A multi-dimensional clipping aptamer-based malachite green label-free proportional biosensor

Abstract

The application discloses a malachite green label-free proportional biosensor based on multi-dimensional cutting aptamer, which comprises the following steps: (1) a performance-improved malachite green aptamer sequence; (2) a label-free proportional malachite green sensor sequence; and (3) malachite green detection. The biosensor is constructed by using various aptamer sequences, and the ideal sensing effect can be realized through the competition balance principle. The label-free proportional sensor can present a good linear relationship in the range of 5nM-4muM, and has a low detection limit of nanomolar level, which indicates that the sensor has good malachite green detection potential and can meet the application requirements.

Description

Technical Field

[0001] The present invention belongs to the field of biosensors, in particular to a malachite green label-free ratiometric biosensor based on multi-dimensionally tailored aptamers. Background Art

[0002] In recent years, with the rapid development of society, the high demand for protein-rich foods (such as fish, shrimp, and crab) has accelerated the production of aquatic products and other fishery products. At the same time, the use of chemical agents in aquaculture has increased in many countries to prevent and control diseases in aquatic products. Malachite green (MG), a cationic triphenylmethane compound, is commonly used as a dye, fungicide, or antiparasitic agent in aquaculture. It is widely used in aquaculture worldwide and often remains in aquatic products and environmental water. As an ideal antiparasitic agent for increasing fishery yields, MG can protect various aquatic animals from parasitic diseases in aquaculture. MG is also an effective fungicide for preventing and controlling fungal growth on fish and fish eggs. In a study of 49 compounds tested against fungal growth in fish eggs, MG was found to be the most effective fungicide. Furthermore, MG can effectively control protozoa such as Trichomonas and ciliates. Studies have also shown that MG can prevent and treat helminthic infections. However, MG is carcinogenic, teratogenic, and mutagenic, and is often illegally added to edible aquatic products, posing a serious threat to human health and the social environment. Therefore, the development of new and effective malachite green biosensing methods is urgently needed.

[0003] Aptamers were first identified in vitro by Ellington and Szostak in 1990 through systematic evolution of ligands by exponential enrichment. Over the following three decades, a growing number of aptamers have been isolated, capable of binding to target molecules with both affinity and specificity. In terms of detection, aptamer biosensors have seen rapid development in recent years, capable of generating output signals in a target-responsive manner and exhibiting stability, making them easy to transport and store.

[0004] The present invention optimizes and transforms the aptamer of MG to comprehensively improve its properties, and develops a MG label-free ratiometric biosensor based on the principle of competitive equilibrium, ultimately achieving label-free, rapid, low-cost, and ultra-sensitive fluorescence detection of MG. Summary of the Invention

[0005] Based on this, the present invention proposes a malachite green performance-enhancing aptamer based on multi-dimensional tailoring, and successfully constructs a label-free ratiometric malachite green biosensor.

[0006] On the one hand, the present application proposes a malachite green nucleic acid aptamer with improved performance, the sequence of which is shown in any one of SEQ ID NOs: 2 to 6, SEQ ID NO: 11, and SEQ ID NOs: 15 to 32.

[0007] Application of the above-mentioned nucleic acid aptamer sequence in the development of malachite green detection method.

[0008] Application of the above nucleic acid aptamer sequence in a malachite green food safety or environmental detection kit.

[0009] On the other hand, the present application proposes a label-free ratiometric malachite green biosensor, characterized in that the biosensor comprises: (1) a malachite green sensor sequence; (2) detection of malachite green;

[0010] The sequence of the above biosensor is shown in SEQ ID NO: 5 or SEQ ID NO: 31.

[0011] The signal reporting molecules of the above biosensor are thioflavin T and malachite green molecules.

[0012] The buffer composition of the above-mentioned biosensor contains 20 mM Tris-HCl.

[0013] The pH of the buffer solution of the above biosensor is 7.4.

[0014] Specific standard curve establishment:

[0015] Different concentrations of MG and thioflavin T (ThT) were added to Tris-HCl buffer (20 mM, pH 7.4) containing 1 μM aptamer. Immediately after mixing, the fluorescence spectra of malachite green and thioflavin T were recorded using a fluorescence spectrophotometer. A standard curve was plotted with the ratio of their maximum fluorescence values ​​as the ordinate and the malachite green concentration as the abscissa.

[0016] On the other hand, the present invention proposes the use of the above-mentioned biosensor or method in the development of a malachite green detection method.

[0017] On the other hand, the present invention proposes the use of the above-mentioned biosensor or method in a malachite green food safety or environmental detection kit.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Through multi-dimensional system tailoring, the present invention obtains a malachite green DNA nucleic acid aptamer with comprehensively improved fluorescence performance, stability, affinity, and binding ability;

[0020] 2. The present invention utilizes optimized nucleic acid aptamers to construct a label-free, ratiometric, sensitive, and low-cost biosensor based on the competitive equilibrium effect;

[0021] 3. The malachite green biosensor proposed in this invention exhibits excellent detection capabilities, showing good linearity in the target range of 5nM to 4μM and a low detection limit at the nanomolar level;

[0022] 4. The biosensor proposed in the present invention can realize rapid, low-cost, stable and ultra-sensitive malachite green detection, and has certain versatility and industrialization potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 1 shows the initial trimming and loop optimization of the malachite green aptamer. A shows the secondary structures of different MG DNA aptamer variants after initial trimming and loop optimization, along with the corresponding minimum free energy and base pairing probability. Purple nucleotides indicate optimized positions. B shows the fluorescence activation effects of different MG DNA aptamer variants after initial trimming and loop optimization. The dashed line represents the fluorescence intensity of the original sequence (set to 1× fluorescence). The three-dimensional structures of the binding sites of MG with MC (C), MC-DA (E), and MC-DA-L (G) sequences and the two-dimensional schematic diagrams of the molecular docking results are shown. The enlarged box highlights the MG binding pocket. D shows the circular dichroism spectra of the original sequence and the MC-DA-LG sequence. F shows the melting curve analysis of the original sequence, MC, MC-DA, and MC-DA-LG sequences. The dashed lines indicate the temperature and fluorescence intensity at which fluorescence reaches half its maximum value during DNA melting. The Tm values ​​of each sequence are indicated by the numbers in the figure.

[0024] Figure 2 is the circular dichroism spectrum of the original sequence and the MC sequence.

[0025] Figure 3 are the circular dichroism spectra of the original sequence and the MC-DA sequence.

[0026] Figure 4The "zipper" strategy experiment. A is a schematic diagram of the "zipper" strategy (left), and the secondary structures of different MG DNA aptamer variants after applying the "zipper" trimming strategy and optimizing the top loop structure (right), with accompanying minimum free energy and base pairing probability. Purple nucleotides indicate optimized nucleotide positions; B is the fluorescence activation effect of different MG DNA aptamer variants after applying the "zipper" trimming strategy and top loop modification. The dotted line represents the fluorescence intensity of the original sequence (set to 1× fluorescence); C is the circular dichroism spectrum of the MC-DA-LG and CAG-T-ML sequences; D is the three-dimensional structure of the MC-DA-ML sequence-MG complex, with the magnified box highlighting its interaction pocket; E is a two-dimensional schematic diagram of the molecular docking of the MG molecule within the MC-DA-ML sequence binding site.

[0027] Figure 5 The secondary structures of different MG DNA aptamer variants during the top loop size optimization process are shown, along with the minimum free energy and base pairing probability for each structure. Purple bases indicate optimized positions.

[0028] Figure 6 The secondary structures of different MG DNA aptamer variants during the top loop sequence optimization process are shown, along with the minimum free energy and base pairing probability of each structure. Purple bases indicate optimized positions.

[0029] Figure 7 The secondary structures of different MG DNA aptamer variants during top stem optimization are shown, along with the minimum free energy and base pairing probability for each structure. Purple bases indicate optimized positions.

[0030] Figure 8 Fluorescence intensity of different MG DNA aptamer variants during the optimization process for the top stem.

[0031] Figure 9 This is the molecular docking result of CAG-T-ML-S4 sequence and MG.

[0032] Figure 10 Figure 2. Stoichiometric ratio evaluation of different sequences. A is the original sequence; B is the MC-DA-LG sequence; C is the CAG-T-ML-S4 sequence.

[0033] Figure 11 To verify the affinity of the aptamer using the MST method. A is the original sequence; B is the MC-DA sequence; C is the MC-DA-LG sequence.

[0034] Figure 12 To verify the affinity of the aptamer using the colloidal gold method. A is the original sequence; B is the MC-DA sequence; C is the MC-DA-LG sequence.

[0035] Figure 13The schematic diagram of detection mechanism.

[0036] Figure 14 The molecular docking results of MC-DA-LG sequence and ThT. A is the three-dimensional structure of the sequence-ThT complex and its display after rotating 90°; B is the two-dimensional schematic diagram of molecular docking of ThT molecule in the sequence binding site; C is the three-dimensional schematic diagram of ThT binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0037] Figure 15 The molecular docking results of CAG-T-ML-S4G sequence and ThT. A is the three-dimensional structure of the sequence-ThT complex and its display after rotating 90°; B is the two-dimensional schematic diagram of molecular docking of ThT molecule in the sequence binding site; C is the three-dimensional schematic diagram of ThT binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0038] Figure 16 The MG detection waveform diagram. A is the detection waveform diagram of MC-DA-LG sequence; B is the detection waveform diagram of CAG-T-ML-S4 sequence.

[0039] Figure 17 The linear relationship between the fluorescence signal and different concentrations of MG. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0041] Example 1. Preliminary trimming and loop optimization

[0042] All the nucleotide sequences involved in the experiment are shown in Table 1.

[0043] Table 1 Nucleotide sequences involved in the experiment

[0044]

[0045]

[0046] Previous studies have reported that the T7, A19, A20 and other bases of the original sequence of MG DNA aptamer can interact with MG, indicating that the middle loop and adjacent complementary stem of the aptamer may both participate in the binding of MG. Therefore, in order to make the middle loop and the bottom stem of the MG DNA aptamer more stable, T23 was mutated to C23 at the 3' end to obtain the MC sequence ( Figure 1 A) The results showed that the minimum free energy of MC sequence was -2.09 kcal / mol, lower than that of the original sequence (-1.52 kcal / mol), indicating that the mutation of C base could make the higher conformation of aptamer more stable. Moreover, the MG fluorescence of MC sequence was higher, about 1.7 times of the original sequence fluorescence Figure 1 B) The binding pocket of the original sequence was mainly concentrated in the upper loop region, and the molecular docking results confirmed that the mutation of C base made the interaction pocket move to the 3' and 5' ends, increasing the number of interacting bases in the binding pocket and making the nucleic acid structure more stable. Among them, G5 interacts with MG through electrostatic interaction, T18, T22 through hydrogen bonding, and T2, A4, C8, T9, A10, A19, A21 through van der Waals force Figure 1 C) The point mutation made the MG binding pocket more stable, thereby enhancing the fluorescence. Further, after removing the excess "bubble" A4 base, a more typical mismatch-stem loop structure, MC-DA, was formed, and the sequence was more stable (the minimum free energy was -4.77 kcal / mol) Figure 1 A) Molecular docking simulation showed that the interaction pocket position of MC-DA sequence was basically unchanged compared with MC sequence, in which T6 interacted with MG through hydrogen bonding, and T2, A5, C7, A19, A20, T21, C22 interacted with MG through van der Waals force Figure 1 E) Fluorescence experiments also proved that MC-DA sequence could more obviously excite MG fluorescence, which was 1.8 times of the original sequence Figure 1 B).

[0047] After preliminary pruning, further optimization of the base type of the middle loop was carried out, that is, based on the MC-DA sequence, the type of the 5' end mismatch base was further optimized. In order to retain the mismatch base pair, the 5' end mismatch base C7 was mutated to A7 or G7, respectively, obtaining MC-DA-LA and MC-DA-LG sequences Figure 1 A) The results showed that MC-DA-LG sequence significantly enhanced the fluorescence sequence, which was nearly 2 times higher than the original sequence Figure 1 B) MC-DA-LG sequence had a significantly stable higher structure than MC-DA-LA sequence, with minimum free energies of -8.77 and -5.53 kcal / mol, respectively. After mutation to G (MC-DA-LG sequence), the interaction pocket position was basically unchanged, and on the basis of T6 interaction, the ring G sequence also increased the MG interaction of G7 nucleotide Figure 1G), which may lead to a higher fluorescence intensity of the sequence. Based on the MC-DA-LG sequence, the type of mismatched base on the right side of the middle loop (base 19) was further optimized. In order to retain the ring structure, the GA mismatched base pair was mutated to GT or GG mismatch, respectively, to obtain CAG-RT and CAG-RG sequences. The results showed that the fluorescence activation effect of the GA mismatched sequence (MC-DA-LG) was still the best ( Figure 1 A to B). Among the current aptamer variants, the MC-DA-LG sequence has the lowest minimum free energy (-8.77 kcal / mol). This structural stability directly affects its binding effect with the MG molecule.

[0048] The original sequence and three fluorescent preferred sequences (MC, MC-DA, MC-DA-LG sequences) were further evaluated. Figure 1 D and Figures 2-3 As shown in the figure, the circular dichroism results show that the original sequence, MC, MC-DA, and MC-DA-LG sequences all have positive peaks at around 275nm and negative peaks at around 250nm, which are characteristic circular dichroism spectra of the typical B-DNA helix. The circular dichroism peaks of the three MG DNA aptamer variants all shift to varying degrees, indicating that deletion and mutation optimization have caused changes in the higher-order structure of the nucleic acid ( Figure 1 D. Figure 2 and Figure 3 ).

[0049] The Tm value is the melting temperature of DNA and is a key indicator for measuring the stability of DNA molecules. Figure 1 As can be seen from Figure 5, the Tm values ​​of the sequences gradually increased as the tailoring progressed, from 42.77°C in the original sequence to 61.35°C, indicating the increasing stability of the MG DNA aptamers. Furthermore, all four sequences maintained highly stable higher-order structures at 37°C, demonstrating that the tailored sequences are suitable for use under physiological conditions.

[0050] Example 2. “Zipper strategy” and top stem-loop optimization

[0051] The experiment further verified the significance of the complementary stem region between the top loop and the middle loop for MG binding. The top stem region of the MC-DA-LG sequence contains two pairs of "AT" bases ( Figure 1 A). On this basis, the "zipper" tailoring strategy was used to explore the significance of the top complementary stem for the high-level topological structure of the aptamer and its role in MG binding. By mutating the "AT" complementary pairs into "CT" mismatched base pairs one by one, the complementarity of the top stem was changed, that is, the sequence was pulled down like a zipper, resulting in fewer and fewer complementary pairs between the top ring and the middle ring ( Figure 4 A). Figure 4As shown in Figure B, the fluorescence results show that the four sequences designed based on the "zipper" cutting strategy (CAG-Zipper 1 to 4) have weaker fluorescence excitation effects on MG than the MC-DA-LG sequence. In addition, the fewer the number of complementary pairs in the top stem, the lower the fluorescence quantum yield for MG excitation. When only one pair of "TA" bases is complementary (CAG-Zipper 3), the sequence is no longer able to form a stable complementary structure, resulting in the same fluorescence collapse (extremely weak fluorescence value) as the completely mismatched sequence (CAG-zipper4). The above results show that the complementary sequence between the top loop and the middle loop is very important. This high-level loop-stem-loop structure can effectively promote the binding and excitation of MG.

[0052] Furthermore, the experiment began to optimize the size of the top loop. Sequence optimization was performed by expanding the top loop by 1 A base, deleting 1 T base, adding 1 T base, or expanding 2 T bases, and four aptamer variants, CAG-CCTATG, CAG-CCTG, CAG-CCTTTG, and CAG-CCTTTTG, were obtained ( Figure 5 ).like Figure 4 As shown in Figure B, the fluorescence results show that when the size of one base is increased, the fluorescence quantum yield of MG is higher. Therefore, the size of the top ring is determined to be a ring formed by 6 bases, that is, it is necessary to add one base to the top ring of the original sequence. This may be because the minimum free energy of the top ring sequence of 6 bases is relatively low and the structure is more stable. The conformation it forms may be more suitable for the embedding and excitation of MG. In addition, the stability of some sequences is poor, which is not conducive to the binding interaction between the aptamer and MG molecules and fluorescence excitation ( Figure 5 ).

[0053] Subsequently, the top base sequence types were optimized. The experiment was conducted with 8 different top sequence combination designs ( Figure 6 In addition, since the middle loop of the aptamer, which consists of mismatches and two pairs of complementary bases, is also 6 nt, which is the same as the optimal size of the top loop, we tried to graft the TGT-AAA loop of the middle loop to the top, and obtained the CAG-T-ML sequence ( Figure 4 A). The results showed that among all aptamer variants, the CAG-T-ML sequence after the tandem intermediate loop significantly stimulated the MG fluorescence intensity, achieving the best excitation efficiency to date, three times that of the original sequence. Circular dichroism results showed that the CAG-T-ML sequence still exhibited a B-DNA helical structure, but its circular dichroism peak shifted compared to the MC-DA-LG sequence, indicating that the higher-order structure of the nucleic acid changed after the tandem TGT-AAA loop. Figure 4C). In addition, molecular docking results also show that the interaction pocket of CAG-T-ML is basically concentrated near the middle ring, and molecular docking results show that G4, G7, and T22 in the sequence interact with MG through hydrogen bonds, and C3, G4, A5, T6, T18, A20, A21, and C23 interact with MG through van der Waals forces ( Figure 4 This demonstrates, to a certain extent, that the early optimization of mismatched bases was effective. The optimized TGT-AAA middle loop can produce specific and superimposable fluorescence activation effects on MG, significantly increasing the fluorescence quantum yield of MG after TGT-AAA is tandemly added to the top.

[0054] Previous experiments optimized the size and sequence of the top loop and demonstrated the importance of the stem between the two loops. Subsequently, experiments optimized the length and sequence of the top stem. The top stem of the original sequence was an AT sequence. Based on the CAG-T-ML sequence, AT complementary base pairs were deleted or added to the top stem, and aptamer structures containing 0, 1, 2, 3, 4, and 5 pairs of top stems were obtained, named CAG-T-ML-S0~5, among which the CAG-T-ML-S2 sequence was the same as the CAG-T-ML sequence ( Figure 7 The results showed that the longer the top stems between the two rings, the more stable the sequence and the stronger the fluorescence. When the number of complementary sequence pairs of the top stems reached 4 or 5 pairs, the sequence showed similar optimal excitation ( Figure 8 This may be because the extension of the stem can reduce the flexibility of the ring region at both ends of the stem, allowing it to stably form the expected ring structure, thereby providing sufficient binding space for MG. Figure 9 As shown in the figure, the molecular docking results also showed that the CAG-T-ML-S4 sequence retained the original binding pocket, in which G4, G7, and T26 interacted with MG through hydrogen bonds, and C3, A5, T6, T21, T22, A24, A25, and C27 interacted with MG through van der Waals forces, and the stability of the sequence was improved.

[0055] Example 3. Performance evaluation of MG DNA aptamer variants

[0056] 1. Stoichiometric ratio

[0057] To explore the binding ratios of aptamer variants to MG, the stoichiometric ratios of MG binding to the original sequence, MC-DA-LG, and CAG-T-ML-S4 sequences were calculated using the classic Job's plot method. Figure 10As shown in the fitting plots for the three sequences, fluorescence peaks appear at 0.510, 0.370, and 0.313, respectively. Calculations show that the molar ratios of MG binding to the three sequences are 0.963:1, 1.706:1, and 2.194:1, respectively. This demonstrates that through sequence optimization and systematic tailoring, a single aptamer molecule can bind to a greater number of MG molecules, thereby achieving enhanced fluorescence excitation of MG.

[0058] Table 2 Stoichiometric ratios of MG DNA aptamer variants binding to MG

[0059]

[0060] 2. Sequence stability

[0061] Furthermore, the melting temperatures of different MG aptamer variants were systematically monitored. As shown in Table 3, the Tm values ​​of the sequences increased with the tailoring process, demonstrating that sequence optimization enhances aptamer stability. Furthermore, the Tm values ​​of the different aptamer variant sequences were all above physiological temperature, demonstrating that the aptamers possess excellent thermal stability and are ideal for in vivo or intracellular applications.

[0062] Table 3 Melting temperatures of MG DNA aptamer variants

[0063]

[0064] 3. Sequence affinity

[0065] The affinity of aptamers is one of their most important properties. We then used a variety of methods to monitor the affinity of different important sequences. Since MG itself is fluorescent and its emission wavelength is in the red light range, we innovatively performed label-free MST measurements, using MG molecules as fluorescent molecules without any modification of the aptamer, thereby avoiding target-induced fluorescence increases and reducing costs. Figure 11 As shown, as the trimming process occurs, the sequence affinity gradually increases, showing a smaller and smaller K d The colloidal gold results also showed the same trend ( Figure 12 Affinity experiments have shown that the affinity of a nucleic acid sequence for MG is correlated with the intensity of its fluorescence excitation. Specifically, the stronger the fluorescence excitation of the sequence, the higher its affinity for the MG molecule. This suggests that the overall performance of the sequence improves with the process of tailoring.

[0066] Example 4. Application of aptamers in malachite green biosensing

[0067] Table 4 Biosensing performance of MG with different sequences

[0068]

[0069]

[0070] In order to explore the application performance of the optimized sequence in biosensing, a label-free ratiometric fluorescence sensor was constructed based on the competitive equilibrium principle to detect malachite green. Figure 13 As shown, the aptamer can bind to the MG molecule and induce MG fluorescence emission. Adding a competing small molecule to the aptamer-MG complex system will compete with MG for the same nucleic acid binding site, causing the fluorescence intensity of the system to change, enabling the sensing and detection of the target substance.

[0071] As a dye molecule, ThT can be embedded in advanced conformations such as G-quadruplexes, double strands, and hairpin structures, thereby achieving enhanced fluorescence excitation. In the MG biosensor, the fluorescence properties of the two dyes MG and ThT were used to construct a ratiometric, label-free, ultra-sensitive, and ultra-fast biosensor. Based on this, the feasibility of the sensor was first systematically evaluated, and the interaction between the representative sequences MC-DA-LG and CAG-T-ML-S4 and ThT was simulated through molecular docking. The results are shown in Figure 2. Figure 14 and 15 The sequence has similar and overlapping interaction binding pockets with ThT and MG, which theoretically proves the feasibility of using ThT molecules to compete with MG molecules.

[0072] The performance of two label-free ratiometric aptamer sensors was evaluated. Different concentrations of MG and ThT were added to a Tris-HCl buffer (20 mM, pH 7.4) containing 1 μM aptamer. Immediately after mixing, the fluorescence spectra of malachite green and thioflavin T were recorded using a fluorescence spectrophotometer. A standard curve was plotted with the ratio of the maximum fluorescence values ​​of the two as the ordinate and the malachite green concentration as the abscissa. Figure 16 As shown in Figure 2, as the concentration of target MG increases, ThT fluorescence gradually decreases and MG fluorescence gradually increases. Figure 17 As shown in Table 4 , the two ratiometric sensors exhibited a good linear relationship in the range of 5 nM to 4 μM, and achieved low detection limits of 2.15 nM (CAG-T-ML-S4 sensor) and 4.99 nM (MC-DA-LG sensor), respectively (Table 4 ), indicating that the sensors exhibited good MG detection potential and could meet application requirements.

Claims

1. A malachite green nucleic acid aptamer with improved performance, characterized in that: The nucleic acid aptamer sequence is shown in any one of SEQ ID NOs: 2 to 6, SEQ ID NO: 11, and SEQ ID NOs: 15 to 32.

2. Application of the nucleic acid aptamer sequence according to claim 1 in the development of a malachite green detection method.

3. Use of the aptamer sequence according to claim 1 in preparing a malachite green food safety or environmental detection kit.

4. A label-free ratiometric malachite green biosensor, characterized in that: The biosensor comprises: (1) a malachite green biosensor sequence; (2) a signal reporter molecule of the malachite green biosensor; The malachite green biosensor sequence is shown in SEQ ID NO: 5 or SEQ ID NO: 31; The signal reporting molecules of the malachite green biosensor are thioflavin T and malachite green molecules.

5. The biosensor according to claim 4, wherein The buffer composition of the biosensor contained 20 mM Tris-HCl.

6. The biosensor according to claim 5, wherein The pH of the biosensor buffer was 7.

4.

7. The method for quantitative detection of malachite green by a biosensor according to any one of claims 4 to 6, characterized in that: Including the establishment of standard curve: Different concentrations of malachite green and thioflavin T were added to 20 mM Tris-HCl buffer containing 1 µM aptamer. Immediately after mixing, the fluorescence spectra of malachite green and thioflavin T were recorded using a fluorescence spectrophotometer. A standard curve was plotted with the ratio of their maximum fluorescence values ​​as the ordinate and the malachite green concentration as the abscissa.

8. Use of the biosensor according to any one of claims 4 to 6 or the method according to claim 7 in the development of a malachite green detection method.

9. Use of the biosensor according to any one of claims 4 to 6 or the method according to claim 7 in preparing a malachite green food safety or environmental detection kit.

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