Multidimensional performance-enhanced aptamer and application thereof in biosensing and mild bacteriostasis

Abstract

The application discloses a multi-dimensional performance-improved aptamer, and applies the aptamer to a malachite green label-free proportional biosensor and long-acting, mild and slow-release bacteriostatic regulation. Specifically, two kinds of malachite green aptamer biosensors with superior detection performance are constructed through a competition balance principle, and the two kinds of biosensors have a wide detection range and a low detection limit. In addition, the aptamer can slow release malachite green in the bacteriostatic process, and help to realize long-acting and mild bacteriostatic process. Application of the multi-dimensional performance-improved aptamer to the slow-release bacteriostatic field can effectively alleviate the toxic side effects of bactericides on non-target microorganisms or host cells, and is beneficial to realize long-acting control and reduce drug resistance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to an aptamer with improved multi-dimensional performance and application thereof in biosensing and mild bacteriostasis. BACKGROUND

[0002] Malachite Green (MG) is a triphenylmethane compound, which has been widely used in aquaculture for antibacterial and antifungal treatment and has a significant bactericidal effect. However, studies have found that malachite green has potential mutagenicity, carcinogenicity and accumulation toxicity, and therefore has been banned in food-related fields in many countries and regions. Despite this, due to its low cost and convenient use, malachite green is still illegally used in some areas, which seriously threatens public health and food safety. Therefore, it is of great significance to develop a high-sensitivity, high-specificity, convenient and efficient detection method for monitoring malachite green residues.

[0003] As an artificially screened single-stranded DNA or RNA molecule, the nucleic acid aptamer has the ability to bind to target molecules with high affinity and high specificity, and is widely used in small molecule detection, biological imaging and targeted drug delivery. The aptamer for malachite green is one of the current research hotspots. However, the existing aptamer still has deficiencies in affinity, sequence stability and signal response strength, which limits its application effect in the detection of actual complex samples. Therefore, it is necessary to optimize the structure, screening strategy and signal coupling mechanism of the aptamer in multiple dimensions to realize efficient recognition and signal amplification of malachite green, and meet the needs of rapid, accurate and on-site detection.

[0004] On the other hand, although malachite green has a rapid and efficient bacteriostatic effect in bactericidal application, its release is relatively violent and lacks controlled release, which can easily cause toxic side effects to non-target microorganisms or host cells, and is not conducive to long-term control. In addition, the unstable release of the bactericidal mechanism may exacerbate the occurrence of drug resistance. Therefore, the construction of a functional module capable of specific binding to malachite green and regulating its release behavior, such as an aptamer-nano-carrier composite system, not only helps to prolong the bactericidal effect time and reduce toxicity, but also provides technical support for the development of a mild and controllable bactericidal system. SUMMARY

[0005] Based on this, the application provides an aptamer with improved multi-dimensional performance and application thereof in biosensing and mild bacteriostasis.

[0006] In one aspect, the application provides a malachite green nucleic acid aptamer with improved performance, as shown in any one of SEQ ID NO: 2-28.

[0007] In another aspect, the present application provides application of the nucleic acid aptamer sequence in development of a detection method for malachite green.

[0008] In another aspect, the present application provides application of the nucleic acid aptamer sequence in a detection kit for malachite green in food safety or environment.

[0009] In one aspect, the present application provides a label-free proportional malachite green biosensor, comprising: (1) a malachite green biosensor sequence; and (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 molecule of the biosensor is thioflavin T (ThT) and malachite green (MG) molecule.

[0012] The buffer pH of the biosensor is 5.4-8.4.

[0013] In another aspect, the present application provides application of the biosensor in development of a detection method for malachite green.

[0014] In another aspect, the present application provides application of the biosensor in a detection kit for malachite green in food safety or environment.

[0015] In another aspect, the present application provides application of a malachite green aptamer in mild bacteriostasis, 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 application also provides application of a malachite green aptamer in prolonging the effective period of a bacteriostatic agent, wherein the aptamer is any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 26.

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

[0018] 1. The present application obtains a malachite green DNA nucleic acid aptamer with improved fluorescence performance, stability, affinity and binding capacity through multi-dimensional system tailoring.

[0019] 2. The present application constructs a label-free, proportional, sensitive and low-cost biosensor based on the optimized nucleic acid aptamer and competition balance effect, and the sensor can present a good linear relationship in the range of 5nM-8μM target, and has a low detection limit of nanomolar level.

[0020] 3. The biosensor can realize rapid, low-cost, stable and super-sensitive malachite green detection, and has certain universality and industrialization potential.

[0021] 4. The aptamer with multi-dimensional performance improvement is applied to the mild bacteriostatic field, can effectively alleviate the toxic side effects of bactericides on non-target microorganisms or host cells, and is beneficial to realize long-acting control and reduce drug resistance. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 For stem cutting schematic diagram. A is the secondary structure of different MG DNA aptamer variants in the stem optimization process, which shows the minimum free energy and base pairing probability. The purple nucleotide indicates the optimized position; B is the circular dichroism spectrum of the stem trimming preferred sequence; C is the fluorescence activation effect of different MG DNA aptamer variants after stem trimming. The dotted line indicates the fluorescence intensity of the original sequence (set as 1x fluorescence); D-G are the three-dimensional structures of CAG-T-ML-S4, CAG-T-ML-S4-TA, CAG-T-ML-S4-TA-AT and CAG-T-ML-S4-TA-AT+1 and MG complex and the view after rotating 90° (upper); The middle enlarged frame highlights the MG binding pocket; The bottom is the two-dimensional schematic diagram of molecular docking of the MG binding site.

[0023] Figure 2 For the secondary structure of different MG DNA aptamer variants in the top stem optimization process, the minimum free energy and base pairing probability of each structure are attached. The purple base indicates the optimized position.

[0024] Figure 3 For the secondary structure of different MG DNA aptamer variants in the bottom stem sequence optimization process, the minimum free energy and base pairing probability of each structure are attached. The purple base indicates the optimized position.

[0025] Figure 4 For the secondary structure of different MG DNA aptamer variants in the bottom stem length optimization process, the minimum free energy and base pairing probability of each structure are attached. The purple base indicates the optimized position.

[0026] Figure 5Figure 6. Schematic diagram of loop section tandem cutting. A is the fluorescence activation effect of different MG DNA aptamer variants in the process of loop section tandem optimization. The dotted line represents the fluorescence intensity of the original sequence (set as 1 x fluorescence). The inset shows the agarose gel electrophoresis results of these sequences; B is the secondary structure of different MG DNA aptamer variants after loop section tandem optimization, with the minimum free energy and base pairing probability. The purple nucleotides represent the optimized positions; C is the top ten molecular docking models of the CAG-T-ML-S4-TA-AT+1-1L sequence and the three-dimensional structure of the MG complex.

[0027] Figure 6 Figure 7. Molecular docking results of the CAG-T-ML-S4-TA-AT+1-1L sequence and MG. A is the three-dimensional structure of the sequence and the MG complex and its display after rotating 90°; B is the two-dimensional schematic diagram of molecular docking of the MG molecule in the sequence binding site; C is the three-dimensional schematic diagram of the MG binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0028] Figure 7 Figure 8. Molecular docking results of the CAG-T-ML-S4-TA-AT+1-3L sequence and MG. A is the three-dimensional structure of the sequence and the MG complex and its display after rotating 90°; B is the two-dimensional schematic diagram of molecular docking of the MG molecule in the sequence binding site; C is the three-dimensional schematic diagram of the MG binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0029] Figure 8 Figure 9. Molecular docking results of the CAG-T-ML-S4-TA-AT+1-4L sequence and MG. A is the three-dimensional structure of the sequence and the MG complex and its display after rotating 90°; B is the two-dimensional schematic diagram of molecular docking of the MG molecule in the sequence binding site; C is the three-dimensional schematic diagram of the MG binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0030] Figure 9 Figure 10. Molecular docking results of the CAG-T-ML-S4-TA-AT+1-5L sequence and MG. A is the three-dimensional structure of the sequence and the MG complex and its display after rotating 90°; B is the two-dimensional schematic diagram of molecular docking of the MG molecule in the sequence binding site; C is the three-dimensional schematic diagram of the MG binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0031] Figure 10 Figure 11. Molecular docking results of the CAG-T-ML-S4-TA-AT+1-6L sequence and MG. A is the three-dimensional structure of the sequence and the MG complex and its display after rotating 90°; B is the two-dimensional schematic diagram of molecular docking of the MG molecule in the sequence binding site; C is the three-dimensional schematic diagram of the MG binding pocket in the sequence. The enlarged box highlights the binding pocket.

[0032] Figure 11 Evaluation of the loop-tandem sequence structure. A~F are Job's curves of CAG-T-ML-S4-TA-AT+1-1L~6L sequences, respectively. The dotted line indicates the intersection of the two fitted lines, and the horizontal coordinate value of the intersection is labeled in the figure; G~K are the three-dimensional structures of the MG binding pocket (left) and the two-dimensional molecular docking diagram of the corresponding MG binding site (right) in CAG-T-ML-S4-TA-AT+1-1L, -3L, -4L, -5L and -6L sequences, respectively; L~Q are circular dichroism spectra of CAG-T-ML-S4-TA-AT+1-1L~6L sequences.

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

[0034] Figure 13 Verification of the affinity of the aptamer by colloidal gold method. A is CAG-T-ML-S4-TA-AT+1 sequence; B is CAG-T-ML-S4-TA-AT+1-4L sequence.

[0035] Figure 14 Biosensor results. A is the circular dichroism spectra of CAG-T-ML-S4-TA-AT+1-4L sequence after interaction with MG or ThT; B is the fluorescence detection results of cation species optimization in the biosensor; C is the circular dichroism spectra of CAG-T-ML-S4-TA-AT+1-4L sequence under different cation conditions; D is the fluorescence detection results of pH condition optimization in the biosensor; E is the detection waveform diagram of CAG-T-ML-S4-TA-AT+1-4L sequence biosensor; F is the linear relationship of CAG-T-ML-S4-TA-AT+1 and CAG-T-ML-S4-TA-AT+1-4L sequence biosensor.

[0036] Figure 15 Detection specificity of the MG ratio-type sensor.

[0037] Figure 16 Detection waveform diagram of CAG-T-ML-S4-TA-AT+1 sequence biosensor.

[0038] Figure 17To evaluate the bactericidal effect of aptamer on MG at the microbial level. A is the picture of the inhibition zone of S. aureus on the culture plate after treatment with MG and different nucleic acid sequences; B is the quantitative analysis of the diameter of the inhibition zone; C is the quantitative result of the fluorescence level of reactive oxygen species (ROS) under different treatment conditions; D is the quantitative result of the proportion of microbial apoptosis under different treatment conditions; E-H are scanning electron microscope (SEM) images of S. aureus under different treatment conditions. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to 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.

[0040] Example 1. Aptamer stem trimming

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

[0042] Table 1 Nucleotide sequences involved in the experiment

[0043]

[0044]

[0045] The stem sequence of the MG DNA aptamer was replaced and optimized as the initial aptamer (Original sequence). The original base of the stem of the CAG-T-ML-S4 sequence was A-T complementary base pair, which was replaced by T-A, G-C, C-G, A-T intercalation, C-G intercalation ( Figure 1 A, Figure 2 ) respectively. The results showed that after replacing all the top stems with T-A base pairs, the fluorescence excitation of MG was significantly enhanced ( Figure 1 C). In addition, after mutation to G-C base pairs, the fluorescence excitation of the sequence to MG was significantly reduced. This indicates that the top stem not only provides the role of secondary structure support, but also participates in the formation of the MG specific binding pocket itself. In addition, the molecular docking results show that after replacing the top stem with T-A base pairs, the binding pocket of MG moves to the top end, and T-A stem participates in the interaction of MG ( Figure 1 E). This indicates that the CAG-T-ML-S4-TA sequence can interact with MG in a more specific and specific manner, and the stability of its higher structure 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 were five aptamer variants, i.e. original bottom stem (CAG-T-ML-S4-TA), A-T, T-A, G-C, C-G bottom stem. Figure 1 A, Figure 3 ). It was found that the CAG-T-ML-S4-TA-AT sequence had the best fluorescence excitation effect on MG, but other sequences also had relatively superior fluorescence excitation ability of MG ( Figure 1 C). It is possible that the bottom stem sequence does not significantly affect the binding of MG due to its distance from the binding pocket of MG. The bottom stem is likely to only provide a role of stabilizing the high-level conformation and topology of the aptamer. The molecular docking results show that the A-T base pair at the bottom of the CAG-T-ML-S4-TA-AT sequence increases the interaction bases with MG, and the interaction bases and interaction pockets are mainly concentrated in the top ring, in which A24 interacts with MG by hydrophobic force, G7 and T9 by hydrogen bond, and T8, T10, A21-A23 and A25 by van der Waals force. The bottom stem does not interact with the MG molecule, which also explains why the type of bottom stem sequence has less effect on the fluorescence excitation of MG ( Figure 1 F).

[0047] The number of base pairs of the bottom stem was further optimized. Based on the CAG-T-ML-S4-TA-AT sequence, 1-5 complementary base pairs of A-T were added or deleted to obtain CAG-T-ML-S4-TA-AT+1-5 and CAG-T-ML-S4-TA-AT-1-5 sequences ( Figure 1 A, Figure 4 ). As shown in Figure 1 C, the fluorescence results show that the fewer the number of complementary base pairs of the bottom stem, the weaker the fluorescence, indicating that its secondary structure has been destroyed, so MG cannot be embedded in the fluorescence pocket, and fluorescence cannot be excited. In addition, the CAG-T-ML-S4-TA-AT+1 sequence has the best fluorescence excitation effect on MG, and increasing 2-5 pairs of A-T base pairs has a decreasing trend of fluorescence. This is likely due to the complementary being too long, causing the bottom stem to pair too stably, resulting in a decrease in sequence flexibility, lack of conformation folding space, and resulting in the high-level structure of the sequence being 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 of the top T-A stem 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)] differs from that of the B-DNA helical structure. The former exhibits an unusual B'-DNA structure, showing a positive peak around 280 nm, a negative peak around 245 nm, and a positive peak around 220 nm. For example... Figure 1 As shown in Figure B, with the increase of AT and TA base pairs in the sequence, the positive peak at 220 nm in the CD spectrum of the sequence becomes higher and higher. With the increase of (A+T) content, the negative band of the CD spectrum of the sequence becomes deeper, and the conformational variability increases.

[0049] Furthermore, the hairpin structure is an intramolecular variant of B-DNA. In addition to the double helix stem, it also contains a single-stranded loop, thus exhibiting a shoulder peak at approximately 260 nm. Compared to the CAG-T-ML-S4 sequence, the other three optimized sequences obtained through stem optimization all showed varying degrees of shoulder peaks at 260 nm, indicating a change in the higher-order topology of the optimized sequences. Moreover, CD spectroscopy produced significant hairpin structure features, suggesting different hairpin structure formation efficiencies among different sequences. Figure 1 B).

[0050] Example 2. Optimized trimming of the ring-shaped part of the aptamer

[0051] The successful replication of the TGT-AAA loop suggests that experiments should further explore tandem loops. During the trimming process, the top loop was replaced with the original ligand loop, yielding a single-loop 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 tandem multivalent TGT-AAA loops yielded trivalent, tetravalent, pentavalent, and hexavalent sequences, namely the CAG-T-ML-S4-TA-AT+3L~6L sequences. Figure 5 B). For example Figure 5 As shown in A and B, the fluorescence results indicate that the sequence stability increases with the increase of the number of tandem TGT-AAA rings, and the fluorescence excitation of the sequence pair with MG gradually strengthens. When tandemly connected to four rings, the fluorescence of the CAG-T-ML-S4-TA-AT+1-4L sequence essentially reaches a plateau. This demonstrates that the TGT-AAA ring formed based on GA mismatch and TA complementarity can indeed specifically enhance MG excitation, and this effect can be superimposed through the tandem connection of functional units. Of course, the TA complementarity stem around the TGT-AAA ring cannot be ignored; besides stabilizing the secondary structure of the aptamer, it also forms the binding pocket of MG, providing dye insertion sites. Furthermore, as... Figure 1 G, Figure 5C、 Figures 6-10 and Figure 11 The docking results of the molecules shown in G~K showed that MG preferentially binds to the top of the loop closest to the 5' and 3' end loop. Moreover, the TA stem between every two loops is actually a potential MG binding pocket. The tandem TGT-AAA loops provide more binding sites for MG, leading to a change in the stoichiometric ratio of the aptamer and target interaction, thus leading to an increasingly high fluorescence quantum yield.

[0052] The circular dichroism results show that Figure 11 L~Q), with the increase in the number of tandem loops, the sequence still maintains the positive peaks at 220 and 280 nm and the negative peak at 245 nm, and the shoulder peak at 260 nm becomes more and more obvious. The above results show that the tandem loop sequence has a typical mixed structure of hairpin and B-DNA helix.

[0053] Example 3. Identification of the binding performance of the aptamer

[0054] 1. Stoichiometric ratio evaluation

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

[0056]

[0057] The experiment further tested the binding molar ratio of MG to the six sequences by the Job's plot method. The results showed that the fluorescence peak in the fitting graph 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~F), respectively. With the increase in the number of intermediate loops in the tandem, the X value corresponding to the intersection of the two fitting straight lines gradually decreased, showing an increasingly high chemical binding stoichiometric ratio (Table 2). From the CAG-T-ML-S4-TA-AT+1-1L sequence to the -5L sequence, one sequence can bind to about 2~4 MG molecules, and one six-loop sequence can bind to 7.636 MG molecules. The significantly improved chemical binding stoichiometric ratio also explains why the fluorescence is stronger with more tandem loops in the loop part.

[0058] 2. Affinity evaluation

[0059] The affinity of the aptamer is one of its most important properties. We immediately monitored the affinity of CAG-T-ML-S4-TA-AT+1 and CAG-T-ML-S4-TA-AT+1-4L sequences using various methods. According to the literature, the affinity Kd value of the original sequence (Original) is 2.43 μM. As 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 gradually reduced K d values. The colloidal gold results also showed the same trend Figure 13 . Both affinity determination methods showed that the affinity of the trimmed sequences was much better than that of the original sequence.

[0060] Example 4. Multi-dimensional performance-enhanced aptamer for malachite green biosensing

[0061] The potential of the trimmed sequences in detection was further explored. A label-free ratiometric aptamer sensor was constructed using the principle of competition equilibrium, i.e., the aptamer can bind to MG molecules and induce fluorescence emission of MG, and the addition of a competitive fluorescent small molecule, ThT, to the aptamer-MG complex system will cause the fluorescence intensity of the system to change, realizing the sensing and detection of malachite green.

[0062] ThT, as a dye molecule, can be embedded in G-quadruplexes, double-stranded, hairpin structures, and other higher-order conformations, thereby realizing enhanced fluorescence excitation. In the MG sensor, a ratiometric, label-free, ultra-sensitive and ultra-fast biosensor was constructed using two dyes, MG and ThT. 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. Circular dichroism results showed that the CD spectra of CAG-T-ML-S4-TA-AT+1-4L sequence appeared different degrees of shift after adding MG and ThT, respectively, and obvious shoulder peaks appeared, indicating that both small molecules can interact with the aptamer Figure 14 A).

[0063] Further, to obtain the optimal sensing performance, the experimental conditions were systematically optimized. The sensor showed different fluorescence signals under different ionic conditions Figure 14 B). This is because ionic conditions can have a large impact on nucleic acid conformation. CD spectra showed that the nucleic acid can still maintain its higher-order structure without being destroyed when no ions are added or potassium ions, sodium ions and magnesium ions are present in the system. However, when lead ions are present in the system, the higher-order conformation of the nucleic acid is completely collapsed, which explains the reason for the sharp drop in the fluorescence intensity of the dye molecule under this system Figure 14B and C). However, the ratio of the fluorescence peak values of MG and ThT changed little under different ionic conditions, which also highlighted the unique advantages of the ratiometric sensor and showed the anti-interference ability of the sensor. The fluorescence intensity and the ratio of the fluorescence peak values of the two dye molecules were combined to optimize the detection conditions without introducing ions. In addition, the ratiometric sensor also had excellent anti-interference ability for pH, and could have a relatively stable signal value in the pH range of 5.4-8.4. Finally, the mild condition of pH 7.4 was selected as the pH of the acid-base condition of the sensing process. Figure 14 F). It was evaluated that the ratiometric sensor constructed with CAG-T-ML-S4-TA-AT+1-4L could exhibit a good linear relationship in a wide range of 5 nM-8 μM, and a lower detection limit of 1.12 nM was obtained. Figure 14 E and F). In addition, the constructed ratiometric sensor also had excellent specificity for crystal violet (CV), rhodamine B (RB), coomassie brilliant blue (CBB) and leucomalachite green (LMG) fuels. Figure 15 ) The ratiometric sensor constructed with the CAG-T-ML-S4-TA-AT+1 sequence also exhibited good MG sensing potential, and the detection performance of different sequences could meet the application requirements. Figure 14 F, Figure 16 Table 3 MG biosensing performance of different sequences

[0064] Table 3 MG biosensing performance of different sequences

[0065]

[0066] In the spiked recovery test of real samples of lake water and fish meat, the proposed ratiometric biosensor showed good recovery rate, proving its applicability in real sample analysis and its adaptability to common sample matrix and potential interference (Table 4). These results showed the application potential of different aptamer variants and the universality of the applied biosensing strategy.

[0067] Table 4 Real sample recovery test results

[0068]

[0069] Example 5. Application of multi-dimensional performance improved aptamer in mild bacteriostasis

[0070] "Mild bacteriostasis" is a bacteriostasis mode different from the traditional strong bactericidal strategy, which emphasizes minimizing the damage to the host environment, microecosystem or non-target microorganisms while inhibiting the excessive growth or infection of microorganisms. This strategy usually does not aim to completely kill the target microorganisms in a short time, but achieves a long-acting, slow-releasing, micro-damaging and low-inducing resistance bacteriostasis mechanism by controlled release of bacteriostatic factors, reducing the instantaneous concentration peak of bacteriostatic agents or masking their activity.

[0071] Mild bacteriostasis has important significance. Slow down the development of drug resistance: strong 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 of microorganisms by reducing transient drug pressure, thereby reducing the generation of drug-resistant strains at the source. Maintain the balance of microecology: mild bacteriostasis retains the activity of beneficial or neutral microorganisms in the environment while not completely eliminating the target bacterial population, which helps to maintain the functional integrity of complex microecological systems such as the intestinal tract and skin. Reduce host toxicity and side effects: high-concentration bacteriostatic agents can cause side effects such as oxidative stress and membrane damage to host cells (such as skin cells and intestinal epithelial cells), and mild bacteriostasis can help reduce non-target damage to host cells, especially for long-term contact or chronic disease states. More suitable for the management of chronic and low-grade infections: For some non-acute infection states (such as wound treatment, skin inflammation, oral plaque control, and medical device surface management), mild bacteriostasis can provide a physiologically friendly management method.

[0072] The "mild bacteriostasis" effect has many potential application scenarios. Medical dressings or implant materials: by using aptamer-controlled release of bacteriostatic agents, prevent infection of surgical areas or implant surfaces, while avoiding cytotoxicity or tissue irritation. Daily care products: In areas such as skin care and oral care, mild bacteriostasis 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 bacteriostasis and avoiding irritation or damage to newly formed tissue. Biological regulation in microbial culture / synthetic biology platforms: controlled inhibition of contaminating bacteria or regulation of population density can be used for high-precision synthetic biological regulation and microbial community design. Agricultural and food safety fields: can be used in fruit and vegetable surfaces, packaging materials or hydroponic systems to provide low-residue and environmentally friendly bacteriostatic protection.

[0073] Based on this background, the experiment further expands the potential of aptamers with multi-dimensional performance improvement to the field of microbial applications, trying to use the binding ability of aptamers and MG to achieve sustained and slow-release bacteriostatic effect. The experiment selects MC-DA, MC-DA-LG, CAG-T-ML-S4, 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 negative bacteria. From the perspective of aptamer binding, the aptamer can be used to control the release of MG, thereby achieving the effect of sustained and slow-release bacteriostasis. 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 inhibition zone on the first day, indicating that MG can indeed significantly inhibit the growth of Staphylococcus aureus. The addition of the aptamer sequence resulted in a significantly smaller inhibition zone compared to the MG-only group. A smaller inhibition zone diameter indicates a certain degree of reduced bactericidal effect. Furthermore, on the first day, as the affinity between the aptamer sequence and MG increased, the diameter of the inhibition zone gradually decreased. Figure 17 (A and B). This indicates that a tighter binding of the aptamer to MG can enhance the masking of the bactericidal effect of MG. In all groups, the inhibition zone gradually decreased from day 1 to day 5, but the decrease in the inhibition zone on day 5 was less pronounced than that on day 4. On day 5, the inhibition zone in the aptamer group was smaller than that in the MG group, indicating that the aptamer can protect microorganisms from the potent damage of the bactericide over a period of several days, thus providing a slow-release and long-lasting bactericidal process.

[0075] It is evident that the aptamer forms a complex upon binding with MG, which can mask the damaging effects of MG on microorganisms. As the aptamer gradually degrades in the system, more and more naked MG molecules are exposed, thus achieving a slow-release and long-lasting bactericidal effect. This effect can be applied to various scenarios; that is, using the same amount of bactericide may help achieve a milder and longer-lasting bactericidal effect.

[0076] Furthermore, the effects of MG aptamer sequences on microbial redox stress and survival were investigated. Figure 17 As shown in Figure C, the addition of the antibacterial agent MG significantly increased the ROS level of the microorganisms, which was 2.68 times that of the control group. The addition of different aptamer sequences significantly reduced the ROS level of the microorganisms. With further aptamer optimization, the sequence affinity became stronger, and the ROS level decreased. The ROS level of the microorganisms in the MG@CAG-T-ML-S4-TA-AT+1-4L sequence group was not significantly different from that of the control group, indicating that the stronger the aptamer affinity, the more effectively it can alleviate and reduce the MG-mediated oxidative stress effect.

[0077] A similar trend was observed when assessing the apoptosis rate of Staphylococcus aureus. The addition of the aptamer sequence could alleviate MG-mediated microbial apoptosis in the short term; however, the apoptosis rates in the MG@ML-S4 and MG@CAG-T-ML-S4-TA-AT+1-4L sequence treatment groups were not significantly different from 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] 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 at the morphological level. For example...Figure 17 E, the morphology of S. aureus in the control group presents a regular spherical appearance, the individual morphology is uniform, the surface is smooth and the outline is clear, the structure is complete, there is no phenomenon such as damage, collapse or abnormal protrusion, which indicates that the structure of the microorganism maintains good integrity and presents healthy morphological characteristics. After the addition of MG, the morphology of the coccus is significantly changed, and the coccus no longer presents a regular spherical shape, part of the cells appear collapse, deformation, and even present irregular cystic structure. The morphology difference between individuals is large, and the uniformity of the group is destroyed. Moreover, the surface of the coccus is rough, and there may be damage to the membrane structure, which leads to the fact that the cell morphology cannot be maintained normally, and the basic structure has been completely destroyed, presenting an unhealthy physiological state (F and G). After the addition of aptamer, the morphology of the microorganism is greatly relieved, and presents a similar morphological structure to the control group (H). This experiment proves from the perspective of morphology that the aptamer has a certain protective effect on the microorganism to which the bacteriostatic agent is applied. Figure 17 Figure 17 H). This experiment proves from the perspective of morphology that the aptamer has a certain protective effect on the microorganism to which the bacteriostatic agent is applied.

[0079] In summary, the aptamer sequence with multi-dimensional performance improvement is tested on the microbial culture, and it is found that it can effectively combine with the MG molecule, so that the MG can be released controllably and gradually in a period of time. This sustained and long-acting sustained release mechanism enables the aptamer to exert a lasting bacteriostatic effect, providing a new method for reducing microbial infection, while minimizing the risk of drug resistance.​

Claims

1. A performance-enhanced malachite green aptamer, characterized in that, The nucleic acid aptamer sequence is shown as SEQ ID NO:

26.

2. The nucleic acid aptamer of claim 1 for use in the development of a method for detecting malachite green, which is not for the purpose of disease diagnosis and treatment.

3. The nucleic acid aptamer of claim 1 for use in the preparation of a food safety or environmental detection kit for malachite green.

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

26. The signal reporting molecule of the biosensor is sulfur yellow T and malachite green molecule.

5. The biosensor of claim 4, wherein, The buffer pH of the biosensor is 5.4-8.

4.

6. The biosensor of any one of claims 4-5 for use in the development of a method for detecting malachite green, which is not for the purpose of disease diagnosis and treatment.

7. The biosensor of any one of claims 4-5 for use in the preparation of a food safety or environmental detection kit for malachite green.

8. Use of a malachite green aptamer for extending the shelf life of a bacteriostatic agent, characterized in that, The aptamer is SEQ ID NO: 26, and the bacteriostatic agent is malachite green.

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