Malachite green label-free proportional biosensor based on multi-dimensional cutting aptamer

Abstract

The invention discloses a malachite green label-free proportional biosensor based on a multi-dimensional cutting aptamer. The malachite green label-free proportional biosensor comprises (1) a malachite green aptamer sequence with improved performance; (2) a label-free proportional malachite green sensor sequence; and (3) detection of malachite green. The biosensor is constructed by utilizing multiple aptamer sequences, and an ideal sensing effect can be realized through a competitive balance principle. The label-free proportional sensor can present a good linear relationship in a range of 5nM-4mu M, and has a nanomole-level low detection limit value, which shows that the sensor shows good malachite green detection potential, and can meet application requirements.

Description

Technical Field

[0001] The present invention belongs to the field of biosensors, and particularly relates to a label-free ratio-type biosensor for malachite green based on a multi-dimensionally tailored aptamer. Background Art

[0002] In recent years, with the rapid development of society, the high demand for protein-rich foods (such as fish, shrimps, and crabs) has accelerated the production of aquatic products and other fishery products. At the same time, in order to prevent and control the occurrence of diseases in aquatic products, the use of chemical drugs in aquaculture has increased in many countries. Malachite green (MG) is a cationic triphenylmethane compound, which is often used as a dye, fungicide, or antiparasitic drug. It is widely used in the global aquaculture industry and is commonly residues in aquatic products and environmental water. As an ideal antiparasitic agent for increasing fishery production, MG can protect various aquatic animals from diseases caused by parasites in aquaculture. MG is also an effective fungicide for preventing and controlling the growth of fungi on fish and fish eggs. Among 49 compounds tested for fish egg fungi, MG was found to be the most effective fungicide. In addition, MG can also be used for the efficient control of protozoa, such as trichomonas and ciliates. Moreover, studies have shown that MG can also prevent and control worm infections. However, MG has toxicities such as carcinogenicity, teratogenicity, and mutagenicity, and is often illegally added in the field of edible aquatic products, posing a serious threat to human health and the social environment. Therefore, there is an urgent need to develop an effective new method for the biosensing of malachite green.

[0003] Nucleic acid aptamers were first isolated in vitro by Ellington and Szostak in 1990 through the systematic evolution of ligands by exponential enrichment (SELEX) technology. In the subsequent thirty-year development, more and more nucleic acid aptamers have been isolated, which can bind to target molecules in an affinity and specific manner. In terms of detection, nucleic acid aptamer biosensors have developed rapidly in recent years, which can generate output signals in a target-responsive mode, and their properties are stable, facilitating transportation and storage.

[0004] The present invention optimizes and transforms the aptamer of MG, comprehensively improves its properties, and develops a label-free ratio-type biosensor for MG based on the principle of competitive equilibrium, ultimately realizing label-free, rapid, low-cost, and ultrasensitive fluorescence detection of MG. Summary of the Invention

[0005] Based on this, the present invention proposes an aptamer for improving the performance of malachite green based on multi-dimensional tailoring, and successfully constructs a label-free ratio-type malachite green biosensor.

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

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

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

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

[0010] The sequence of the above biosensor is shown as 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 biosensor contains 20 mM Tris-HCl.

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

[0014] Specific establishment of the standard curve:

[0015] Add different concentrations of MG and thioflavin T (ThT) to Tris-HCl buffer (20 mM, pH 7.4) containing 1 μM aptamer; immediately record the fluorescence spectra of malachite green and thioflavin T using a fluorescence spectrophotometer after mixing; plot a standard curve with the ratio of the maximum fluorescence values of the two as the ordinate and the malachite green concentration as the abscissa.

[0016] On the other hand, the present invention proposes the application of the above biosensor or method in the development of malachite green detection methods.

[0017] On the other hand, the present invention proposes the application of the above biosensor or method in malachite green food safety or environmental detection kits.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. Through multi-dimensional systematic trimming, 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 the optimized nucleic acid aptamer to construct a label-free, proportional, sensitive, and low-cost biosensor based on the competitive equilibrium effect;

[0021] 3. The malachite green biosensor proposed by the present invention exhibits excellent detection capabilities, can present a good linear relationship within the target range of 5 nM to 4 μM, and has a low detection limit at the nanomolar level;

[0022] 4. The biosensor proposed by the present invention can achieve rapid, low-cost, stable, and ultrasensitive detection of malachite green, and has certain generality and industrialization potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 For the preliminary trimming and loop optimization of the malachite green aptamer. A shows the secondary structures of different MG DNA aptamer variants after preliminary trimming and loop structure optimization, as well as the corresponding minimum free energy and base pairing probability. Purple nucleotides indicate the optimized positions; B shows the fluorescence activation effects of different MG DNA aptamer variants after preliminary trimming and loop structure optimization. The dashed line represents the fluorescence intensity of the original sequence (set as 1× fluorescence); Two-dimensional schematic diagrams of the three-dimensional structures and molecular docking results of the binding sites of MG with MC (Figure C), MC-DA (Figure E), and MC-DA-L (Figure G) sequences. The enlarged box highlights the binding pocket of MG; 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 line indicates the temperature and fluorescence intensity when the fluorescence reaches half of the maximum value during DNA melting. The Tm values of each sequence are indicated by the numbers in the figure.

[0024] Figure 2 For the circular dichroism spectra of the original sequence and the MC sequence.

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

[0026] Figure 4For the "zipper" strategy experiment. A shows the schematic diagram of the "zipper" strategy (left), and the secondary structures of different MG DNA aptamer variants after applying the "zipper" cutting strategy and optimizing the top loop structure (right), along with the minimum free energy and base pairing probability. The purple nucleotides indicate the optimized nucleotide positions; B shows the fluorescence activation effects of different MG DNA aptamer variants after adopting the "zipper" cutting strategy and top loop modification. The dashed line represents the fluorescence intensity of the original sequence (set as 1× fluorescence); C shows the circular dichroism spectra of the MC-DA-LG and CAG-T-ML sequences; D shows the three-dimensional structure of the MC-DA-ML sequence complex with MG, and the enlarged box highlights its interaction pocket; E shows the two-dimensional schematic diagram of the molecular docking of the MG molecule within the binding site of the MC-DA-ML sequence.

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

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

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

[0030] Figure 8 The fluorescence intensities of different MG DNA aptamer variants during the top stem optimization process.

[0031] Figure 9 The molecular docking results of the CAG-T-ML-S4 sequence with MG.

[0032] Figure 10 For the stoichiometry 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 For verifying the aptamer affinity 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 For verifying the aptamer affinity 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 13Schematic diagram of the detection mechanism.

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

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

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

[0039] Figure 17 Linear relationship between fluorescence signal and different concentrations of MG. Detailed implementation manners

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0041] Example 1. Preliminary trimming and optimization of the loop part

[0042] All 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 bases such as T7, A19, and A20 of the original sequence of the MG DNA aptamer (Original sequence) can interact with MG, indicating that the middle loop part and the adjacent complementary stems of the aptamer may all participate in the binding of MG. Therefore, in order to make the middle loop part 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). It was found that the minimum free energy of the MC sequence was -2.09 kcal / mol, lower than that of the original sequence (-1.52 kcal / mol), indicating that mutating the C base could make the higher-order conformation of the nucleic acid aptamer more stable. Moreover, the MG fluorescence of the MC sequence was higher, about 1.7 times that of the original sequence ( Figure 1 B). The binding pocket of the original sequence was mainly concentrated in the upper loop region. The molecular docking results confirmed that mutating the C base caused the interaction pocket to move towards 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 interacted with MG through electrostatic force, T18 and T22 through hydrogen bonds, and T2, A4, C8, T9, A10, A19, and A21 through van der Waals forces. Figure 1 C). The point mutation made the MG binding pocket more stable, thus enhancing the fluorescence. Further, after removing the redundant "bubble" A4 base, a more typical mismatch-stem-loop structure, MC-DA, was formed, and this sequence had stronger stability (the minimum free energy was -4.77 kcal / mol). ( Figure 1 A). The molecular docking simulation showed that the position of the interaction pocket of the MC-DA sequence was basically unchanged compared with that of the MC sequence. Among them, T6 interacted with MG through hydrogen bonds, and T2, A5, C7, A19, A20, T21, and C22 interacted with MG through van der Waals forces. Figure 1 E). The fluorescence experiment also proved that the MC-DA sequence could more significantly excite the MG fluorescence, which was 1.8 times that of the original sequence. Figure 1 B).

[0047] After preliminary trimming, the base types of the middle loop were further optimized, that is, on the basis of the MC-DA sequence, the base types of the 5'-end mismatched bases were further optimized. In order to retain the mismatched base pairs, the 5'-end mismatched base C7 was mutated to A7 or G7 respectively, and the MC-DA-LA and MC-DA-LG sequences were obtained. Figure 1 A). The results showed that the MC-DA-LG sequence significantly enhanced the fluorescence sequence, which was nearly twice that of the original sequence. Figure 1 B). The MC-DA-LG sequence had a significantly more stable higher-order structure than the MC-DA-LA sequence, with minimum free energies of -8.77 and -5.53 kcal / mol respectively. After mutating to G (MC-DA-LG sequence), the position of the interaction pocket was basically unchanged, and on the basis of the interaction of T6, the loop G sequence also increased the MG interaction involving the G7 nucleotide. Figure 1G), which may result in a sequence with higher fluorescence intensity. Based on the MC-DA-LG sequence, the types of mismatched bases on the right side of the middle loop (the 19th base) were further optimized. To retain the loop structure, the G-A mismatched base pairs were mutated to G-T or G-G mismatches respectively, resulting in the CAG-RT and CAG-RG sequences. It was found that the fluorescence activation effect of the G-A mismatch sequence (MC-DA-LG) was still the best ( Figure 1 A–B). Among the aptamer variants at the current stage, the MC-DA-LG sequence has the lowest minimum free energy (-8.77 kcal / mol), and this structural stability directly affects its binding effect with the MG molecule.

[0048] The original sequence and three fluorescence-optimized sequences (MC, MC-DA, and MC-DA-LG sequences) were further evaluated. As Figure 1 D and Figures 2 - 3 shown, it can be seen from the circular dichroism results that positive peaks appeared at around 275 nm and negative peaks appeared at around 250 nm for the original sequence, MC, MC-DA, and MC-DA-LG sequences. This is the characteristic circular dichroism spectrum of the typical B-DNA helix. The circular dichroism peaks of the three MG DNA aptamer variants all shifted to varying degrees, indicating that deletion and mutation optimization changed 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 to measure the stability of DNA molecules. As Figure 1 F shows, with the progress of trimming, the Tm value of the sequence gradually increases. It increases from 42.77 °C of the original sequence to 61.35 °C, indicating that the stability of the MG DNA aptamer is getting stronger and stronger. Moreover, the four sequences still maintain a very stable higher-order structure at 37 °C, indicating that the trimmed sequences can be applied under physiological conditions.

[0050] Example 2. "Zipper strategy" and optimization of the top stem-loop

[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 "A-T" bases ( Figure 1 A). On this basis, the "zipper" trimming strategy was used to explore the significance of the top complementary stem for the higher-order topology of the aptamer and its role in MG binding. By mutating the "A-T" complementary pairs into "C-T" mismatched base pairs one by one, the complementary situation 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 loop and the middle loop ( Figure 4 A). As Figure 4As shown in Figure B, the fluorescence results indicate that for the four sequences (CAG-Zipper 1-4) designed based on the "zipper" truncation strategy, their fluorescence excitation effects on MG are weaker than those of the MC-DA-LG sequence. Moreover, the fewer the number of complementary base pairs in the top stem, the lower the fluorescence quantum yield for MG excitation. When there is only one pair of "T-A" base complementarity (CAG-Zipper 3), the sequence can hardly form a stable complementary structure, resulting in fluorescence collapse (extremely weak fluorescence value) similar to that of the completely mismatched sequence (CAG-zipper4). The above results illustrate that the complementary sequence between the top loop and the middle loop is very important, and this loop-stem-loop higher-order structure can well promote the binding and excitation effects of MG.

[0052] Furthermore, the experiment began to optimize the size of the top loop. The sequence was optimized by expanding one A base, deleting one T base, adding one T base, or expanding two T bases in the top loop respectively, and four aptamer variants ( Figure 5 ) were obtained. As Figure 4 shown in Figure B, the fluorescence results indicate that when expanding the size by one base, the fluorescence quantum yield of MG is relatively high. Therefore, it is determined that the size of the top loop is composed of a ring formed by 6 bases, that is, one base needs to be added on the basis of the original top loop of the sequence. This may be because the minimum free energy of the 6-base top loop sequence is relatively low, the structure is more stable, and the conformation formed may be more suitable for the embedding and excitation of MG. Moreover, the stability of individual sequences is poor, which is not conducive to the binding interaction between the aptamer and the MG molecule and fluorescence excitation ( Figure 5 ).

[0053] Subsequently, the sequence types of the top bases were optimized. The experiment carried out 8 different designs of top sequence combinations ( Figure 6 ). In addition, since the loop composed of mismatches and two pairs of complementary bases in the middle of the aptamer is also 6nt, the same as the optimal size of the top loop, the TGT-AAA loop in the middle loop was tried to be grafted to the top to obtain the CAG-T-ML sequence ( Figure 4 Figure A). The results show that among all aptamer variants, the CAG-T-ML sequence after concatenating the middle loop significantly excites the fluorescence intensity of MG, achieving the optimal excitation efficiency so far, which is 3 times that of the original sequence. The circular dichroism results show that the CAG-T-ML sequence still presents a B-DNA helical structure, but its circular dichroism peak is shifted compared with that of the MC-DA-LG sequence, indicating that the higher-order structure of the nucleic acid has changed after concatenating the TGT-AAA loop ( Figure 4C). In addition, the molecular docking results also showed that the interaction pockets of CAG-T-ML were mainly concentrated near the middle loop, and the molecular docking results indicated that G4, G7, and T22 in this sequence interacted with MG through hydrogen bonds, and C3, G4, A5, T6, T18, A20, A21, and C23 interacted with MG through van der Waals forces. Figure 4 D, E). This to a certain extent proved that the previous optimization of the mismatched bases was effective. The optimized middle loop of TGT-AAA could produce specific and superimposable fluorescence activation effects on MG, resulting in a significant increase in the fluorescence quantum yield of MG after TGT-AAA was tandemly connected at the top.

[0054] In the previous experiment, the size and sequence of the top loop were optimized, and the importance of the stem between the two loops was demonstrated. Subsequently, the length and sequence of the top stem were optimized separately. The top stem of the original sequence was an A-T sequence. Based on the CAG-T-ML sequence, A-T complementary base pairs were deleted or added at the top stem to obtain aptamer structures containing 0, 1, 2, 3, 4, and 5 pairs of top stems, named CAG-T-ML-S0 to 5, where the CAG-T-ML-S2 sequence was the same as the CAG-T-ML sequence. Figure 7 ) It was found that the longer the number of top stems between the two loops and the more stable the sequence, the stronger the fluorescence. When the number of complementary sequence pairs of the top stem 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 loop regions at both ends of the stem, enabling it to stably form the expected circular structure, thereby providing sufficient binding space for MG. As Figure 9 shown, the molecular docking results also indicated that the CAG-T-ML-S4 sequence retained the original binding pocket, where 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. Stoichiometry

[0057] To explore the binding ratio of the aptamer variant to MG, the stoichiometry of the binding of MG to the original sequence, MC-DA-LG, and the CAG-T-ML-S4 sequence was calculated by the classical Job’s plot method. As shown in Table 2 and Figure 10As shown, the fluorescence peaks in the fitting graphs of the three sequences appear at 0.510, 0.370, and 0.313 respectively. Through calculation, the binding molar ratios of the MG molecule to the three sequences are 0.963:1, 1.706:1, and 2.194:1 respectively. It can be seen that through sequence optimization and system trimming, an aptamer molecule can bind more MG molecules, thereby achieving enhanced fluorescence excitation of MG.

[0058] Table 2 Stoichiometry of the binding of MG DNA aptamer variants 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, as the trimming process progresses, the Tm values of the sequences continuously increase, demonstrating that the stability of the aptamer is enhanced through sequence optimization. Moreover, the Tm values of different aptamer variant sequences are all higher than the physiological temperature, indicating that the aptamer has good thermal stability and can be ideally applied in vivo or intracellularly.

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

[0063]

[0064] 3. Sequence affinity

[0065] The affinity of the aptamer is one of its most important properties. Subsequently, the affinities of different important sequences were monitored using various methods. Since MG itself has fluorescence and its emission wavelength is in the red light range. Therefore, an innovative label-free MST measurement was carried out, that is, using the MG molecule as the fluorescent molecule and not modifying the aptamer, thereby avoiding the increase in fluorescence induced by the target and reducing costs. As Figure 11 shown, as the trimming process occurs, the sequence affinity gradually increases, showing an increasingly smaller K d value. The colloidal gold results also showed the same trend ( Figure 12 ). The affinity experiment proved that the degree of affinity of the nucleic acid sequence for MG is correlated with the fluorescence excitation intensity for MG, that is, the stronger the fluorescence excitation of the sequence for MG, the higher its affinity for the MG molecule. That is to say, as the trimming progresses, the comprehensive performance of the sequence is improved.

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

[0067] Table 4 Malachite green biosensing performance of different sequences

[0068]

[0069]

[0070] To explore the application performance of the optimized sequence in biosensing, a label-free proportional fluorescence sensor was constructed based on the competitive equilibrium principle for the detection of malachite green. The detection principle is as Figure 13 shown. The aptamer can bind to MG molecules and induce the fluorescence emission of MG. When a competitive small molecule is added to the aptamer-MG complex system, the small molecule will compete with MG for the same nucleic acid binding site, thereby causing a change in the fluorescence intensity of the system and realizing the sensing detection of the target substance.

[0071] As a dye molecule, ThT can intercalate into higher-order conformations such as G-quadruplexes, double-stranded, and hairpin structures, thereby achieving enhanced fluorescence excitation. In the MG biosensor, a proportional, label-free, ultrasensitive, and ultrafast biosensor was constructed using the fluorescence properties of two dyes, MG and ThT. 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 by molecular docking. The results are as Figure 14 and 15 shown. The sequences have similar and overlapping interaction binding pockets with ThT and MG, theoretically demonstrating the feasibility of using ThT molecules to compete with MG molecules.

[0072] The performance of two label-free proportional aptamer sensors was evaluated. Different concentrations of MG and ThT were added to the 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 concentration of malachite green as the abscissa. As Figure 16 shown, as the concentration of the target MG increases, the fluorescence of ThT gradually decreases and the fluorescence of MG gradually increases. As Figure 17 and Table 4 show, the two proportional sensors can show a good linear relationship in the range of 5 nM to 4 μM, and low detection limits of 2.15 nM (CAG-T-ML-S4 sensor) and 4.99 nM (MC-DA-LG sensor) were obtained respectively (Table 4), indicating that the sensors exhibit good MG detection potential and can both meet the application requirements.

Claims

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

2. Use 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 a malachite green food safety or environmental detection kit.

4. A label-free proportional malachite green biosensor, characterized in that, The biosensor includes: (1) a malachite green biosensor sequence; (2) detection of malachite green. The malachite green biosensor sequence is as shown in SEQ ID NO: 5 or SEQ ID NO:

31.

5. The biosensor according to claim 4, characterized in that, The signal reporter molecules of the biosensor are thioflavin T and malachite green molecules.

6. The biosensor according to claim 4, characterized in that, The buffer component of the biosensor contains 20 mM Tris-HCl.

7. The biosensor according to claim 4, characterized in that, The buffer pH of the biosensor is 7.

4.

8. The method for quantitatively detecting malachite green by the biosensor according to claims 4 to 7, characterized in that, Establishment of a standard curve: Add different concentrations of malachite green and thioflavin T to a 20 mM Tris-HCl buffer containing 1 μM aptamer; immediately record the fluorescence spectra of malachite green and thioflavin T using a fluorescence spectrophotometer after mixing; plot a standard curve with the ratio of the maximum fluorescence values of the two as the ordinate and the malachite green concentration as the abscissa.

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

10. Use of the biosensor according to any one of claims 4-7 or the method according to claim 8 in a malachite green food safety or environmental detection kit.

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