step B3 are 35℃ for 60 min; the concentration of the S*-Ru chain is 1 μM, the concentration of the IT chain is 800 nM, the concentration of the recognition unit AT is 100 nM, the concentration of the blocking chain B is 125 nM, the concentration of the hairpin probe H1 is 200 nM, the concentration of the hairpin probe H2 is 200 nM, and the concentration of the exonuclease Exo-III is 0.4 U / μL.

[0022] According to a fourth aspect of the present invention, a Cd reaction system for a feedback-type HCR-Exo as described above is proposed. 2+ The detection kit, wherein the Cd 2+ The detection kit includes target DNA (T), hairpin probe H1, hairpin probe H2, S* / IT complex, exonuclease Exo-III, recognition unit AT, and blocking strand B.

[0023] According to a fifth aspect of the present invention, a feedback-type HCR-Exo reaction system as described above is proposed for the detection of trace Cd in food. 2+ Applications in [the field].

[0024] The beneficial effects of this invention are:

[0025] This invention improves the reaction system of target DNA and Cd by designing a feedback-type HCR-Exo reaction system. 2+ The detection sensitivity was improved, and further signal amplification was achieved through the synergy of HCR and Exo. The ECL signal change value of the system was correlated with the target DNA and Cd. 2+ The concentrations of these components are positively correlated, thus enabling the targeting of DNA and Cd. 2+ The detection method has the advantages of stability, high sensitivity and good selectivity. Attached Figure Description

[0026] Figure 1 The schematic diagram (A) of the feedback-type HCR-Exo reaction system for DNA detection and for Cd is shown in a specific embodiment of the present invention. 2+ Schematic diagram of the detection principle (B);

[0027] Figure 2 The response (A) of ECL under different conditions in a specific embodiment of the present invention is shown, where a represents S* / IT, b represents H1+H2+S* / IT+Exo-III, c represents T+H1+H2+S* / IT, and d represents T+H1+H2+S* / IT+Exo-III.

[0028] (B) represents the intensity of ECL under different conditions, where a represents S* / IT, b represents H1+H2+S* / IT+Exo-III, c represents T+H1+H2+S* / IT, and d represents T+H1+H2+S* / IT+Exo-III.

[0029] (C) is a PAGE image of the feedback HCR-Exo reaction system, where the image is a 20bp DNA label, a represents S*, b represents IT, c represents S* / IT, d represents H1+H2, e represents T+H1+H2, f represents H1+H2+S* / IT, g represents T+H1+H2+S* / IT, h represents T+H1+H2+S* / IT+Exo-III, and i represents S* / IT+H1+H2+Exo-III;

[0030] (D) is an AFM image of dsDNA nanowires generated in a feedback-type HCR-Exo reaction system;

[0031] Figure 3 ECL response (A) of the feedback HCR-Exo reaction system at 100 nM T at different time points in a specific embodiment of the present invention;

[0032] (B) shows the change in ECL intensity over time for the feedback-type HCR-Exo reaction system in the absence of T and in the presence of 100 nM T;

[0033] (C) represents the ECL intensity of the feedback-type HCR-Exo reaction system with different concentrations T, where a to l are 0 pM, 1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 50 nM and 100 nM respectively;

[0034] (D) ECL intensity of the feedback HCR-Exo reaction system at different concentrations (1 pM to 100 nM) T was detected, with the inset showing the calibration curve between ECL intensity and T concentration;

[0035] (E) ECL intensity obtained for detecting different mutant T analytes and 0 nM T in the feedback HCR-Exo reaction system;

[0036] Figure 4 In a specific embodiment of the present invention, there is no Cd. 2+ and has Cd 2+ Time-dependent ECL intensity (A) of the feedback-type HCR-Exo reaction system in the presence of [a specific substance], with the inset showing the ECL intensity at 1 μMcd. 2+ ECL response of the feedback-type HCR-Exo reaction system at different times under the presence of [condition];

[0037] (B) Stability of the feedback-type HCR-Exo reaction system at different concentrations;

[0038] (C) represents different concentrations of Cd. 2+ When present, the ECL intensity of the feedback-type HCR-Exo reaction system, where from a to j are 0 pM, 1 pM, 10 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 100 nM and 1 μM respectively;

[0039] (D) To detect different concentrations of Cd 2+ The ECL intensity of the feedback-type HCR-Exo reaction system is shown in the inset, where the ECL intensity is a function of Cd. 2+ Calibration curves between concentrations;

[0040] Figure 5 Analysis of Cd in the feedback-type HCR-Exo reaction system in a specific embodiment of the present invention 2+ Schematic diagram with other metal ions (A);

[0041] (B) Analysis of 1 μM Cd in a feedback-type HCR-Exo reaction system 2+ 10 μM other metal ions (Ag) + K + Ni 2+ Pb 2 + Fe 3+ Zn 2+ and Cu 2+ And the ECL response of all the above metal ion mixtures;

[0042] (C) Analysis of 1 μM Cd in a feedback-type HCR-Exo reaction system 2+ 10 μM other metal ions (Ag) + K + Ni 2+ Pb 2 + Fe 3+ Zn 2+ and Cu 2+ ) and the ECL strength when all the above metal ions are mixed. Detailed Implementation

[0043] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0044] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0045] The sequences of the hairpin probe H1, as described in the following embodiments, are shown in SEQ ID NO.1; the sequences of the hairpin probe H2, as described in SEQ ID NO.2; the sequences of the S*, as described in SEQ ID NO.3; the sequences of the IT strand, as described in SEQ ID NO.4; and the sequences of the target DNA (T), as described in SEQ ID NO.5. The reaction system further includes a recognition unit AT and a blocking strand B; the recognition unit AT is composed of a T(a*-b*) initiating sequence and a Cd... 2+ The aptamer sequence is composed of: the blocking chain B partially hybridizes with the recognition unit AT to prevent signal leakage caused by the release of the trigger chain T; the sequence of the recognition unit AT is shown in SEQ ID NO.6; the sequence of the blocking chain B is shown in SEQ ID NO.7; wherein, Ru(bpy)3 2+ The modification is placed at the 5' end of the S* chain.

[0046] Example 1

[0047] Hairpin probe design: Hairpin probes were designed using NUPACK software, and the relevant nucleic acid sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. In the absence of the target analyte, each hairpin was designed to have sufficient complementary base pairing at its stem end to maintain its stability. However, in the presence of the target analyte, it was designed to trigger a feedback HCR-Exo reaction, significantly amplifying the ECL signal and enabling the detection of the target analyte. All hairpin probe powders were first dissolved in phosphate buffer, and their absorbance was measured using a UV spectrophotometer to calculate the accurate concentration. Then, all hairpin probes were prepared to 4 μM using 10 mM Tris-HCl buffer (10 mM Tris, 100 mM KCl, 2 mM MgCl2, pH 7.2). The probes were then incubated in PCR at 95°C for 5 min and 25°C for 2 h to allow them to form stable hairpins. All reactions were performed in 10 mM Tris-HCl buffer.

[0048] Figure 1 (A) is a schematic diagram of the feedback-type HCR-Exo reaction system for DNA detection. S* (1 μM), IT (800 nM), H1 (200 nM), H2 (200 nM), and Exo-III (0.4 U / μL) are added to 10 mM Tris-HCl buffer, followed by ECL detection in 20 mM TPrA solution. The detection voltage is set from 0.6 V to 1.6 V, and the scan rate is set to 100 mV / s. Figure 2In (A) and (B), which exist only in homogeneous ECL systems of Ru-S* / IT, the observed ECL signal is negligible. Figure 2 (Curve a in the figure). Similarly, in the absence of a target molecule (T), the system is not induced, and the ECL signal is negligible. Figure 2 Curve b) shows that the DNA probe in solution can coexist stably without significant signal leakage. The ECL signal was significantly enhanced after the addition of the target molecule (T). Figure 2 Curve d in the figure demonstrates that the sensor can only be activated in the presence of the target molecule (T). Finally, to prove that Exo-III plays a crucial role in this sensor, we specifically designed a control group with the target molecule (T) added but without Exo-III added. Figure 2 Curve c) in the diagram clearly shows the ECL signal base of curve c, further verifying that the product chain T-(H1-H2-S*) generated by the target molecule (T) is... N With a large amount of negative charge, it cannot approach the ITO electrode surface, and in the absence of Exo-III, Ru-S* cannot be hydrolyzed and digested, thus failing to generate a significant ECL signal.

[0049] HCR-Exo products were characterized by gel electrophoresis. DNA mixtures with different lanes (S* / IT; H1+H2; T+H1+H2; H1+H2+S* / IT; T+H1+H2+S* / IT; T+H1+H2+S* / IT+Exo-III and S* / IT+H1+H2+Exo-III; S* 1 μM; IT 800 nM) were mixed in 10 mM Tris-HCl buffer and incubated at 35 °C for 60 min. Next, 10 μL of each of the above DNA mixtures was mixed with 2 μL of loading buffer and added to a 12% natural polyacrylamide gel. The electrophoresis apparatus voltage was set to 80 V. After 1 h, the gel was removed and stained with GelRed. Finally, the DNA was visualized under ultraviolet light using a chemiluminescence imaging system. The electrophoresis results are shown below. Figure 2As shown in (C), lanes a, b, and d indicate that all probes can coexist stably. Clear HCR product bands are visible in lane g (T+H1+H2+S* / IT), indicating that the S* in S* / IT hybridizes with the S in H2 and releases IT to further initiate new HCR reactions. In lane h (T+H1+H2+S* / IT+Exo-III), the addition of Exo-III results in less HCR product compared to lane g. This is because Exo-III digests and hydrolyzes the SS* on the H2 in the HCR product chain, yielding a macromolecular product with a slightly lower molecular weight than that in lane g. These results demonstrate the successful construction of our proposed feedback HCR-Exo reaction system.

[0050] The HCR-Exo products were characterized using atomic force microscopy (AFM). A mixture of the feedback-type HCR-Exo reaction system (H1, 200 nM; H2, 200 nM; S* / IT (S*, 1 μM; IT, 800 nM); and Exo-III, 0.4 U / μL) was mixed with 100 nM of the target molecule (T) and incubated in Tris-HCl buffer at 35 °C for 60 min. Freshly peeled mica sheets were pretreated with 90 μL of (3-aminopropyl)trimethoxysilane (APTES) and 30 μL of N,N-diisopropylethylamine (DIPEA) vapor for 2 h to impart a positive charge; this process was performed in a desiccator. DNA samples diluted to 20 nM were dropped onto mica sheets, washed three times with ultrapure water after 15 min, dried under nitrogen, and scanned using an AFM. Results are shown below. Figure 2 As shown in (D), a polymer nanowire with a height of approximately 1.7 nm can be observed from the characterization results. In summary, ECL experiments, polyacrylamide gel electrophoresis, and AFM characterization all demonstrate that the sensor can only be efficiently assembled and trigger the long DNA nanowire as expected in the presence of the target DNA; otherwise, the sensor cannot be activated, proving that the sensor we constructed has the ability to detect analytes.

[0051] Example 2

[0052] DNA detection based on feedback-based HCR-Exo reaction

[0053] ssDNA S* (1 μM) and IT (800 nM) were reacted in 10 mM Tris-HCl buffer at 35 °C for 30 min to form an S* / IT complex. Subsequently, hairpin H1 (200 nM), H2 (200 nM), Exo-III (0.4 U / μL), and different concentrations of T (0 pM, 1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 50 nM, and 100 nM) were added to the S* / IT mixture, and the mixture was reacted at 35 °C for 60 min. Finally, ECL detection was performed in 20 mM TPrA solution at a detection voltage ranging from 0.6 V to 1.6 V and a scan rate of 100 mV / s.

[0054] Figure 1 (A) is a schematic diagram of the feedback-type HCR-Exo reaction system for target DNA detection. Figure 3 As shown in (A) and 3(B), the target DNA in the HCR-Exo system tends to stabilize after 60 min of incubation. Figure 3 (C) and Figure 3 (D) As can be seen, in the feedback HCR-Exo reaction system without the addition of target DNA, each DNA probe can maintain its own stability, and the ECL intensity of the system only changes slightly. When different concentrations of target DNA are added, the change in ECL intensity is positively correlated with the concentration of target DNA, which can be used to detect the target DNA. Figure 3 (D) It can be seen that as the concentration of target DNA increases, the change value of ECL signal has a good linear relationship with the concentration of target DNA in the range of 1pM to 100pM, and the detection limit is 0.098pM, realizing rapid and highly sensitive detection of target DNA.

[0055] To demonstrate the selectivity of this method for detecting target DNA (T), single-base (T1), double-base (T2), and triple-base (T3) mutations were selected as interfering components for investigation. Figure 3 (E) It can be seen that the ECL signal of the system only changes significantly when it interacts with the target DNA (T). The changes in ECL signal caused by single base (T1), double base (T2) and triple base (T3) mutations are very small. The above results show that the method of the present invention has good selectivity for the detection of target DNA.

[0056] Example 3

[0057] Cd based on feedback-type HCR-Exo reaction 2+ Detection

[0058] First, ssDNA S* (1 μM) and IT (800 nM) were mixed in 10 mM Tris-HCl buffer and reacted at 35 °C for 30 min to form the S* / IT complex. Then, AT (100 nM) and blocking strand B (125 nM) were reacted at 35 °C for 30 min to form the AT / B complex. Next, different concentrations of Cd... 2+ ECL values ​​(0 pM, 1 pM, 10 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 100 nM, and 1 μM) were added to a feedback-type HCR-Exo reaction system (H1, 200 nM; H2, 200 nM; S* / IT (S*, 1 μM; IT, 800 nM); AT / B (AT, 100 nM; B, 125 nM); and Exo-III (0.4 U / μL), and reacted at 35 °C for 60 min. ECL measurements were recorded in a Tris-HCl buffer solution containing 20 mM TPrA from 0.6 V to 1.6 V.

[0059] Figure 1 (B) is a feedback-type HCR-Exo reaction system used for Cd 2+ A schematic diagram of the detection principle. (e.g.) Figure 4 (A) and Figure 4 As shown in (B), 1 μM Cd 2+ In the HCR-Exo system, the incubation time tended to stabilize after 60 min, and the incubation time was stable for three different concentrations (5 nM, 100 nM, and 1 μM) of Cd. 2+ During eight consecutive cyclic scans, the ECL signal showed no significant change. Figure 4 (C) and Figure 4 (D) It can be seen that no Cd was added in the feedback-type HCR-Exo reaction system. 2+ Each DNA probe maintains its own stability, and the ECL strength of the system changes only slightly when different concentrations of Cd are added. 2+ At that time, the change in ECL intensity was related to Cd. 2+ The concentration of Cd is positively correlated, and this can be used to study the concentration of Cd. 2+ Conduct testing. (By...) Figure 4 (D) It can be seen that, with Cd 2+ The change in ECL signal with increasing concentration is related to Cd. 2+ The concentration exhibits good linearity in the range of 1 pM to 1 nM, with a detection limit of 0.13 pM, achieving effective detection of Cd. 2+ Rapid and highly sensitive detection.

[0060] To demonstrate the effectiveness of this method for Cd 2+ Selectivity of detection, selecting Ag + K +Ni 2+ Pb 2+ Fe 3+ Zn 2+ and Cu 2+ The interfering components were investigated. Figure 5 It is evident that only with Cd 2+ The ECL signal of the system will only change significantly when Ag is applied. + K + Ni 2+ Pb 2+ Fe 3+ Zn 2+ and Cu 2+ The resulting change in the ECL signal is very small. The above results indicate that the method of this invention is effective for Cd... 2+ The detection has good selectivity.

[0061] Table 1. DNA probes designed using NUPACK software.

[0062]

[0063]

[0064] In Table 1 above, Ru(bpy)3 2+ The modification is placed at the 5' end of the S* chain.

[0065] Example 4

[0066] Application of cadmium ion analysis method based on feedback HCR-Exo reaction in large yellow croaker and prawn samples

[0067] Select edible parts, mince and mix them thoroughly, accurately weigh 100mg of large yellow croaker and prawn food samples, and add 5.62μg / kg and 28.10μg / kg of Cd to the 100mg food samples respectively. 2+ The standard solution was processed using microwave digestion according to national standards. First, the pretreated food sample was treated with 8 mL of 65% (w / w) nitric acid, 1 mL of hydrochloric acid, and 1 mL of hydrofluoric acid. Next, the mixture was further digested in a constant-temperature drying oven. Finally, after cooling to room temperature, it was diluted to 25 mL with ultrapure water to complete the deacidification process. 10 μL of the pretreated food sample solution was added to 190 μL of ECL probe solution and reacted at 35 °C for 60 min for Cd... 2+ Determination. The proposed feedback-type HCR-Exo reaction system and standard ICP-MS method were used to determine the Cd content in the above food samples. 2+ Conduct testing and comparison.

[0068] Analysis of different concentrations of Cd added2+ The HCR-Exo detection system was evaluated using standard samples of large yellow croaker and tiger prawns to assess its application potential in real-world samples. As shown in Table 2, at different spike levels in large yellow croaker and tiger prawns, Cd... 2+ The recoveries were 92.70%–97.54% and 98.54%–103.38%, respectively, with relative standard deviations (RSD) below 5%. These results demonstrate that our proposed HCR-Exo system can reliably determine Cd in real samples. 2+ The flexibility and programmability of the HCR-Exo sensing system help explore various biomolecules in real samples, and it has great application potential in the field of trace analysis.

[0069] Table 2. HCR-Exo reaction system and ICP-MS detection of Cd in large yellow croaker and shrimp. 2+ Spiked recovery results

[0070]

[0071] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A reaction system based on feedback HCR-Exo, characterized in that, The reaction system comprises target DNA (T), hairpin probe H1, hairpin probe H2, S* / IT complex and exonuclease Exo-III; the target DNA (T) is composed of a*-b*; the hairpin probe H1 comprises b*, c, b and a; the hairpin probe H2 comprises c*, b, c and d; the S* / IT complex comprises an S*-Ru chain and an IT chain, wherein the S*-Ru chain comprises a, d* and c*, and the IT chain comprises a base sequence identical to the target DNA (T).

2. The reaction system of the feedback HCR-Exo according to claim 1, characterized in that, The b* and b in the hairpin probe H1 are complementary to form a double strand as the stem of the hairpin probe H1, c is the loop of the hairpin structure, and a is the 3' sticky end extending from the stem of the hairpin structure; the c* and c in the hairpin probe H2 are complementary to form a double strand as the stem of the hairpin probe H2, b is the loop of the hairpin structure, and d is the 3' sticky end extending from the stem of the hairpin structure; the 5' end of the S*-Ru strand is labeled with Ru(bpy)3 2+ modification.

3. The reaction system of feedback HCR-Exo according to claim 2, characterized in that, The sequence of the hairpin probe H1 is shown as SEQ ID NO. 1; the sequence of the hairpin probe H2 is shown as SEQ ID NO. 2; the sequence of the S* is shown as SEQ ID NO. 3; the sequence of the IT chain is shown as SEQ ID NO. 4; and the sequence of the target DNA (T) is shown as SEQ ID NO.

5. 4.The reaction system of the feedback HCR-Exo according to claim 1, characterized in that, The reaction system further comprises a recognition unit AT and a blocking chain B; the recognition unit AT is initiated by T(a*-b*) and Cd 2+ The sequence of the aptamer is shown in SEQ ID NO. 6; the sequence of the blocking chain B is shown in SEQ ID NO.

7.

5. A method for detecting a nucleic acid of a reaction system of the feedback-type HCR-Exo according to any one of claims 1 to 4, characterized by, The method comprises the following steps: A1, reacting the S*-Ru chain and the IT chain under the condition of Tris-HCl buffer solution to obtain the S* / IT complex; A2, then adding the hairpin probe H1, the hairpin probe H2, the exonuclease Exo-III and different concentrations of the target DNA (T) into the S* / IT complex to react; A3, then performing ECL detection in a TPrA solution, the detection voltage is 0.6V-1.6V, and the scanning rate is set to 100mV / s. 6.The method according to claim 5, wherein, The reaction condition in step A1 is 35℃ for 30min, and the reaction condition in step A2 is 35℃ for 60min; the concentration of the S*-Ru chain is 1μM, the concentration of the IT chain is 800nM, the concentration of the hairpin probe H1 is 200nM, the concentration of the hairpin probe H2 is 200nM, and the concentration of the exonuclease Exo-III is 0.4U / μL.

7. A Cd of a reaction system of the feedback HCR-Exo according to any one of claims 1 to 4. 2+ An analysis method characterized by, The method comprises the following steps: B1, reacting the S*-Ru chain and the IT chain under the condition of Tris-HCl buffer solution to obtain the S* / IT complex; B2, reacting the recognition unit AT and the blocking chain B to obtain an AT / B complex; B3, mixing the S* / IT complex and the AT / B complex, adding the hairpin probe H1, the hairpin probe H2, the exonuclease Exo-III and different concentrations of Cd 2+ reaction; B4, then performing ECL detection in a TPrA solution, the detection voltage is 0.6V-1.6V, and the scanning rate is set to 100mV / s.

8. The Cd of the reaction system of feedback type HCR-Exo according to claim 7. 2+ An analysis method characterized by, The reaction condition in step B1 and step B2 is 35℃ for 30min, and the reaction condition in step B3 is 35℃ for 60min; the concentration of the S*-Ru chain is 1μM, the concentration of the IT chain is 800nM, the concentration of the recognition unit AT is 100nM, the concentration of the blocking chain B is 125nM, the concentration of the hairpin probe H1 is 200nM, the concentration of the hairpin probe H2 is 200nM, and the concentration of the exonuclease Exo-III is 0.4U / μL.

9. A Cd of a reaction system of the feedback HCR-Exo according to any one of claims 1 to 4. 2+ A test kit characterized in that, The Cd 2+ The detection kit comprises target DNA (T), hairpin probe H1, hairpin probe H2, S* / IT complex, exonuclease Exo-III, recognition unit AT and blocking strand B.

10. Use of the reaction system of feedback HCR-Exo according to any one of claims 1-4 in detecting trace Cd in food. 2 + ​

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