MC-LR rapid detection biosensor based on exonuclease III, detection kit and detection method

Abstract

The invention relates to an exonuclease III-based MC-LR rapid detection biosensor, a detection kit and a detection method. The exonuclease III-based MC-LR rapid detection biosensor comprises an Aptamer / Blocker compound, a hairpin probe H and exonuclease III, wherein the Aptamer / Blocker compound comprises a specific aptamer A and a blocking agent B of MC-LR, and two ends of the hairpin probe H are respectively modified by a fluorophore and a quencher. The detection kit comprises the MC-LR rapid detection biosensor based on exonuclease III and a buffer solution, wherein the buffer solution comprises a PBS buffer solution and an exonuclease III buffer solution. The exonuclease Exo III with the MC-LR aptamer as the recognition probe is used for assisting in catalyzing target recovery, operation is easy and convenient, sensitivity is high, and good recovery rate and accuracy are achieved in an actual water sample.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a novel MC-LR high-sensitivity detection biosensor based on nuclease III, a detection kit and a detection method. Background Art

[0002] With climate change, cyanobacterial blooms are increasing worldwide. Microcystiins (MCs) are heptapeptide toxins containing two variable amino acids and five constant amino acids, mainly produced by cyanobacteria, posing a global threat to water quality and human health. So far, more than 300 MCs homologues have been discovered. Among them, MC-LR is the most toxic and common variant of microcystin, and its wide distribution and high toxicity have attracted increasing attention from the scientific community. MC-LR can cause acute or chronic damage to humans and threaten organs such as the liver, intestines and kidneys. Considering long-term health and human safety, the World Health Organization (WHO) has proposed a safe limit of 1 μg / L for MC-LR in drinking water. Therefore, there is still an urgent need to develop new MC-LR detection methods in environmental water samples.

[0003] A variety of analytical methods have been reported for MC-LR determination. Although some chemical analytical methods, including high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), have excellent accuracy and sensitivity, they usually require bulky instruments, expensive equipment, complex pretreatment procedures, and well-trained operators. Enzyme-linked immunosorbent assay (ELISA) and protein phosphatase inhibition assay (PPIA) are limited by their flexibility and sensitivity. Therefore, finding a simple detection method with satisfactory detection performance is of great significance for MC-LR analysis.

[0004] Aptamers are short nucleic acids selected by systematic evolution of ligands by exponential enrichment (SELEX). Due to their high specificity and strong affinity, they have been widely used as recognition elements to sense targets. As early as 2001, researchers developed the first aptamer for MC-LR detection. The MC-LR-specific aptamer screened by researchers in 2012 is currently widely used. In recent years, various biosensors have been developed for MC-LR analysis, including colorimetry, fluorescence, electrochemistry, electrochemiluminescence (ECL), and surface-enhanced Raman spectroscopy (SERS). In order to improve the detection limit of sensing methods, signal amplification strategies are used as powerful tools for the development of ultrasensitive biosensors, including hybridization chain reaction (HCR), catalytic hairpin assembly (CHA), DNAzyme-mediated loop cleavage, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Among them, exonuclease ExoⅢ, as an amplified exonuclease, is widely used in the cyclic amplification strategy in the field of sensing. Exonuclease ExoⅢ can only act on double-stranded DNA with blunt or recessed 3' ends, and is inactive on other double-stranded DNA and single-stranded DNA. Unlike other amplification strategies, signal amplification assisted by exonuclease ExoⅢ is a sequence-independent amplification method that can simplify sensor design. Therefore, exonuclease ExoⅢ is particularly suitable for use as a high-performance DNA amplifier in biosensing.

[0005] Chinese patent document CN 105548555 A discloses a preparation and detection method of a non-enzyme immunosensor for microcystin-LR (MC-LR), wherein carboxylated carbon nanofibers and polyethylene glycol are assembled layer by layer on the surface of a glassy carbon electrode to construct a high-density MC-LR antigen sensing interface, synthesize gold nanoparticles and prepare gold nanoparticle probes, and adopt the principle of indirect competitive immunoassay to coat MC-LR antigen and MC-LR target antigen to compete with MC-LR antibody to form an immune complex, react the gold nanoparticle probe with the immune complex, and oxidize the gold nanoparticles bonded to the electrode surface to generate AuCl 4- , by electrochemical determination of AuCl 4- Restoring the peak-to-peak current value can achieve the purpose of detecting MC-LR, but the accuracy is not high.

[0006] Therefore, it is necessary to develop a MC-LR rapid detection biosensor, detection kit and detection method based on nuclease III, which uses the MC-LR aptamer as the recognition probe for nuclease ExoⅢ to assist in catalytic target recovery. It is easy to operate, highly sensitive, and has good recovery rate and accuracy in actual water samples. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a new detection system, which is a MC-LR rapid detection biosensor based on nuclease III, and the nuclease ExoⅢ with MC-LR aptamer as the recognition probe assists in catalytic target recovery. The system is simple to operate, highly sensitive, and has good recovery rate and accuracy in actual water samples.

[0008] In order to solve the above technical problems, the technical solution of the present invention is: the MC-LR rapid detection biosensor based on nuclease III includes an Aptamer / Blocker complex, a hairpin probe H (Hairpin probe) and nuclease III; wherein the Aptamer / Blocker complex includes a specific aptamer A and a blocker B of MC-LR, and the two ends of the hairpin probe H are modified with a fluorophore (FAM) and a quencher (BHQ), respectively.

[0009] The above technical scheme is adopted to assist the target recovery by using the exonuclease ExoⅢ with the aptamer as the recognition probe. In the presence of MC-LR, due to the high affinity between MC-LR and the aptamer, the specific aptamer A in the Aptamer / Blocker complex will specifically bind to MC-LR, thereby releasing the blocker B from the Aptamer / Blocker complex. Then the blocker B reacts with the hairpin probe H to form a special structure that can be recognized by the exonuclease ExoⅢ, thereby inducing the target recycling reaction assisted by the exonuclease ExoⅢ. The hairpin probe H is modified with a fluorophore (FAM) and a quencher (BHQ), and a high fluorescence signal is generated by the separation of the fluorophore (FAM) and the quencher (BHQ), which amplifies the detection signal and improves the detection sensitivity; the sensor is easy to operate, highly sensitive, and has a detection limit of 0.37ng / L. This method has good recovery and accuracy in actual water samples, and provides a new candidate method for monitoring MC-LR in the environment.

[0010] Preferably, the sequence of the specific aptamer A of the MC-LR is as shown in SEQ ID NO.1, and SEQ ID NO.1 is: GGC GCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTCCGC TTT TTT;

[0011] The sequence of the blocker B is shown in SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5, and SEQ ID NO.2 is: GCG GAG ATG GGG CAT TTT TTT;

[0012] SEQ ID NO.3 is: GCG GAG ATG GGG CAT AAT TTT TT;

[0013] SEQ ID NO.4 is: GCG GAG ATG GGG CAT AAT GTT TTT T;

[0014] SEQ ID NO.5 is: GCG GAG ATG GGG CAT AAT GAG TTT TTT;

[0015] The sequence of the hairpin probe H is as shown in SEQ ID NO.6, and SEQ ID NO.6 is: FAM-AGA CTA GAC CGGAAC GAC GGT CTA GTC T (BHQ1)-ATG CCC CAT CTC CGC.

[0016] Preferably, the sequence of the blocker B is as shown in SEQ ID NO.4, and SEQ ID NO.4 is GCG GAG ATGGGG CAT AAT GTT TTT T (25nt).

[0017] The technical problem that the present invention also solves is to provide a detection kit for detecting MC-LR by signal amplification, and the detection kit is prepared with a fluorescent aptamer sensor assisted by nuclease ExoⅢ, which is used for high-sensitivity detection of MC-LR, with accurate detection and strong specificity.

[0018] In order to solve the above technical problems, the technical solution of the present invention is: the detection kit for signal amplification and detection of MC-LR includes a MC-LR rapid detection biosensor based on nuclease III and a buffer solution, and the buffer solution includes a PBS buffer solution and a nuclease ExoⅢ buffer solution.

[0019] Preferably, the formula of the PBS buffer solution is: 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4, and its pH = 7.4.

[0020] The composition, type and pH of the buffer may fluctuate slightly, but are generally stable. Exonuclease ExoⅢ buffer was purchased from New England Biolabs (NEB) and Beijing Polymer Biotech Co., Ltd., and the exonuclease Ⅲ buffer was purchased from Life Science Reagents (NEB) with the model number M0206; buffer: NEBuffer 1 (10x); purchased from Beijing Polymer Biotech Co., Ltd.; model: MF466-01; buffer: 10×Klenow Buffer; oligonucleotide sequences purified by HPLC were purchased from Sangon Biotech (Shanghai) Co., Ltd. MC-LR, microcystin-rr (MC-RR), microcystin-yr (MC-YR) and microcystin-la (MC-LA) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; exonuclease ExoⅢ was purchased from New England Biolabs (NEB) and Beijing Polymer Biotech Co., Ltd.; all reagents were not further purified.

[0021] The technical problem that the present invention also solves is to provide a method for quantitative detection of MC-LR based on a MC-LR rapid detection biosensor of nuclease exonuclease III, and to perform quantitative detection of MC-LR using a fluorescent aptamer sensor assisted by nuclease exonuclease ExoⅢ, which has high detection sensitivity and strong selectivity.

[0022] In order to solve the above technical problems, the technical solution of the present invention is: the method for quantitative detection of MC-LR by the MC-LR rapid detection biosensor based on nuclease III, the specific steps are:

[0023] S1: First, the aptamer / blocker complex is synthesized by using the MC-LR specific aptamer A and blocker B;

[0024] S2: pre-hybridizing the probe H and then annealing to form the structure of the hairpin probe H;

[0025] S3: Add different concentrations of MC-LR to the Aptamer / Blocker complex and incubate, then add the hairpin probe H for reaction, and finally add exonuclease ExoⅢ and exonuclease ExoⅢ buffer for incubation again to perform exonuclease ExoⅢ-mediated digestion reaction;

[0026] S4: Fluorescence signal detection after the exonuclease ExoⅢ mediated digestion reaction.

[0027] Using the above technical solution, the method for quantitative detection of MC-LR based on the MC-LR rapid detection biosensor of nuclease III is a reliable method for detecting trace MC-LR, with good sensitivity and selectivity, and the detection limit (LOD) is as low as 0.37ng / L. In addition, the method has strong practicality and can be effectively used for water samples with a satisfactory recovery rate (95.04% to 107.01%). The sensor has excellent sensitivity, high selectivity, efficient signal amplification and fluorescence readout capabilities, providing a new and reliable alternative for the detection of trace MC-LR in the environment and early warning of algal toxins.

[0028] Preferably, the mixing ratio of the MC-LR specific aptamer A and the blocker B in step S1 is 1:1 to 4:1.

[0029] Preferably, the specific steps of step S1 are: mixing 200 nM MC-LR specific aptamer A with 100 nM blocker B, that is, the mixing ratio of MC-LR specific aptamer A to blocker B is 2:1; heating at 95°C for 5 minutes, and then slowly cooling to room temperature to obtain an Aptamer / Blocker complex.

[0030] Preferably, in step S2, 100 nM probe H is pre-hybridized at 97° C. for 4 min and slowly cooled to room temperature to form a hairpin structure, wherein the probe has been modified with a fluorophore (FAM) and a quencher (BHQ) before pre-hybridization, and pre-hybridization enables it to form a stable hairpin structure.

[0031] Preferably, the specific steps of step S3 are: adding different concentrations of MC-LR to the Aptamer / Blocker complex, incubating at 37°C for 0 to 120 min, adding 100 nM probe H, incubating at 37°C for 30 min, then adding 1 unit / μL of nuclease ExoⅢ, adding nuclease ExoⅢ buffer at the same time, and finally adding PBS buffer solution to form a 100 μL system, incubating at 37°C for 30 min, thereby completing the enzymatic cleavage reaction mediated by nuclease ExoⅢ.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) A fluorescent aptamer sensor assisted by exonuclease ExoⅢ was used for high-sensitivity detection of MC-LR; the detection method had high sensitivity, strong selectivity, and a low limit of detection (LOD) of 0.37 ng / L;

[0034] (2) This method has strong specificity, with obvious fluorescence signal response only to the target MC-LR, and low fluorescence signal response to other substances. Experiments have shown that the sensing system also has a good recovery rate (95.04% to 107.01%), and can be successfully applied to actual water samples to achieve accurate determination of MC-LR under different environmental conditions;

[0035] (3) The present invention proposes a new fluorescence strategy for detecting MC-LR and realizes a new monitoring system, specifically: the sensor is designed based on the high affinity of the MC-LR aptamer, and the fluorescent probe technology and the signal amplification of ExoIII are integrated for the first time to form a new detection system, which further improves the detection efficiency of the sensor; the detection results are good, the detection performance is satisfactory, the operation is simple, the sensitivity is high, and it has good recovery rate and accuracy in actual water samples;

[0036] (4) The MC-LR rapid detection biosensor based on nuclease III has strong specificity and high sensitivity, and also has efficient signal amplification and fluorescence readout functions. It provides a simple and reliable method for the detection of trace MC-LR in the environment and the early warning of algal toxin hazards. It also provides valuable directions for the application of fluorescence sensing and nuclease ExoⅢ in the field of algal toxin immunoassay. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A diagram showing the sensing mechanism of MC-LR detection by the MC-LR rapid detection biosensor based on nuclease III of the present invention;

[0038] Figure 2 The fluorescence spectra of the feasibility analysis and verification of MC-LR detection by the MC-LR rapid detection biosensor based on nuclease III of the present invention are shown in FIG. 1 ; wherein (A) is a fluorescence spectra of the hairpin probe H chain under different reaction conditions; (B) is a fluorescence spectra of the presence and absence of MC-LR in the sensing system;

[0039] Figure 3 The figure is a PAGE electrophoresis diagram for feasibility analysis and verification of MC-LR detection by the MC-LR rapid detection biosensor based on nuclease III of the present invention; wherein (A) is the PAGE pattern corresponding to different sequences, lane 1: MC-LR specific aptamer A; lane 2: blocker B; lane 3: A / B complex; lane 4: A / B complex + MC-LR; wherein (B) is the PAGE pattern analysis mediated by ExoⅢ, lane 1: B; lane 2: hairpin probe H; lane 3: B+H+ExoⅢ;

[0040] Figure 4This is a diagram showing the optimization results of the number of bases of the blocker used in MC-LR detection in the MC-LR rapid detection biosensor based on nuclease III of the present invention;

[0041] Figure 5 This is a diagram showing the optimization results of the A / B concentration ratio in step S1 of MC-LR detection in the MC-LR rapid detection biosensor based on nuclease III of the present invention;

[0042] Figure 6 This is a diagram showing the optimization results of the reaction time in step S3 of MC-LR detection by the MC-LR rapid detection biosensor based on nuclease III of the present invention;

[0043] Figure 7 The spectrum and linear range of MC-LR quantification for MC-LR detection by the MC-LR rapid detection biosensor based on nuclease III of the present invention; wherein (A) is fluorescence spectrum analysis using different concentrations of MC-LR (0, 0.001, 0.01, 0.1, 1 and 10 μg / L); (B) is the linear relationship between the increase in fluorescence intensity (ΔF) and the logarithm of MC-LR concentration, error bar = ±SD, n = 3;

[0044] Figure 8 This is a graph showing the specificity of MC-LR detected by the MC-LR rapid detection biosensor based on nuclease III of the present invention, error bars = ± SD, n = 3. DETAILED DESCRIPTION

[0045] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with embodiments. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0046] Embodiment: The MC-LR rapid detection biosensor based on nuclease III includes an Aptamer / Blocker complex, a hairpin probe H (Hairpin probe) and nuclease III; wherein the Aptamer / Blocker complex includes a specific aptamer A of MC-LR and a blocker B, and the two ends of the hairpin probe H are modified with a fluorophore (FAM) and a quencher (BHQ), respectively. The sequence of the specific aptamer A of MC-LR is shown in SEQ ID NO.1, which is: GGCGCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGCTTT TTT;

[0047] The sequence of the blocker B is shown in SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5, and SEQ ID NO.2 is: GCG GAG ATG GGG CAT TTT TTT (21 nt);

[0048] SEQ ID NO.3 is: GCG GAG ATG GGG CAT AAT TTT TT (23nt);

[0049] SEQ ID NO.4 is: GCG GAG ATG GGG CAT AAT GTT TTT T (25nt);

[0050] SEQ ID NO.5 is: GCG GAG ATG GGG CAT AAT GAG TTT TTT (27 nt);

[0051] The sequence of the hairpin probe H is as shown in SEQ ID NO.6, and SEQ ID NO.6 is: FAM-AGA CTA GAC CGGAAC GAC GGT CTA GTC T (BHQ1)-ATG CCC CAT CTC CGC.

[0052] After optimization experiments, it was found that the sequence of the blocker B was as shown in SEQ ID NO.4, and SEQ ID NO.4 was GCGGAG ATG GGG CAT AAT GTT TTT T (25nt).

[0053] The reaction principle is as follows:

[0054] Schematic diagram of the fluorescent biosensor used for quantitative detection of MC-LR. Figure 1 As shown; the sensor system includes an Aptamer / Blocker complex (abbreviated as A / B complex), a hairpin probe H, and an exonuclease ExoⅢ. The two ends of the probe H are modified with a fluorophore (FAM) and a quencher (BHQ), respectively. According to the principle of fluorescence resonance energy transfer (FRET), the close proximity of the fluorophore / quencher will lead to the effective quenching of the FAM fluorophore. When MC-LR interacts with its specific aptamer A, the strong interaction between the two leads to the formation of the A / MC-LR complex, and the blocker B will be released from the double-stranded structure of A / B ( Figure 1(A) in the figure). Then, the released blocker B can hybridize with the 3' end of probe H to form a B / H complex with a blunt 3' end. Since the exonuclease ExoⅢ can catalyze the cleavage of the single nucleotide at the end, the blocker B and DNA fragments are released, and the FAM and BHQ at both ends are separated, thus completing the signal amplification ( Figure 1 (B) in the figure). The free blocker B can continuously trigger the next signal amplification reaction assisted by the exonuclease ExoⅢ, produce abundant DNA fragments, and continuously amplify the fluorescence signal for MC-LR detection. When there is no MC-LR, its binding reaction with the specific aptamer A and the cleavage reaction mediated by the exonuclease ExoⅢ are inhibited, resulting in a weak background signal.

[0055] The detection kit for signal amplification detection of MC-LR includes a MC-LR rapid detection biosensor based on nuclease III and a buffer solution, wherein the buffer solution includes a PBS buffer solution and a nuclease ExoIII buffer solution. The formula of the PBS buffer solution is: 136.89mM NaCl, 2.67mM KCl, 8.1mM Na2HPO4, 1.76mM KH2PO4, and its pH is 7.4. The composition, type and pH of the buffer solution may fluctuate within a small range, but are generally stable. Exonuclease ExoⅢ was purchased from Beijing PolyMei Biotechnology Co., Ltd. (M5 HiPer Exonuclease Ш (E.coIi)), and the raw materials were derived from purified E.coli K-12, BE257 / pSGR3 strain; the components included: 25 μL of M5 HiPer Exonuclease Ш and 21 mL of 10xMei5Buffer 1.1, Exonuclease ExoⅢ buffer was purchased from New England Biolabs (NEB) and Beijing PolyMei Biotechnology Co., Ltd., and the oligonucleotide sequence purified by HPLC was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; MC-LR, microcystin-rr (MC-RR), microcystin-yr (MC-YR) and microcystin-la (MC-LA) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Exonuclease ExoⅢ was purchased from New England Biolabs (NEB) and Beijing PolyMei Biotechnology Co., Ltd.; all reagents were not further purified.

[0056] The method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on nuclease III comprises the following specific steps:

[0057] S1: firstly synthesizing an Aptamer / Blocker complex by using a specific aptamer A of MC-LR and a blocker B; the specific steps of step S1 are: the mixing ratio of the MC-LR specific aptamer A and the blocker B in step S1 is 1:1 to 4:1; heating at 95°C for 5 minutes, and then slowly cooling to room temperature to obtain an Aptamer / Blocker complex;

[0058] S2: pre-hybridizing the probe H and then annealing it to form a hairpin probe H structure; in the step S2, 100 nM probe H is pre-hybridized at 97° C. for 4 min and slowly cooled to room temperature to form a hairpin structure; wherein the probe has been modified with a fluorophore (FAM) and a quencher (BHQ) respectively before pre-hybridization, and pre-hybridization enables it to form a stable hairpin structure;

[0059] S3: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating, then adding the hairpin probe H for reaction, and finally adding the exonuclease ExoⅢ and the exonuclease ExoⅢ buffer for incubation again, and performing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction; the specific steps of step S3 are: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating at 37°C for 90 minutes, adding 100nM probe H, incubating at 37°C for 30 minutes, then adding 1unit / μL of exonuclease ExoⅢ, adding the exonuclease ExoⅢ buffer at the same time, and finally adding PBS buffer solution to prepare a 100μL system, incubating at 37°C for 30 minutes, thereby completing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction;

[0060] S4: After the restriction digestion reaction mediated by exonuclease ExoⅢ, fluorescence signal detection was performed. A multifunctional microplate reader (Berteng Instruments, Inc., USA) was used to record the fluorescence signal (excitation wavelength: 460 nm, emission wavelength range: 500-620 nm, maximum emission wavelength: 525 nm).

[0061] To obtain the best analytical results for MC-LR sensing, several experimental parameters (blocker length, A / B ratio, and reaction time) were systematically optimized.

[0062] Specific embodiment 1: Optimizing the base length of the blocker B chain, the sequence of the blocker B is shown in SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5,

[0063] SEQ ID NO.2 is: GCG GAG ATG GGG CAT TTT TTT (21 nt);

[0064] SEQ ID NO.3 is: GCG GAG ATG GGG CAT AAT TTT TT (23nt);

[0065] SEQ ID NO.4 is: GCG GAG ATG GGG CAT AAT GTT TTT T (25nt);

[0066] SEQ ID NO.5 is: GCG GAG ATG GGG CAT AAT GAG TTT TTT (27 nt);

[0067] After optimization experiments, it was found that the sequence of the blocker B was as shown in SEQ ID NO.4, and SEQ ID NO.4 was GCGGAG ATG GGG CAT AAT GTT TTT T (25nt).

[0068] Specific Example 2: The optimized blocker B chain base length of 25 nt is used to further optimize the concentration ratio of A:B; the method for quantitative detection of MC-LR based on the MC-LR rapid detection biosensor of nuclease III, the specific steps are:

[0069] S1: first, synthesizing an Aptamer / Blocker complex by using a specific aptamer A of MC-LR and a blocker B; the specific steps of step S1 are: the mixing ratios of the MC-LR specific aptamer A and the blocker B in step S1 are 1:1, 2:1, 3:1, and 4:1, respectively; heating at 95°C for 5 minutes, and then slowly cooling to room temperature to obtain an Aptamer / Blocker complex;

[0070] S2: pre-hybridizing the probe H and then annealing it to form a hairpin probe H structure; in the step S2, 100 nM probe H is pre-hybridized at 97° C. for 4 min and then slowly cooled to room temperature to form a hairpin structure; wherein the modification of the fluorophore FAM and the quencher BHQ is handed over to the bioengineering company for synthesis in advance together with other sequences; before pre-hybridization, the probe H has been modified with the fluorophore (FAM) and the quencher (BHQ), respectively;

[0071] S3: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating, then adding the hairpin probe H for reaction, and finally adding the exonuclease ExoⅢ and the exonuclease ExoⅢ buffer for incubation again, and performing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction; the specific steps of step S3 are: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating at 37°C for 90 minutes, adding 100nM probe H, incubating at 37°C for 30 minutes, then adding 1unit / μL of exonuclease ExoⅢ, adding the exonuclease ExoⅢ buffer at the same time, and finally adding PBS buffer solution to prepare a 100μL system, incubating at 37°C for 30 minutes, thereby completing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction;

[0072] S4: After the restriction digestion reaction mediated by exonuclease ExoⅢ, fluorescence signal detection was performed. A multifunctional microplate reader (Berteng Instruments, Inc., USA) was used to record the fluorescence signal (excitation wavelength: 460 nm, emission wavelength range: 500-620 nm, maximum emission wavelength: 525 nm).

[0073] Finally, the optimal A:B (25nt) concentration ratio was 2:1.

[0074] Specific Example 3: Using the optimal B chain base length of 25nt and the optimal A:B (25nt) concentration ratio of 2:1, the incubation time of step S3 is further optimized. In this specific example, the incubation time of step S3 is 2.5h.

[0075] The method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on exonuclease III comprises the following specific steps:

[0076] S1: firstly synthesizing an Aptamer / Blocker complex by using a specific aptamer A of MC-LR and a blocker B; the specific steps of step S1 are as follows: the mixing ratios of the MC-LR specific aptamer A and the blocker B in step S1 are 1:1, 2:1, 3:1, and 4:1, respectively; heating at 95°C for 5 minutes, and then slowly cooling to room temperature to obtain an Aptamer / Blocker complex;

[0077] S2: pre-hybridizing the probe H and then annealing it to form a hairpin probe H structure; in the step S2, 100 nM probe H is pre-hybridized at 97° C. for 4 min and slowly cooled to room temperature to form a hairpin structure; wherein the modification of the fluorophore FAM and the quencher BHQ is handed over to the bioengineering company for synthesis in advance together with other sequences; before pre-hybridization, the probe H has been modified with the fluorophore (FAM) and the quencher (BHQ), respectively;

[0078] S3: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating, then adding the hairpin probe H for reaction, and finally adding the exonuclease ExoⅢ and the exonuclease ExoⅢ buffer for incubation again, and performing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction; the specific steps of step S3 are: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating at 37°C for 90 minutes, adding 100nM probe H, incubating at 37°C for 30 minutes, then adding 1unit / μL of exonuclease ExoⅢ, adding the exonuclease ExoⅢ buffer at the same time, and finally adding PBS buffer solution to prepare a 100μL system, incubating at 37°C for 30 minutes, thereby completing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction;

[0079] S4: After the restriction digestion reaction mediated by exonuclease ExoⅢ, fluorescence signal detection was performed. A multifunctional microplate reader (Berteng Instruments, Inc., USA) was used to record the fluorescence signal (excitation wavelength: 460 nm, emission wavelength range: 500-620 nm, maximum emission wavelength: 525 nm).

[0080] Specific Example 4: Using the optimal B chain base length of 25nt and the optimal A:B (25nt) concentration ratio of 2:1, the incubation time of step S3 is further optimized. In this specific example, the incubation time of step S3 is 1h.

[0081] The method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on exonuclease III comprises the following specific steps:

[0082] S1: firstly synthesizing an Aptamer / Blocker complex by using a specific aptamer A of MC-LR and a blocker B; the specific steps of step S1 are as follows: the mixing ratios of the MC-LR specific aptamer A and the blocker B in step S1 are 1:1, 2:1, 3:1, and 4:1, respectively; heating at 95°C for 5 minutes, and then slowly cooling to room temperature to obtain an Aptamer / Blocker complex;

[0083] S2: pre-hybridizing the probe H and then annealing it to form a hairpin probe H structure; in the step S2, 100 nM probe H is pre-hybridized at 97° C. for 4 min and slowly cooled to room temperature to form a hairpin structure; wherein the modification of the fluorophore FAM and the quencher BHQ is handed over to the bioengineering company for synthesis in advance together with other sequences; before pre-hybridization, the probe H has been modified with the fluorophore (FAM) and the quencher (BHQ), respectively;

[0084] S3: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating, then adding the hairpin probe H for reaction, and finally adding the exonuclease ExoⅢ and the exonuclease ExoⅢ buffer for incubation again, and performing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction; the specific steps of step S3 are: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating at 37°C for 0 min, adding 100 nM probe H, incubating at 37°C for 30 min, then adding 1 unit / μL of the exonuclease ExoⅢ, adding the exonuclease ExoⅢ buffer at the same time, and finally adding the PBS buffer solution to prepare a 100 μL system, incubating at 37°C for 30 min, thereby completing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction;

[0085] S4: After the restriction digestion reaction mediated by exonuclease ExoⅢ, fluorescence signal detection was performed. A multifunctional microplate reader (Berteng Instruments, Inc., USA) was used to record the fluorescence signal (excitation wavelength: 460 nm, emission wavelength range: 500-620 nm, maximum emission wavelength: 525 nm).

[0086] Specific Example 5: Using the optimal blocker B chain base length of 25nt and the optimal A:B (25nt) concentration ratio of 2:1, the incubation time of step S3 is further optimized. In this specific example, the incubation time of step S3 is 1.5h.

[0087] The method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on exonuclease III comprises the following specific steps:

[0088] S1: firstly synthesizing an Aptamer / Blocker complex by using a specific aptamer A of MC-LR and a blocker B; the specific steps of step S1 are as follows: the mixing ratios of the MC-LR specific aptamer A and the blocker B in step S1 are 1:1, 2:1, 3:1, and 4:1, respectively; heating at 95°C for 5 minutes, and then slowly cooling to room temperature to obtain an Aptamer / Blocker complex;

[0089] S2: pre-hybridizing the probe H and then annealing it to form a hairpin probe H structure; in the step S2, 100 nM probe H is pre-hybridized at 97° C. for 4 min and slowly cooled to room temperature to form a hairpin structure; wherein the modification of the fluorophore FAM and the quencher BHQ is handed over to the bioengineering company for synthesis in advance together with other sequences; before pre-hybridization, the probe H has been modified with the fluorophore (FAM) and the quencher (BHQ), respectively;

[0090] S3: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating, then adding the hairpin probe H for reaction, and finally adding the exonuclease ExoⅢ and the exonuclease ExoⅢ buffer, and incubating again for the exonuclease ExoⅢ-mediated enzymatic cleavage reaction; the specific steps of step S3 are: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating at 37°C for 30 minutes, adding 100nM probe H, incubating at 37°C for 30 minutes, then adding 1unit / μL of the exonuclease ExoⅢ, adding the exonuclease ExoⅢ buffer at the same time, and finally adding the PBS buffer solution to prepare a 100μL system, incubating at 37°C for 30 minutes, thereby completing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction;

[0091] S4: After the restriction digestion reaction mediated by exonuclease ExoⅢ, fluorescence signal detection was performed. A multifunctional microplate reader (Berteng Instruments, Inc., USA) was used to record the fluorescence signal (excitation wavelength: 460 nm, emission wavelength range: 500-620 nm, maximum emission wavelength: 525 nm).

[0092] Specific Example 6: Using the optimal blocker B chain base length of 25nt and the optimal A:B (25nt) concentration ratio of 2:1, the incubation time of step S3 is further optimized. In this specific example, the incubation time of step S3 is 2h.

[0093] The method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on exonuclease III comprises the following specific steps:

[0094] S1: firstly synthesizing an Aptamer / Blocker complex by using a specific aptamer A of MC-LR and a blocker B; the specific steps of step S1 are as follows: the mixing ratios of the MC-LR specific aptamer A and the blocker B in step S1 are 1:1, 2:1, 3:1, and 4:1, respectively; heating at 95°C for 5 minutes, and then slowly cooling to room temperature to obtain an Aptamer / Blocker complex;

[0095] S2: pre-hybridizing the probe H and then annealing it to form a hairpin probe H structure; in the step S2, 100 nM probe H is pre-hybridized at 97° C. for 4 min and slowly cooled to room temperature to form a hairpin structure; wherein the modification of the fluorophore FAM and the quencher BHQ is handed over to the bioengineering company for synthesis in advance together with other sequences; before pre-hybridization, the probe H has been modified with the fluorophore (FAM) and the quencher (BHQ), respectively;

[0096] S3: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating, then adding the hairpin probe H for reaction, and finally adding the exonuclease ExoⅢ and the exonuclease ExoⅢ buffer, and incubating again for the exonuclease ExoⅢ-mediated enzymatic cleavage reaction; the specific steps of step S3 are: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating at 37°C for 60 minutes, adding 100nM probe H, incubating at 37°C for 30 minutes, then adding 1unit / μL of exonuclease ExoⅢ, adding the exonuclease ExoⅢ buffer at the same time, and finally adding PBS buffer solution to prepare a 100μL system, incubating at 37°C for 30 minutes, thereby completing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction;

[0097] S4: After the restriction digestion reaction mediated by exonuclease ExoⅢ, fluorescence signal detection was performed. A multifunctional microplate reader (Berteng Instruments, Inc., USA) was used to record the fluorescence signal (excitation wavelength: 460 nm, emission wavelength range: 500-620 nm, maximum emission wavelength: 525 nm).

[0098] Specific Example 7: Using the optimal blocker B chain base length of 25nt and the optimal A:B (25nt) concentration ratio of 2:1, the incubation time of step S3 is further optimized. In this specific example, the incubation time of step S3 is 3h.

[0099] The method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on exonuclease III comprises the following specific steps:

[0100] S1: firstly synthesizing an Aptamer / Blocker complex by using a specific aptamer A of MC-LR and a blocker B; the specific steps of step S1 are as follows: the mixing ratios of the MC-LR specific aptamer A and the blocker B in step S1 are 1:1, 2:1, 3:1, and 4:1, respectively; heating at 95°C for 5 minutes, and then slowly cooling to room temperature to obtain an Aptamer / Blocker complex;

[0101] S2: pre-hybridizing the probe H and then annealing it to form a hairpin probe H structure; in the step S2, 100 nM probe H is pre-hybridized at 97° C. for 4 min and slowly cooled to room temperature to form a hairpin structure; wherein the modification of the fluorophore FAM and the quencher BHQ is handed over to the bioengineering company for synthesis in advance together with other sequences; before pre-hybridization, the probe H has been modified with the fluorophore (FAM) and the quencher (BHQ), respectively;

[0102] S3: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating, then adding the hairpin probe H for reaction, and finally adding the exonuclease ExoⅢ and the exonuclease ExoⅢ buffer, and incubating again for the exonuclease ExoⅢ-mediated enzymatic cleavage reaction; the specific steps of step S3 are: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating at 37°C for 120min, adding 100nM probe H, incubating at 37°C for 30min, then adding 1unit / μL of exonuclease ExoⅢ, adding the exonuclease ExoⅢ buffer at the same time, and finally adding PBS buffer solution to prepare a 100μL system, incubating at 37°C for 30min, thereby completing the exonuclease ExoⅢ-mediated enzymatic cleavage reaction;

[0103] S4: After the restriction digestion reaction mediated by exonuclease ExoⅢ, fluorescence signal detection was performed. A multifunctional microplate reader (Berteng Instruments, Inc., USA) was used to record the fluorescence signal (excitation wavelength: 460 nm, emission wavelength range: 500-620 nm, maximum emission wavelength: 525 nm).

[0104] After the above optimization examples, the best reaction time was 2.5 h (Specific Example 3).

[0105] The final optimized results are expressed as mean ± standard deviation; the optimal blocker B base sequence after final optimization is 25 nt, the A / B ratio is 2:1, and the optimal reaction time is 2.5 h (Specific Example 3, such as Figure 4-6 shown).

[0106] (1) The feasibility verification experiment is carried out under the optimal conditions. The specific feasibility verification process is as follows:

[0107] In order to verify the feasibility of the experimental device disclosed in the present invention, the fluorescence spectra of the sensing system with and without MC-LR were first detected. Figure 2 As shown in (A), in the presence of MC-LR, the fluorescence intensity of the sensing system increased significantly, which preliminarily demonstrated the feasibility of the fluorescence sensing system. Next, the fluorescence signal of the sensing system under different conditions was tested. Figure 2As shown in (B), only when H, A / B, exonuclease ExoⅢ and MC-LR are present at the same time, the tested solution can show a significant increase in fluorescence intensity (blue curve). This indicates that MC-LR can release blocker B from the A / B double-stranded structure, thereby further activating the signal amplification reaction assisted by exonuclease ExoⅢ. Solutions under other conditions all show weaker fluorescence signals (black, purple and green curves), because the cleavage reaction cannot be initiated in the absence of MC-LR and / or blocker B is still blocked. These results indicate that the proposed fluorescence sensing platform mediated by exonuclease ExoⅢ can be used for sensitive detection of MC-LR.

[0108] (2) PAGE experiment was used to further verify the feasibility. The specific steps were as follows:

[0109] Native polyacrylamide gel electrophoresis (15% PAGE) was used. 3.8 mL ultrapure water, 5 mL of 30% Acr-bis (29:1), 1 mL of 10×TBE buffer, 100 μL of APS (10%), and 4 μL of TEMED were mixed at 37°C for 1 h to prepare the gel; then, 10 μL of different test solutions were mixed with 2 μL of 100×SYBR Green solution and 2 μL of 6× loading buffer and loaded; the gel was placed in 1×TBE buffer (pH=8.0) and electrophoresed at 45 V for 30 min, then switched to 90 V for another 90 min. Finally, the PAGE gel was scanned using a gel image analysis system (Bio-Rad, USA).

[0110] The electrophoresis results are as follows Figure 3 As shown in (A), lanes 1-3 represent MC-LR specific aptamer A, blocker B and A / B double-stranded complex respectively; when MC-LR is incubated with A / B complex, three bands can be observed in lane 4, which represent A / B complex, A / MC-LR complex and blocker B released during the reaction. These PAGE results show that MC-LR can indeed release blocker B from A / B complex. Figure 3 As shown in (B), lane 1 and lane 2 represent blocker B and hairpin probe H, respectively; after blocker B, hairpin probe H and nuclease ExoⅢ were mixed together for reaction, two bands could be clearly observed in lane 3, which were blocker B and a small chain, indicating that free blocker B successfully activated the signal amplification reaction assisted by nuclease ExoⅢ and produced DNA fragments that were cleaved after the reaction; all PAGE results successfully verified the feasibility of the sensor system for MC-LR detection.

[0111] (3) Evaluation of MC-LR detection performance

[0112] 1) Quantification of MC-LR:

[0113] Under the optimized conditions, different concentrations of MC-LR were incubated with the sensor to investigate the linear range of the sensor and evaluate the quantitative analysis ability of the method for MC-LR. As the concentration of MC-LR increased, the fluorescence signal at 525 nm gradually increased (e.g. Figure 7 As shown in (A)). Figure 7 As shown in (B), at 1×10 -3 Within the dynamic range of MC-LR of -10μg / L, the curve of fluorescence intensity changing with the logarithm of MC-LR concentration showed a good linear relationship, and the linear equation was ΔF = 75.39 × LogC MC-LR +279.39(R 2 =0.99), where ΔF is the change in fluorescence intensity relative to blank. According to the 3σ rule, we measured the detection limit of the sensor for MC-LR to be 0.37 ng / L. The fluorescent aptamer sensor we developed has high sensitivity and is superior to some previously reported methods for detecting MC-LR (as shown in Table 1). The excellent performance of this sensing platform is due to the powerful signal amplification function of the reaction mediated by the exonuclease ExoⅢ.

[0114] 2) Specificity experiments:

[0115] In order to evaluate the selectivity of the MC-LR fluorescence sensing system, three common MC analogs (MC-RR, MC-YR, and MC-LA) and four heavy metal ions (Cu 2+ 、Cd 2+ , Pb 2+ , Mn 2+ ) as a control. Compared with the blank experiment, these potential interfering substances at concentrations of 0.1 or 1 μg / L did not cause significant changes in fluorescence signals (such as Figure 8 As shown in Figure 2, the sensor only produces a significant fluorescence signal response to the target MC-LR, which has good specificity. In addition, when these potential interfering substances are mixed with MC-LR in a solution, the fluorescence intensity is close to that generated by the MC-LR sample (as shown in Figure 2). Figure 8 The above results all indicate that the proposed exonuclease ExoⅢ-assisted sensing platform has the potential to selectively quantify MC-LR levels in environmental water samples.

[0116] 3) Real water sample testing:

[0117] In order to verify the practicality of the designed fluorescence sensor, the sensor system was used to detect MC-LR in actual water samples. As shown in Table 1, different concentrations of MC-LR were added to different actual water samples for determination (0.1, 1 and 10 μg / L). The calculated sample recovery rate was 95.04% to 107.01%, and the relative standard deviation (RSD) was 1.87% to 4.38%. The above results show that the sensor has good accuracy and is suitable for on-site detection of MC-LR in environmental water bodies.

[0118] Table 1 Determination results of MC-LR in environmental water samples by fluorescence sensing system (n=3)

[0119]

[0120] In summary, the present invention successfully constructed a new type of fluorescent biosensor based on aptamers and combined with a signal amplification reaction mediated by exonuclease ExoⅢ to detect MC-LR in the environment. When MC-LR is present, it binds to the specific aptamer A, resulting in the release of blocker B, and triggers the next step of enzymatic cleavage mediated by exonuclease ExoⅢ, accumulating a large amount of fluorescent signals. The constructed fluorescent aptamer sensor has a wide linear range, good specificity, and a detection limit as low as 0.37ng / L. The sensor can be successfully applied to the detection of MC-LR in actual water samples, with a recovery rate of 95.04% to 107.01%. In general, the sensor has good detection performance, and has the advantages of simple operation, high sensitivity, and fluorescence readout, providing a new candidate method for monitoring MC-LR in environmental water bodies.

[0121] For those skilled in the art, the above embodiments are merely illustrative descriptions of the present invention, and the above embodiments are preferred implementations of the present invention. Obviously, the specific implementation of the present invention is not limited to the above methods, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are within the protection scope of the present invention.

Claims

1. A MC-LR rapid detection biosensor based on exonuclease III, characterized in that: The MC-LR rapid detection biosensor based on nuclease III comprises an Aptamer / Blocker complex, a hairpin probe H and nuclease III; wherein the Aptamer / Blocker complex comprises a specific aptamer A and a blocker B of MC-LR, and both ends of the hairpin probe H are modified with a fluorophore FAM and a quencher BHQ, respectively.

2. The MC-LR rapid detection biosensor based on exonuclease III according to claim 1, characterized in that: The sequence of the specific aptamer A of the MC-LR is shown in SEQ ID NO.1, and SEQ ID NO.1 is: GGC GCC AAACAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGC TTT TTT; The sequence of the blocker B is shown in SEQ ID NO.2 or SEQ ID NO.3 or SEQ ID NO.4 or SEQ ID NO.5, and SEQ ID NO.2 is: GCG GAG ATG GGG CAT TTT TTT; SEQ ID NO.3 is: GCG GAG ATG GGG CAT AAT TTT TT; SEQ ID NO.4 is: GCG GAG ATG GGG CAT AAT GTT TTT T; SEQ ID NO.5 is: GCG GAG ATG GGG CAT AAT GAG TTT TTT; The sequence of the hairpin probe H is as shown in SEQ ID NO.6, SEQ ID NO.6 is: FAM-AGA CTA GAC CGG AAC GAC GGT CTA GTC T (BHQ1)-ATG CCC CATCTC CGC。 3. The MC-LR rapid detection biosensor based on exonuclease III according to claim 2, characterized in that: The sequence of the blocker B is shown in SEQ ID NO.4, and SEQ ID NO.4 is GCG GAG ATG GGG CAT AATGTT TTT T.

4. A detection kit for signal amplification detection of MC-LR, characterized in that: The invention comprises a MC-LR rapid detection biosensor based on nuclease III and a buffer solution, wherein the buffer solution comprises a PBS buffer solution and a nuclease III buffer solution.

5. The detection kit for signal amplification and detection of MC-LR according to claim 4, characterized in that: The formula of the PBS buffer solution is: 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4, and its pH = 7.

4.

6. A method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on exonuclease III, comprising the following steps: S1: First, the aptamer / blocker complex is synthesized by using the MC-LR specific aptamer A and blocker B; S2: pre-hybridizing the probe H and then annealing to form the structure of the hairpin probe H; S3: Add different concentrations of MC-LR to the Aptamer / Blocker complex and incubate, then add the hairpin probe H for reaction, and finally add exonuclease ExoⅢ and exonuclease ExoⅢ buffer for incubation again to perform exonuclease ExoⅢ-mediated digestion reaction; S4: Fluorescence signal detection after the exonuclease ExoⅢ mediated digestion reaction.

7. The method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on exonuclease III according to claim 6, characterized in that: The mixing ratio of the MC-LR specific aptamer A and the blocker B in step S1 is 1:1 to 4:

1.

8. The method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on exonuclease III according to claim 7, characterized in that: The specific steps of step S1 are: mixing 200 nM MC-LR specific aptamer A with 100 nM blocker B, that is, the mixing ratio of MC-LR specific aptamer A to blocker B is 2:1; heating at 95° C. for 5 minutes, and then slowly cooling to room temperature to obtain an Aptamer / Blocker complex.

9. The method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on exonuclease III according to claim 7, characterized in that: In step S2, 100 nM probe H is pre-hybridized at 97° C. for 4 min and slowly cooled to room temperature to form a hairpin structure.

10. The method for quantitative detection of MC-LR using a MC-LR rapid detection biosensor based on exonuclease III according to claim 7, characterized in that: The specific steps of step S3 are: adding MC-LR of different concentrations to the Aptamer / Blocker complex, incubating at 37°C for 0 to 120 minutes, adding 100nM probe H, incubating at 37°C for 30 minutes, then adding 1unit / μL of nuclease ExoⅢ, adding nuclease ExoⅢ buffer at the same time, and finally adding PBS buffer solution to make a 100μL system, incubating at 37°C for 30 minutes, thereby completing the enzymatic cleavage reaction mediated by nuclease ExoⅢ.

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