Aptamer with improved multi-dimensional performance and application thereof in biosensing and mild bacteriostasis

Abstract

The invention discloses an aptamer with improved multi-dimensional performance. The aptamer is applied to a malachite green label-free proportional biosensor and long-acting, mild and slow-release antibacterial regulation and control. Specifically, two malachite green aptamer biosensors with excellent detection performance are constructed through a competitive balance principle, and the malachite green aptamer biosensors have a wide detection range and a low detection limit. In addition, the aptamer can slowly release malachite green in the bacteriostasis process, and the long-acting and mild bacteriostasis process is assisted. The aptamer with improved multi-dimensional performance is applied to the field of slow-release bacteriostasis, can effectively relieve toxic and side effects of a bactericide on non-target microorganisms or host cells, and is beneficial to realizing long-acting control and relieving drug resistance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an aptamer with improved multidimensional performance and its application in biosensing and mild bacteriostasis. Background Art

[0002] Malachite green (MG), a triphenylmethane compound, was once widely used in aquaculture for antibacterial and antifungal treatments, demonstrating significant bactericidal efficacy. However, studies have shown MG to have potential mutagenicity, carcinogenicity, and cumulative toxicity, leading to its ban in food-related applications in several countries and regions. Despite this, MG's illegal use persists in some regions due to its low cost and ease of use, posing a serious threat to public health and food safety. Therefore, the development of highly sensitive, specific, convenient, and efficient detection methods is crucial for the regulation of malachite green residues.

[0003] Aptamers, artificially screened single-stranded DNA or RNA molecules, have the ability to bind to target molecules with high affinity and specificity, and are widely used in small molecule detection, bioimaging, and targeted drug delivery. Aptamers targeting malachite green are currently a hot topic of research. However, existing aptamers still have deficiencies in affinity, sequence stability, and signal response intensity, which limits their application in actual complex sample detection. Therefore, it is necessary to improve aptamers in multiple dimensions from aspects such as structural optimization, screening strategies, and signal coupling mechanisms to achieve efficient recognition and signal amplification of malachite green, meeting the needs of rapid, accurate, and on-site detection.

[0004] On the other hand, although malachite green has a rapid and efficient antibacterial effect in bactericidal applications, its release pattern is relatively violent and lacks sustained-release regulation, which can easily cause toxic side effects on non-target microorganisms or host cells and is not conducive to achieving long-term control. In addition, the unstable release of the bactericidal mechanism may aggravate the occurrence of drug resistance. Therefore, constructing functional modules that can specifically bind to malachite green and regulate its release behavior, such as aptamer-nanocarrier composite systems, will not only help prolong its bactericidal effect time and reduce toxicity, but also provide technical support for the development of mild and controllable bactericidal systems. Summary of the Invention

[0005] Based on this, the present invention proposes an aptamer with improved multidimensional performance and its application in biosensing and mild antibacterial properties.

[0006] In one aspect, the present invention provides a malachite green nucleic acid aptamer with improved performance, as shown in any one of SEQ ID NOs: 2 to 28.

[0007] On the other hand, the present invention provides the use of the above-mentioned nucleic acid aptamer sequence in the development of a malachite green detection method.

[0008] On the other hand, the present invention provides the use of the above-mentioned nucleic acid aptamer sequence in a malachite green food safety or environmental detection kit.

[0009] In one aspect, the present invention provides a label-free ratiometric malachite green biosensor comprising: (1) a malachite green biosensor sequence; (2) detection of malachite green;

[0010] The malachite green biosensor sequence is shown in SEQ ID NO: 14 or SEQ ID NO: 26.

[0011] The signal reporter molecules of the above biosensor are thioflavin T (ThT) and malachite green (MG) molecules.

[0012] The pH of the buffer solution of the above biosensor is 5.4 to 8.4.

[0013] In another aspect, the present invention provides the use of the above-mentioned biosensor in the development of a malachite green detection method.

[0014] On the other hand, the present invention provides the use of the above-mentioned biosensor in a malachite green food safety or environmental detection kit.

[0015] On the other hand, the present invention provides a use of a malachite green aptamer in mild antibacterial activity, wherein the aptamer is any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 26.

[0016] The present invention also provides an application of a malachite green aptamer in extending the effective period of an antibacterial agent, wherein the aptamer is any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 26.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 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;

[0019] 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. The sensor exhibits good linearity within the target range of 5nM to 8μM and has a low detection limit at the nanomolar level.

[0020] 3. The biosensor proposed in this invention can achieve rapid, low-cost, stable and ultra-sensitive malachite green detection, and has certain versatility and industrialization potential;

[0021] 4. The present invention applies aptamers with multi-dimensional performance improvements to the field of mild antibacterial effects, which can effectively alleviate the toxic side effects of fungicides on non-target microorganisms or host cells, and is conducive to achieving long-term control and reducing drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of stem trimming. A shows the secondary structures of different MG DNA aptamer variants during stem optimization, showing the minimum free energy and base pairing probability. Purple nucleotides indicate optimized positions; B shows the circular dichroism spectra of the optimal stem trimming sequence; C shows the fluorescence activation effects of different MG DNA aptamer variants after stem trimming. The dashed line indicates the fluorescence intensity of the original sequence (set to 1× fluorescence); D to G show the three-dimensional structures of the CAG-T-ML-S4, CAG-T-ML-S4-TA, CAG-T-ML-S4-TA-AT, and CAG-T-ML-S4-TA-AT+1 complexes with MG, respectively, and a 90° rotation (top); the magnified box in the middle highlights the MG binding pocket; bottom shows a two-dimensional schematic of the molecular docking of the MG binding site.

[0023] Figure 2 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.

[0024] Figure 3 The secondary structures of different MG DNA aptamer variants during the bottom stem sequence optimization process are shown, along with the minimum free energy and base pairing probability of each structure. Purple bases indicate optimized positions.

[0025] Figure 4 The secondary structures of different MG DNA aptamer variants during the bottom stem length optimization process are shown, along with the minimum free energy and base pairing probability for each structure. Purple bases indicate optimized positions.

[0026] Figure 5Schematic diagram of loop tandem tailoring. A shows the fluorescence activation effects of different MG DNA aptamer variants during loop tandem optimization. The dashed line represents the fluorescence intensity of the original sequence (set to 1× fluorescence). The inset shows the corresponding agarose gel electrophoresis results for these sequences. B shows the secondary structures of different MG DNA aptamer variants after loop tandem optimization, with minimum free energy and base pairing probability. Purple nucleotides indicate optimized positions. C shows the top ten molecular docking models of the three-dimensional structure of the CAG-T-ML-S4-TA-AT+1-1L to 6L sequence in complex with MG.

[0027] Figure 6 Molecular docking results of the CAG-T-ML-S4-TA-AT+1-1L sequence with MG. A shows the three-dimensional structure of the sequence-MG complex and its 90° rotation; B shows a two-dimensional docking diagram of the MG molecule within the sequence's binding site; C shows a three-dimensional schematic of the MG binding pocket within the sequence. The enlarged box highlights the binding pocket.

[0028] Figure 7 Molecular docking results of the CAG-T-ML-S4-TA-AT+1-3L sequence with MG. A shows the three-dimensional structure of the sequence-MG complex and its 90° rotation; B shows a two-dimensional docking diagram of the MG molecule within the sequence's binding site; C shows a three-dimensional schematic of the MG binding pocket within the sequence. The enlarged box highlights the binding pocket.

[0029] Figure 8 Molecular docking results of the CAG-T-ML-S4-TA-AT+1-4L sequence with MG. A shows the three-dimensional structure of the sequence-MG complex and its 90° rotation; B shows a two-dimensional docking diagram of the MG molecule within the sequence's binding site; C shows a three-dimensional schematic of the MG binding pocket within the sequence. The enlarged box highlights the binding pocket.

[0030] Figure 9 Molecular docking results of the CAG-T-ML-S4-TA-AT+1-5L sequence with MG. A shows the three-dimensional structure of the sequence-MG complex and its 90° rotation; B shows a two-dimensional docking diagram of the MG molecule within the sequence's binding site; C shows a three-dimensional schematic of the MG binding pocket within the sequence. The enlarged box highlights the binding pocket.

[0031] Figure 10 Molecular docking results of the CAG-T-ML-S4-TA-AT+1-6L sequence with MG. A shows the three-dimensional structure of the sequence-MG complex and its 90° rotation; B shows a two-dimensional docking diagram of the MG molecule within the sequence's binding site; C shows a three-dimensional schematic of the MG binding pocket within the sequence. The enlarged box highlights the binding pocket.

[0032] Figure 11 Evaluation of the structural properties of the tandem loop sequences. A–F are Job's curves for the CAG-T-ML-S4-TA-AT+1-1L–6L sequence. The dashed line represents the intersection of the two fitted lines, with the abscissa value of the intersection noted in the figure. G–K are the three-dimensional structures of the MG binding pocket in the CAG-T-ML-S4-TA-AT+1-1L, -3L, -4L, -5L, and -6L sequences (left), and the corresponding two-dimensional molecular docking diagrams of the MG binding site (right). L–Q are the circular dichroism spectra of the CAG-T-ML-S4-TA-AT+1-1L–6L sequence.

[0033] Figure 12 To verify the affinity of the aptamer using the MST method. A is the CAG-T-ML-S4-TA-AT+1 sequence; B is the CAG-T-ML-S4-TA-AT+1-4L sequence.

[0034] Figure 13 Aptamer affinity verification using the colloidal gold method. A is the CAG-T-ML-S4-TA-AT+1 sequence; B is the CAG-T-ML-S4-TA-AT+1-4L sequence.

[0035] Figure 14 Biosensing results. A: Circular dichroism spectra of the CAG-T-ML-S4-TA-AT+1-4L sequence after interaction with MG or ThT; B: Fluorescence detection results of the optimized cation species in the biosensor; C: Circular dichroism spectra of the CAG-T-ML-S4-TA-AT+1-4L sequence under different cation conditions; D: Fluorescence detection results of the optimized pH conditions in the biosensor; E: Detection waveform of the CAG-T-ML-S4-TA-AT+1-4L sequence biosensor; F: The linear relationship between the CAG-T-ML-S4-TA-AT+1 and CAG-T-ML-S4-TA-AT+1-4L sequence biosensors.

[0036] Figure 15 The detection specificity of the MG ratiometric sensor.

[0037] Figure 16 This is the detection waveform of the CAG-T-ML-S4-TA-AT+1 sequence biosensor.

[0038] Figure 17Evaluation of the sustained-release and long-lasting bactericidal effects of aptamers on MG at the microbial level. A: Photograph of the inhibition zone of Staphylococcus aureus on a culture plate after treatment with MG and different nucleic acid sequences; B: Quantitative analysis of the inhibition zone diameter; C: Quantitative results of reactive oxygen species (ROS) fluorescence levels under different treatment conditions; D: Quantitative results of the proportion of microbial apoptosis under different treatment conditions; E–H: Scanning electron microscopy (SEM) images of Staphylococcus aureus under different treatment conditions. DETAILED DESCRIPTION

[0039] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0040] Example 1. Aptamer stem cutting

[0041] All nucleotide sequences involved in the experiment are shown in Table 1.

[0042] Table 1 Nucleotide sequences involved in the experiment

[0043]

[0044]

[0045] Using MG DNA aptamer as the initial aptamer (Original sequence), the stem sequence types were replaced and optimized. The original base of the stem of the CAG-T-ML-S4 sequence was an AT complementary base pair, which was replaced with TA, GC, CG, AT interspersed, CG interspersed ( Figure 1 A. Figure 2 The results showed that the fluorescence excitation of MG was significantly enhanced after all the top stems were replaced with TA base pairs ( Figure 1 C). In addition, after the mutation to GC base pair, the fluorescence excitation of the sequence to MG was significantly reduced. This indicates that the top stem not only provides secondary structural support, but its sequence itself participates in the formation of the MG-specific binding pocket. In addition, molecular docking results show that after the top stem is replaced with TA base pair, the MG binding pocket moves to the top, and the TA stem participates in the interaction with MG ( Figure 1 E). This indicates that the CAG-T-ML-S4-TA sequence can interact with MG in a more affinity and specific manner, and its higher-order structural stability is also better than that of CAG-T-ML-S4 ( Figure 1 A).

[0046] The bottom stem sequence of the aptamer was further optimized. There are five aptamer variants, namely the original bottom stem (CAG-T-ML-S4-TA), AT, TA, GC, CG bottom stem ( Figure 1 A. Figure 3 The results showed that the CAG-T-ML-S4-TA-AT sequence had the best fluorescence excitation effect on MG, but other sequences also had relatively superior MG fluorescence excitation capabilities ( Figure 1 C). Probably because the bottom stem is far away from the binding pocket of MG, the type of bottom stem sequence does not significantly affect the binding of MG. The bottom stem is likely to only provide a function to stabilize the higher-order conformation and topological structure of the aptamer. Molecular docking results show that the AT base pair at the bottom of the CAG-T-ML-S4-TA-AT sequence increases its interactive bases with MG, and the interactive bases and interaction pockets are mainly concentrated in the top ring, among which A24 interacts with MG through hydrophobicity, G7 and T9 through hydrogen bonds, and T8, T10, A21 to A23, and A25 through van der Waals forces. The bottom stem does not interact with the MG molecule, which also explains why the type of bottom stem sequence has little effect on the fluorescence excitation of MG ( Figure 1 F).

[0047] The experiment continued to optimize the number of base pairs in the bottom stem, and 1 to 5 pairs of AT complementary base pairs were added or deleted on the basis of the CAG-T-ML-S4-TA-AT sequence, respectively, to obtain the CAG-T-ML-S4-TA-AT+1~5 and CAG-T-ML-S4-TA-AT-1~5 sequences ( Figure 1 A. Figure 4 ).like Figure 1 As shown in C, the fluorescence results show that the fewer the number of complementary base pairs in the bottom stem, the weaker the fluorescence, indicating that its secondary structure may have been destroyed, so MG cannot be embedded in the fluorescent pocket and the fluorescence cannot be excited. In addition, the CAG-T-ML-S4-TA-AT+1 sequence has the best fluorescence excitation effect on MG, and adding 2 to 5 pairs of AT base pairs will result in a decreasing fluorescence trend. This may be due to the excessive complementarity, which makes the bottom stem pairing too stable, reduces the sequence flexibility, lacks conformational folding space, and causes the higher-level structure of the sequence to be unable to form space for MG to be embedded. Molecular docking also shows that the TA-AT+1 sequence retains the binding pocket and recognition function of the TA stem at the top of the middle ring ( Figure 1 G).

[0048] In terms of circular dichroism (CD) spectroscopy, the CD spectrum of the purine-pyrimidine complex poly[d(A)]·poly[d(T)] is different from that of the B-DNA helical structure. The former has an unusual B'-DNA structure, which has a positive peak at a wavelength of around 280nm, a negative peak at a wavelength of around 245nm, and a positive peak at a wavelength of around 220nm. Figure 1 As shown in Figure B, as the number of AT and TA base pairs in the sequence increases, the positive peak of the CD spectrum of the sequence at 220 nm becomes higher and higher. As the (A+T) content increases, the negative band of the CD spectrum of the sequence becomes deeper and the conformational variability increases.

[0049] In addition, the hairpin structure is an intramolecular variant of B-DNA. In addition to the double helix stem, it also contains a single-stranded loop, so the structure will appear as a shoulder peak at a wavelength of around 260nm. Compared with the CAG-T-ML-S4 sequence, the other three preferred sequences obtained by stem optimization all showed shoulder peaks of varying degrees at 260nm, indicating that the high-level topological structure of the optimized sequence has changed. In addition, the CD spectrum produced significant hairpin structural characteristics, indicating that the hairpin structure formation efficiency of different sequences is different ( Figure 1 B).

[0050] Example 2. Optimization and tailoring of the loop portion of the aptamer in series

[0051] The success of repeating the TGT-AAA loop suggested that the experiment should go further and try to tandem the loops. During the tailoring process, the top loop was replaced with the original ligand loop to obtain a single-ring sequence, CAG-T-ML-S4-TA-AT+1-1L. The CAG-T-ML-S4-TA-AT+1 sequence is equivalent to CAG-T-ML-S4-TA-AT+1-2L. Further multivalent concatenation of the TGT-AAA loops gave three, four, five, and six ring sequences, namely CAG-T-ML-S4-TA-AT+3L~6L sequences ( Figure 5 B). Figure 5 As shown in A and B, the fluorescence results show that as the number of TGT-AAA rings in series increases, the stability of the sequence becomes stronger and stronger, and the fluorescence excitation of the sequence pair and MG gradually increases. When connected in series to four rings, the fluorescence of the CAG-T-ML-S4-TA-AT+1-4L sequence basically reaches a plateau. This proves that the TGT-AAA ring part formed based on the GA mismatch and the TA complementary pair can indeed specifically enhance the excitation of MG, and this effect can be achieved by the series connection of functional units. Of course, the TA complementary stem around the TGT-AAA ring part cannot be ignored. In addition to stabilizing the secondary structure of the aptamer, it also constitutes the binding pocket of MG and provides an embedding site for the dye. In addition, as Figure 1 G. Figure 5C. Figures 6-10 and Figure 11 Molecular docking results (G–K) show that MG preferentially binds to the top of the loop closest to the 5' and 3' ends. Furthermore, the TA stem between each of the two loops represents a potential MG binding pocket. The tandem TGT-AAA loops provide more binding sites for MG, potentially altering the stoichiometric ratio of the aptamer-target interaction and leading to higher fluorescence quantum yields.

[0052] The circular dichroism results show that ( Figure 11 As the number of tandem loops increases, the sequence maintains positive peaks at 220 and 280 nm and a negative peak at 245 nm, while the shoulder peak at 260 nm becomes increasingly pronounced. These results suggest that the tandem loop sequence exhibits a typical hybrid structure of hairpin and B-DNA helix.

[0053] Example 3. Identification of the binding properties of aptamers

[0054] 1. Stoichiometric ratio assessment

[0055] Table 2 Stoichiometric ratios of MG DNA aptamer variants binding to MG

[0056]

[0057] The experiment further tested the molar ratio of MG to the six sequences using the Job's plot method. The results showed that the fluorescence peaks in the fitting plots of the six sequences of CAG-T-ML-S4-TA-AT+1-1L~6L appeared at 0.315, 0.257, 0.215, 0.208, 0.205 and 0.117 ( Figure 11 A to F). As the number of tandem intermediate rings increases, the x-value corresponding to the intersection of the two fitted lines gradually decreases, indicating an increasingly higher stoichiometric binding ratio (Table 2). From the CAG-T-ML-S4-TA-AT+1-1L sequence to the -5L sequence, each sequence can bind to approximately 2 to 4 MG molecules, while a six-ring sequence can bind to 7.636 MG molecules. This significantly improved stoichiometric binding ratio also explains why a greater number of tandem rings leads to stronger fluorescence.

[0058] 2. Affinity Assessment

[0059] The affinity of aptamers is one of their most important properties. We then used various methods to monitor the affinity of the CAG-T-ML-S4-TA-AT+1 and CAG-T-ML-S4-TA-AT+1-4L sequences. According to literature reports, the affinity Kd value of the original sequence is 2.43μM. Figure 12As shown, the sequence affinity of CAG-T-ML-S4-TA-AT+1 and CAG-T-ML-S4-TA-AT+1-4L gradually increased, showing a gradually decreasing K d The colloidal gold results also showed the same trend ( Figure 13 ). Both affinity determination methods show that the affinity of the tailored sequence is much better than that of the original sequence.

[0060] Example 4. Multidimensional performance-enhanced aptamers for malachite green biosensing

[0061] The potential of tailored sequences for detection was further explored. A label-free ratiometric aptamer sensor was constructed using the principle of competitive equilibrium. Specifically, the aptamer binds to the MG molecule and induces MG fluorescence emission. Adding a competitive fluorescent small molecule, ThT, to the aptamer-MG complex system competes with MG for the same nucleic acid binding site, resulting in a change in the system's fluorescence intensity, enabling the sensing of malachite green.

[0062] As a dye molecule, ThT can be embedded in higher-order conformations such as G-quadruplexes, double chains, and hairpin structures, thereby achieving enhanced fluorescence excitation. In the MG sensor, two dyes, MG and ThT, were used to construct a ratiometric, label-free, ultra-sensitive, and ultra-fast biosensor. Since the CAG-T-ML-S4-TA-AT+1-4L sequence has a relatively high fluorescence excitation effect, this sequence was selected for further verification and condition optimization. The circular dichroism results showed that after the addition of MG and ThT, the CD spectra of the CAG-T-ML-S4-TA-AT+1-4L sequence showed varying degrees of shifts, and obvious shoulder peaks appeared, indicating that both small molecules can interact with the aptamer ( Figure 14 A).

[0063] Furthermore, in order to obtain the best sensing performance, the experimental conditions were systematically optimized. The sensor showed different intensities of fluorescence signals under different ionic conditions ( Figure 14 B). This is because ionic conditions can have a significant impact on the conformation of nucleic acids. CD spectra show that when no ions are added or when potassium, sodium, and magnesium ions are present in the system, nucleic acids can still maintain their higher-order structure without being destroyed. However, when lead ions are present in the system, the higher-order conformation of nucleic acids completely collapses, which also explains the sudden drop in the fluorescence intensity of the dye molecules in this system ( Figure 14B and C). However, under different ion conditions, the ratio of the fluorescence peaks of MG and ThT does not change much, which also highlights the unique advantages of the ratiometric sensor and shows the sensor's ability to resist ion interference. Comprehensively considering the fluorescence intensity and the ratio of the fluorescence peaks of the two dye molecules, the optimal condition for detection is to not introduce ions. In addition, the ratiometric sensor also has excellent anti-interference ability for pH, and can have a relatively stable signal value in the pH range of 5.4 to 8.4. Finally, the mild condition of pH 7.4 was selected as the pH of the sensing process ( Figure 14 F). After evaluation, the ratiometric sensor constructed using CAG-T-ML-S4-TA-AT+1-4L was able to show good linearity in a wide range of 5nM to 8μM, and achieved a low detection limit of 1.12nM ( Figure 14 E and F). Moreover, the constructed ratiometric sensor has excellent specificity for crystal violet (CV), rhodamine B (RB), Coomassie brilliant blue (CBB) and leucomalachite green (LMG) fuels ( Figure 15 The ratiometric sensor constructed using the CAG-T-ML-S4-TA-AT+1 sequence also demonstrated good MG sensing potential, and the detection performance of different sequences can meet the application requirements ( Figure 14 F. Figure 16 , Table 3).

[0064] Table 3 Biosensing performance of MG with different sequences

[0065]

[0066] In spike recovery tests with real lake water and fish samples, the proposed ratiometric biosensor showed good recoveries, demonstrating its applicability in real sample analysis and its adaptability to common sample matrices and potential interferences (Table 4). These results demonstrate the application potential of different aptamer variants and the versatility of the applied biosensing strategy.

[0067] Table 4 Real sample recovery test results

[0068]

[0069] Example 5. Application of aptamers with multi-dimensional performance improvement in mild antibacterial

[0070] "Mild inhibition" is a different approach from traditional, aggressive bactericidal strategies. It emphasizes minimizing damage to the host environment, microbial ecosystem, or non-target microorganisms while inhibiting microbial overgrowth or infection. This strategy typically does not aim to completely kill target microorganisms within a short period of time. Instead, it achieves a long-lasting, sustained-release, minimally damaging, and low-resistance-inducing antibacterial mechanism through the controlled release of inhibitory factors, reducing the instantaneous peak concentration of the inhibitor, or masking its activity.

[0071] Mild bacteriostasis is of great significance. Slowing down the development of drug resistance: Intense and high-dose bactericidal pressure is one of the main inducements for bacteria to develop drug resistance. Mild bacteriostasis significantly reduces the risk of stress mutation in microorganisms by reducing instantaneous drug pressure, thereby reducing the generation of drug-resistant strains at the source. Maintaining microecological balance: Mild bacteriostasis retains the activity of beneficial or neutral microorganisms in the environment while not completely eliminating the target bacterial flora, which helps to maintain the functional integrity of complex microecological systems such as the intestine and skin. Reducing host toxicity and side effects: High concentrations of antibacterial agents may cause side effects such as oxidative stress and membrane damage to host cells (such as skin cells and intestinal epithelial cells), while mild bacteriostasis helps reduce non-target damage to host cells, and is especially suitable for use in long-term contact or chronic disease states. More suitable for the management of chronic, low-grade infections: For certain non-acute infection states (such as wound treatment, skin inflammation, oral plaque control, medical device surface management, etc.), mild bacteriostasis can provide a physiologically friendly management method.

[0072] The "mild antibacterial" effect has many potential application scenarios. Medical dressings or implant materials: controlled release of antibacterial agents through aptamers to prevent infection in the surgical area or implant surface, while avoiding cytotoxicity or tissue irritation. Daily care products: In fields such as skin care products and oral care, mild antibacterial strategies can be used to control bacterial growth without destroying the normal microbial barrier. Chronic wound treatment: Especially suitable for chronic wounds such as diabetic foot and bedsores, while achieving antibacterial effect and avoiding irritation or damage to new tissue. Biological regulation in microbial culture / synthetic biology platforms: controlled inhibition of contaminating bacteria or regulation of population density, which can be used for high-precision synthetic biology regulation and microbial community design. Agriculture and food safety fields: It can be used on the surface of fruits and vegetables, packaging materials or hydroponic systems to provide low-residue, environmentally friendly antibacterial protection.

[0073] Based on this background, the experiment further expanded the potential of aptamers with multidimensional performance enhancement to the field of microbial applications, attempting to utilize the binding ability of aptamers to MG to achieve a sustained and slow-release antibacterial effect. The experiment selected MC-DA, MC-DA-LG, CAG-T-ML-S4, and CAG-T-ML-S4-TA-AT+1-4L as the intervention sequence.

[0074] MG is an effective bacteriostatic agent that can significantly inhibit Gram-positive and Gram-negative bacteria. Figure 17As shown in Figure A, adding MG solution to the small well in the center of a culture plate inoculated with Staphylococcus aureus produced a significant zone of inhibition on the first day, demonstrating that MG can indeed significantly inhibit the growth of S. aureus. Adding the aptamer sequence significantly reduced the zone of inhibition compared to that of the MG alone group. A smaller zone of inhibition indicates a reduced bactericidal effect. Furthermore, on the first day, as the affinity of the aptamer sequence to MG increased, the zone of inhibition gradually decreased ( Figure 17 A and B). This suggests that the tighter binding of the aptamer to MG can mask the bactericidal effects of MG. Across all groups, the zone of inhibition gradually decreased from day one to day five, but the extent of the decrease on day five was less pronounced compared to day four. On day five, the zone of inhibition in the aptamer group was smaller than that in the MG group, indicating that the aptamer can protect microorganisms from the potent bactericide over several days, resulting in a sustained-release, long-lasting bactericidal process.

[0075] Aptamers bind to MG to form a complex that masks the damaging effects of MG on microorganisms. As the aptamer gradually degrades within the system, more and more MG molecules are exposed, leading to a sustained release and long-lasting bactericidal effect. This effect can be applied in a variety of scenarios, potentially achieving a milder, longer-lasting bactericidal effect even with the same amount of bactericide.

[0076] Furthermore, the effects of MG aptamer sequences on microbial redox stress and survival rate were investigated. Figure 17 As shown in Figure C, the addition of the antibacterial agent MG significantly increased microbial ROS levels, reaching 2.68 times that of the control group. The addition of different aptamer sequences significantly reduced ROS levels. With further aptamer optimization, the sequence affinity increased and ROS levels decreased. The ROS levels of the microorganisms in the MG@CAG-T-ML-S4-TA-AT+1-4L sequence group were not significantly different from those in the control group, indicating that stronger aptamer affinity is more effective in alleviating and reducing MG-mediated oxidative stress.

[0077] A similar trend was also found when evaluating the apoptosis rate of Staphylococcus aureus. The addition of aptamer sequences can alleviate the MG-mediated microbial apoptosis in the short term. The apoptosis rates of the microorganisms in the MG@ML-S4 and MG@CAG-T-ML-S4-TA-AT+1-4L sequence treatment groups were not significantly different from those in the control group ( Figure 17 D) This further illustrates the role of aptamer sequences in the sustained release and long-lasting bactericidal effect of antibacterial agents.

[0078] In order to systematically evaluate the effect of aptamers on the bactericidal effect mediated by antibacterial agents, we further observed the morphology of Staphylococcus aureus in different treatment groups using SEM. Figure 17 As shown in Figure E, the morphology of Staphylococcus aureus in the control group showed a regular spherical appearance, with uniform individual morphology, smooth surface and clear outline, complete structure, and no damage, collapse or abnormal protrusions, indicating that the microbial structure maintained good integrity and showed healthy morphological characteristics. After the addition of MG, the morphology of the cocci changed significantly, no longer showing a regular spherical shape, and some cells collapsed, deformed, and even showed irregular cystic structures. The morphological differences between individuals were large, and the uniformity of the group was destroyed. In addition, the surface of the cocci was rough, and there may be damage to the membrane structure, which made it impossible for the cell morphology to maintain normal. Its basic structure has been completely destroyed, presenting an unhealthy physiological state ( Figure 17 F and G). After adding the aptamer, the perforation morphology of the microorganism was greatly alleviated, showing a morphological structure similar to that of the control group ( Figure 17 H). This experiment demonstrated from a morphological perspective that the aptamer has a certain protective effect on microorganisms treated with antibacterial agents.

[0079] In summary, the present invention tested the multi-dimensionally enhanced aptamer sequence on microbial cultures and found that it effectively binds to MG molecules, thereby gradually and controllably releasing MG over time. This sustained, long-lasting, and sustained-release mechanism enables the aptamer to exert a lasting antibacterial effect, providing a new approach to mitigate microbial infections while minimizing the risk of drug resistance.

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 28.

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 nucleic acid aptamer sequence according to claim 1 in 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) detection of malachite green; The malachite green biosensor sequence is shown in SEQ ID NO: 14 or SEQ ID NO:

26.

5. The biosensor according to claim 4, wherein The signal reporting molecules of the biosensor are thioflavin T and malachite green molecules.

6. The biosensor according to claim 4, wherein The pH value of the buffer solution of the biosensor is 5.4-8.

4.

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

8. Use of the biosensor according to any one of claims 4 to 6 in a malachite green food safety or environmental detection kit.

9. A use of a malachite green aptamer in mild antibacterial activity, characterized in that: The aptamer is any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:

26.

10. A use of a malachite green aptamer in extending the effective period of an antibacterial agent, characterized in that: The aptamer is any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 26.

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