A biosensor and kit for detecting algal toxins

Abstract

The present invention provides a biosensor and a kit for detecting algae toxins, including an aptamer Apt, a T chain, a waste chain, a primer1 chain, a primer2 chain, an auxiliary chain H, a rolling circle template chain padlock and a hairpin HP, and the nucleotide sequence thereof is shown in SEQ ID NO: 1-8. The sensor of the present invention has good analytical performance, and has the advantages of being visible to the naked eye, fast detection speed, simple operation, low price, low detection limit, and high specificity. It can make up for the defects and shortcomings of the existing detection methods of microcystin, and realize rapid and accurate quantitative detection thereof. The kit prepared by this research has methodological versatility for the detection of microcystin, and has potential application prospects in food samples and environmental monitoring.

Description

Technical Field

[0001] The invention belongs to the technical field of sensors, and in particular relates to a kit based on a colorimetric sensing platform of strand displacement and bidirectional rolling circle triggered by an Apt-T composite probe for efficient detection of algal toxins. Background Art

[0002] Microcystin (MCs) is a class of biologically active cyclic heptapeptide compounds. It has a ring structure and an intervening double bond, which makes it quite stable. Microcystin is the most widely distributed hepatotoxin. Its harm to the aquatic environment and human health has become one of the major environmental issues of global concern. Microcystin is mainly produced by freshwater algae Microcystis aeruginosa ( Microcystis aeruginosa ) and has considerable stability. Microcystin can strongly inhibit the activity of protein phosphatase. When cells rupture or age, toxins are released into the water. It is also a strong liver tumor promoter. China's drinking water hygiene standard limits the content of microcystin in drinking water to 1μg / L. The implementation of this standard has put forward higher requirements on the quality of water sources.

[0003] There are many methods for detecting microcystins, such as bioanalysis, cytotoxicity detection, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), protein phosphatase inhibition, etc. These methods have their own advantages, but also have defects to varying degrees. The biological detection method cannot distinguish the isomers of microcystins, and the workload is very large; the sensitivity of the cytotoxin detection method is low; the pretreatment process of the high-performance liquid chromatography method is cumbersome and the equipment is expensive; the reproducibility of the capillary electrophoresis method is poor; the protein phosphatase inhibition method cannot distinguish specific toxin homologues, and the post-processing is difficult. Therefore, the development of simple, rapid, and highly specific detection technology is of great significance for the prevention and diagnosis of foodborne pathogens. Summary of the invention

[0004] In order to achieve faster and lower-cost detection of microcystins, the present invention proposes a sensor for detecting microcystins. The sensor of the present invention has good analytical performance, and has the advantages of being visible to the naked eye, fast detection speed, simple operation, low price, low detection limit, and high specificity.

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] A biosensor for detecting microcystin, comprising an Apt-T composite probe, a composite structure duplex, a Helper chain (H), a rolling circle template chain (padlock), a crRNA1 chain, a crRNA2 chain, and a C-Ag +-C hairpin, CdTe quantum dots, Cas12a protein, T4 DNA ligase, Phi29 DNA polymerase;

[0007] The Apt-T composite probe is obtained by hybridization of the aptamer Apt chain and the trigger T chain;

[0008] The composite structure duplex chain is obtained by hybridization of waste chain, primer1 chain and primer2 chain;

[0009] The C-Ag + -C hairpin is Ag + Obtained by binding to cytosine (C) of HP3;

[0010] The nucleotide sequences of the aptamer Apt, T strand, Waste strand, Primer1 strand, Primer2 strand, Helper strand, rolling circle template strand padlock and hairpin HP are shown in SEQ ID NOs: 1-8;

[0011] The 5' end of the rolling circle template chain padlock is phosphorylated;

[0012] The method for preparing CdTe quantum dots comprises the following steps:

[0013] (1) CdCl 2 , trisodium citrate, and 3-mercaptopropionic acid (MPA) were mixed and the pH was adjusted to 10.5;

[0014] (2) Put Na 2 TeO 3 and NaBH 4 The solution in step (1) is added to the mixture for reflux reaction, and CdTe quantum dots are obtained by separation and purification.

[0015] The separation and purification step is: adding n-propanol to the reaction solution, and then centrifuging to separate the precipitate. Preferably, the centrifugal speed is 11000 rpm.

[0016] The microstructure of the CdTe quantum dots is spherical, and the average diameter is 3.5 nm.

[0017] The C-Ag + -C hairpin preparation method, comprising the following steps:

[0018] The hairpin HP3 was combined with AgNO 3 The solution was mixed in a buffer and incubated at room temperature in the dark to generate C-Ag + -C hairpin structure.

[0019] A kit comprising the above biosensor.

[0020] A method for detecting microcystin using the above biosensor or kit comprises the following steps:

[0021] (1) Adding Apt-T composite probe, composite structure duplex, Helper chain and Cas12a protein to the test solution and incubating in buffer to obtain incubation solution 1;

[0022] (2) Add rolling circle template chain padlock and T4 DNA ligase to the incubation solution and incubate in a buffer containing ATP to inactivate the enzyme to obtain incubation solution 2;

[0023] (3) Add Phi29 DNA polymerase and dNTP to incubation solution 2, inactivate the enzyme to obtain incubation solution 3;

[0024] (4) Add C-Ag to incubation solution 3 + -C hairpins and CdTe quantum dots were mixed and incubated for fluorescence detection.

[0025] In steps (1) and (3), the incubation temperature is 37° C. In step (2), the incubation temperature is 16° C. In step (4), the incubation temperature is room temperature.

[0026] In steps (2) and (3), the temperature for inactivating the enzyme is 65°C.

[0027] The test solutions in step (1) include algae toxin solutions with a series of concentrations and algae toxin solutions with unknown concentrations.

[0028] Step (4) includes the step of calculating the concentration of algae toxin in a solution with an unknown concentration of algae toxin by using a regression equation based on the fluorescence intensity and the logarithm of the concentration of the algae toxin solution with a series of concentrations.

[0029] The detection principle of the present invention is as follows Figure 1 As shown, the sequences are as follows:

[0030] Apt:

[0031] 5'- GGC GCC AAA CAG G AC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CG C-3';

[0032] T-Chain:

[0033] 5'- GCG GAGATG GGG TTT ATT TCC ACT CAC AAC C CTG TTT GGC GCC -3';

[0034] Waste chain:

[0035] 5'-GGG GGG GGT TAT ACC TCA GTCCCTT GTG AGT GGA AAT AAACCC CAT GAATTA-3'

[0036] Primer1 chain:

[0037] 5'-TTT ATT TCC ACT CAC AA GGG GGC-3';

[0038] Primer2 Chain:

[0039] 5'-TGA GGT ATA ACC CCC TT-3';

[0040] Helper chain:

[0041] 5'-TAA TTC ATG GGG TTT ATT TCC ACT CAC AA GGG AC TGA GGT ATA ACC CCCCCC-3'

[0042] Roller ring template chain padlock:

[0043] 5'-Phosphate-GGA AAT AAACGC TCG ATC TAG TCA ACG AAC TTTTGAATTAA GGGGGT TAT ACC TCATCA ACC TCC ACC TCA CTA AC TTTTGAATTGCC CCC TT GTG AGT-3';

[0044] crRNA1:

[0045] 5'-UAA UUU CUA CUA AGU GUA GAU CGC UCG AUC UAG UCA ACG AAC-3';

[0046] crRNA2:

[0047] 5'-UAA UUU CUA CUA AGU GUA GAU UCA ACC UCC ACC UCA CUC AC-3';

[0048] Hairpin HP:

[0049] 5'-CCC CCC GCA GTT GAT CCT TTG GAT ACC CTG GCC CCC C-3'.

[0050] The system of this kit can undergo the following two reaction processes:

[0051] (1) Apt-T composite probe-triggered chain displacement and bidirectional rolling circle colorimetric sensing platform. The probe consists of the microcystin aptamer Apt and a T chain. In the absence of a target, the probe is in a stable structure. When the target is present, Apt binds to the target to release the T chain. The released T chain reacts with the composite structure duplex to trigger chain displacement. The T chain displaces primer1. At this time, the H chain in the reaction system recognizes the exposed toehold and also displaces primer2. Under the action of T4 ligase and phi29 polymerase, the displaced primer1 and primer2 complete the preparation and amplification of the circular template, and then complete the bidirectional rolling circle amplification reaction. The crRNA1 and crRNA2 generated by the template chain padlock amplification will activate the trans-cleavage activity of Cas12a.

[0052] (2) C-Ag + -C hairpin and CdTe quantum dots QDs, in the presence of the target, the activated Cas12a has trans-cutting activity and can recognize the single strand for cutting, + - Ag wrapped in C + The released molecules undergo ion exchange reactions with CdTe quantum dots QDs, quenching the fluorescence of CdTe quantum dots QDs. In the absence of a target, the trans-cleavage activity of Cas12a will not be activated, so the fluorescence of CdTe quantum dots QDs will not be quenched.

[0053] The present invention has the following advantages:

[0054] The sensor provided by the present invention detects the target microcystin and the specific recognition of the aptamer; the two isothermal amplifications of chain displacement and double rolling circle reaction are used to amplify the signal, which greatly improves the sensitivity and selectivity of the detection. The dual detection sensor has a detection range of 2 orders of magnitude for microcystin, and a detection limit of 87.4ng / L. The kit does not require pretreatment, can be completed in one step, is simple to operate, and can visualize the phenomenon. It has the advantages of fast detection speed, simple operation, low price, low detection limit, high specificity, etc. It can make up for the defects and shortcomings of existing detection methods, and realize rapid and accurate qualitative and quantitative detection of it, providing a new kit for the detection of microcystin. It can make up for the defects and shortcomings of existing detection methods of algae toxins, and realize rapid and accurate quantitative detection of it. The colorimetric biosensor prepared by this kit has methodological versatility for the detection of various algae toxins, and has potential application prospects in food samples and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the schematic diagram of the experiment;

[0056] Figure 2 Optimize the test result graph for reaction time;

[0057] Figure 3 This is a graph showing the results of the reaction temperature optimization test;

[0058] Figure 4 Hairpin C-Ag + -C Synthesis of Ag + Concentration optimization test result diagram;

[0059] Figure 5 This is a graph showing the detection results of microcystin at different concentrations. DETAILED DESCRIPTION

[0060] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0061] Example 1 Construction of biosensor

[0062] According to the nucleotide sequences shown in SEQ ID NO: 1-8, aptamers Apt, T strand, waste strand, primer1 strand, primer2 strand, auxiliary strand H, rolling circle template strand padlock and hairpin HP were synthesized, wherein the 5' of the rolling circle template strand padlock was modified with phosphate.

[0063] (1) Preparation of Apt-T probe:

[0064] Equal volumes of Apt (100 μM) and T (100 μM) were mixed thoroughly, incubated at 95°C in a water bath for 5 min, cooled to room temperature to form Apt-T probe (10 μM), and stored at 4°C until use.

[0065] (2) Preparation of composite structure duplex:

[0066] Equal volumes of waste chain, primer 1 chain and primer 2 chain were mixed thoroughly, incubated at 95°C in a water bath for 5 min, cooled to room temperature to form a composite structure duplex (10 μM), and stored at 4°C for use.

[0067] (3) Preparation of hairpin HP:

[0068] Hairpin HP was prepared by quenching method. 10 μM HP was incubated in a 95°C water bath for 5 min, then quickly placed in an ice water bath to form a hairpin structure, which was then stored at 4°C for later use.

[0069] (4) Hairpin C-Ag + Preparation of -C:

[0070] 1 μM hairpin HP (60 μL) was mixed with 5 μM AgNO 3 The solution (72 μL) was mixed and incubated in 100 mM MOPS buffer (pH 7.0) (300 μL) at room temperature in the dark for 1 h to generate C-Ag + -C hairpin;

[0071] (5) Preparation of CdTe quantum dots (QDs):

[0072] (i) Add CdCl 2 (0.5 mmol) and trisodium citrate (0.2 g) were mixed evenly in 50 mL of solution, 3-mercaptopropionic acid (MPA) (52 μL) was immediately added and mixed, and the pH was adjusted to 10.5 with 1 M NaOH solution;

[0073] (ii) Change Na 2 TeO 3 solution (0.1 mmol) and NaBH 4 (50 mg) was added to the above solution and refluxed for 1 h to obtain a reaction solution;

[0074] (iii) The reaction solution was precipitated with n-propanol (1000 μL) and centrifuged at 11000 rpm for 8 min to obtain a red precipitate, which was spherical CdTe quantum dots with a diameter of about 3.5 nm. Before use, it was redispersed in high-purity water to obtain a QDs suspension.

[0075] (6) Construction of the kit

[0076] 3 μL of algae toxin or PBS buffer (control) was added to 27 μL of Apt-T complex probe (3 μL, 10 μM), complex structure duplex (3 μL, 10 μM), 10×NEBuffer 2.1 buffer (3 μL), H chain (3 μL, 10 μM), Cas12a protein (3 μL) and ddH 2 O (12 μL) and incubated at 37°C for 1 h.

[0077] To the resulting 30 μL solution, padlock (5 μL, 10 μM), 10×T4 ligase buffer (5 μL) and T4 DNA ligase (2 μL, 8 U / L) were added, the reaction was allowed to proceed at 16°C overnight, and then treated at 65°C for 10 minutes. Subsequently, 10×Phi29 buffer (5 μL), 1000 U / mL Phi29 DNA polymerase (2 μL) and 10 mM dNTP (5 μL) were added, the reaction was allowed to proceed at 37°C for 1 hour, and then treated at 65°C for 10 minutes. The resulting solution was buffer A.

[0078] 20 μL of hairpin C-Ag + -C and 20 μL QDs suspension were mixed to obtain a solution that became buffer B.

[0079] The resulting 54 μL buffer A and 40 μL buffer B were mixed and incubated at room temperature in the dark for 30 min. 2 O was added to 100 μL. The excitation wavelength was 558 nm, and the emission wavelength between 575 nm and 750 nm was collected. The slits of the excitation wavelength and emission wavelength used were 10 nm and 7 nm. Compared with the control, the corresponding solution could be observed to have a significant color change after the addition of algae toxins. The decrease in absorbance at 650 nm could be detected by fluorescence spectroscopy analysis, indicating that the constructed biosensor can be used for the detection of algae toxins.

[0080] Example 2 Effect of reaction time optimization on sensor analysis performance

[0081] Take the buffer A, buffer B and algae toxin prepared in Example 1, and then screen the optimal reaction time according to the following steps:

[0082] Take 4 EP tubes and add 54 μL of buffer A prepared by Apt-T composite probe (3 μL, 10 μM), composite structure duplex (3 μL, 10 μM), 10×NEBuffer 2.1 buffer (3 μL), H chain (3 μL, 10 μM), Cas12a protein (3 μL) and ddH2O (12 μL), padlock (5 μL, 10 μM), 10×T4 buffer (5 μL) and T4 DNA ligase (2 μL, 8 U / L), 10×Phi29 buffer (5 μL), 1000 U / mL Phi29 DNA polymerase (2 μL), 10 mM dNTP (5 μL), and then add 40 μL hairpin C-Ag + -C and 20 μL CdTe quantum dots QDs were mixed to obtain buffer B, and incubated at 37°C for 90 min. Subsequently, 3 μL of algae toxin was added and reacted at room temperature for 0 min, 30 min, 60 min, and 90 min. 2 O to 100 μL. The absorbance was measured at 650 nm using fluorescence spectroscopy. Figure 2 As shown in the figure, it can be seen that the absorbance gradually increases with the increase of reaction time, and the absorbance reaches the best when the reaction time is 30 minutes, so 30 minutes is selected as the best reaction time.

[0083] Example 3 Effect of reaction temperature on sensor analysis performance

[0084] Take the buffer A, buffer B and algae toxin prepared in Example 1, and then screen the optimal reaction temperature conditions according to the following steps:

[0085] Take 4 EP tubes and add 54 μL of buffer A prepared by Apt-T composite probe (3 μL, 10 μM), composite structure duplex (3 μL, 10 μM), 10×NEBuffer 2.1 buffer (3 μL), H chain (3 μL, 10 μM), Cas12a protein (3 μL) and ddH2O (12 μL), padlock (5 μL, 10 μM), 10×T4 buffer (5 μL) and T4 DNA ligase (2 μL, 8 U / L), 10×Phi29 buffer (5 μL), 1000 U / mL Phi29 DNA polymerase (2 μL), 10 mM dNTP (5 μL), and then add 40 μL hairpin C-Ag + -C and 20 μL CdTe quantum dots QDs were mixed to obtain buffer B, and incubated at 20°C, 25°C, 37°C, 42°C, and 50°C for 90 min. Subsequently, 3 μL of algae toxin was added and reacted at room temperature for 30 min. 2 O was added to 100 μL. The fluorescence intensity was measured at 650 nm using fluorescence spectroscopy. The results were as follows: Figure 3 As shown in the figure, it can be seen that the fluorescence intensity gradually increases with the increase of the reaction temperature, and the fluorescence intensity is optimal when the reaction temperature is 37°C, so 37°C is selected as the optimal reaction temperature.

[0086] Example 4 Hairpin C-Ag + -C Synthesis of Ag + Effect of concentration on sensor analytical performance

[0087] Take the buffer A, buffer B and algae toxin prepared in Example 1, and then screen the optimal reaction time according to the following steps:

[0088] Take 6 EP tubes and add 54 μL of Apt-T composite probe (3 μL, 10 μM), composite structure duplex (3 μL, 10 μM), 10×NEBuffer 2.1 buffer (3 μL), H chain (3 μL, 10 μM), Cas12a protein (3 μL) and ddH, respectively. 2 O (12 μL), padlock (5 μL, 10 μM), 10×T4 buffer (5 μL) and T4 DNA ligase (2 μL, 8 U / L), 10×Phi29 buffer (5 μL), 1000 U / mL Phi29 DNA polymerase (2 μL), 10 mM dNTP (5 μL) were added to the prepared buffer A, and then 40 μL of hairpin C-Ag+ -C (hairpin HP and Ag + The mixture was mixed with 20 μL CdTe quantum dots (QDs) in a ratio of 4:1, 2:1, 1:1, 1:2, 1:6, and 1:8) and the obtained buffer B, and incubated at 37°C for 90 min. Subsequently, 3 μL of algae toxin was added and reacted at room temperature for 30 min. 2 O to 100 μL. The fluorescence intensity was measured at 650 nm using fluorescence spectroscopy. The results are as follows: Figure 4 As shown in the figure, it can be seen that the hairpin C-Ag + -C hairpin and Ag + The quenching effect of QDs quantum dots gradually improves with the increase of the ratio, reaching the best at 1:6, and does not increase significantly with the increase of the ratio. Therefore, 1:6 is selected as the optimal synthesis ratio.

[0089] Example 5 Detection limit of sensor for microcystin

[0090] Take the buffer A, buffer B and microcystin prepared in Example 1, and then screen the optimal reaction time according to the following steps:

[0091] Take 6 EP tubes and add 54 μL of Apt-T composite probe (3 μL, 10 μM), composite structure duplex (3 μL, 10 μM), 10×NEBuffer 2.1 buffer (3 μL), H chain (3 μL, 10 μM), Cas12a protein (3 μL) and ddH, respectively. 2 O (12 μL), padlock (5 μL, 10 μM), 10×T4 buffer (5 μL) and T4 DNA ligase (2 μL, 8 U / L), 10×Phi29 buffer (5 μL), 1000 U / mL Phi29 DNA polymerase (2 μL), 10 mM dNTP (5 μL) were added to the prepared buffer A, and then 40 μL of hairpin C-Ag + -C and 20 μL CdTe quantum dots QDs were mixed with buffer B and incubated at 37 °C for 90 min. Subsequently, 3 μL of different concentrations of algae toxins were added and reacted at room temperature for 30 min. 2 O to 100 μL. The absorbance was measured at 650 nm using fluorescence spectroscopy. Figure 5As shown in the figure, from 0-5 μmol / L, at 650nm, the fluorescence quenching effect gradually increases, which indicates that the prepared kit is sensitive to changes in the concentration of microcystin. The logarithmic value of the concentration of microcystin has a good linear relationship with the absorption intensity within the range of 0-5 μmol / L. According to the calculation method of the three-fold deviation of the ultraviolet absorption intensity corresponding to the blank sample, the detection limit of the colorimetric sensor is calculated to be 87.4 ng / L.

Claims

1. A biosensor for detecting microcystin, characterized in that: include: Apt-T composite probe, composite structure duplex, Helper chain, rolling circle template chain padlock, crRNA1 chain, crRNA2 chain, C-Ag + -C hairpin, CdTe quantum dots, Cas12a protein, T4 DNA ligase and Phi29 DNA polymerase; The Apt-T composite probe is obtained by hybridization of the aptamer Apt chain and the trigger T chain; The composite structure duplex chain is obtained by hybridization of waste chain, primer1 chain and primer2 chain; The C-Ag + -C hairpin is Ag + Obtained by binding to the cytosine of the hairpin HP; The nucleotide sequences of the aptamer Apt, T strand, Waste strand, Primer1 strand, Primer2 strand, Helper strand, rolling circle template strand padlock and hairpin HP are shown in SEQ ID NOs: 1-8; The 5' end of the rolling circle template chain padlock is phosphorylated; The nucleotide sequence of the crRNA1 chain is: 5'-UAA UUU CUA CUA AGU GUA GAU CGC UCG AUC UAGUCA ACG AAC-3'; The nucleotide sequence of the crRNA2 chain is: 5'-UAA UUU CUA CUA AGU GUA GAU UCA ACC UCC ACCUCA CUC AC-3'.

2. The biosensor according to claim 1, characterized in that The method for preparing CdTe quantum dots comprises the following steps: (1) Mix CdCl2, trisodium citrate and 3-mercaptopropionic acid and adjust the pH to 10.5; (2) Na2TeO3 and NaBH4 are added to the solution of step (1) for reflux reaction, and CdTe quantum dots are obtained by separation and purification.

3. The biosensor according to claim 2, characterized in that The separation and purification step is: adding n-propanol to the reaction solution, and then centrifuging and separating the precipitate.

4. The biosensor according to claim 3, characterized in that The centrifugation speed was 11000 rpm.

5. The biosensor according to claim 1, characterized in that The microstructure of the CdTe quantum dots is spherical, and the average diameter is 3.5 nm.

6. The biosensor according to claim 1, characterized in that The C-Ag + The preparation method of C-Ag hairpin comprises the following steps: mixing the hairpin HP with AgNO3 solution in a buffer solution, incubating at room temperature in the dark, and generating C-Ag + -C hairpin structure.

7. A kit comprising the biosensor according to any one of claims 1 to 6.

8. A method for detecting microcystin using the biosensor according to any one of claims 1 to 6 or the kit according to claim 7, characterized in that: The following steps are involved: (1) Adding Apt-T composite probe, composite structure duplex, Helper chain and Cas12a protein to the test solution and incubating in buffer to obtain incubation solution 1; (2) Add rolling circle template chain padlock and T4 DNA ligase to incubation solution 1 and incubate in a buffer containing ATP to inactivate the enzyme to obtain incubation solution 2; (3) Add Phi29 DNA polymerase and dNTP to incubation solution 2, inactivate the enzyme to obtain incubation solution 3; (4) Add C-Ag to incubation solution 3 + -C hairpin and CdTe quantum dots were mixed and incubated for fluorescence detection.

9. The method according to claim 8, characterized in that In steps (1) and (3), the incubation temperature is 37°C; in step (2), the incubation temperature is 16°C; in step (4), the incubation temperature is room temperature; In steps (2) and (3), the temperature for inactivating the enzyme is 65°C.

10. The method according to claim 8, characterized in that The test solution in step (1) includes a microcystin solution with a series of concentrations and a solution with an unknown microcystin concentration; Step (4) includes the step of calculating the concentration of microcystin in a solution with an unknown concentration of microcystin by using a regression equation based on the fluorescence intensity and the logarithm of the concentration of microcystin solutions with a series of concentrations.

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