Label-free proportional staphylococcus aureus enterotoxin A luminous aptamer biosensor

Abstract

The invention discloses a label-free proportional type luminous aptamer biosensor for staphylococcus aureus enterotoxin A (SEA). The label-free proportional type luminous aptamer biosensor comprises the following steps: (1) advanced evolution screening of a complex matrix high-adaptability aptamer; (2) rationally cutting the aptamer assisted by computer simulation; (3) a construction strategy of the label-free proportional biosensor; (4) a label-free proportional SEA bifunctional biosensor sequence; (5) optimizing conditions of the label-free proportional SEA biosensor; and (6) SEA detection. The design principle is that fluorescence of SEA and ThT is excited to different degrees on the basis of a competitive binding effect between an SEA target and a small molecule nucleic acid dye thioflavin T (ThT) as well as a luminous nucleic acid aptamer, so that the ratio of fluorescence signals of the SEA and the ThT is in a linear relationship with the target concentration, and finally, rapid, label-free and low-cost proportional fluorescence detection of the SEA is realized.

Description

Technical Field

[0001] The invention belongs to the field of biosensors, in particular to a label-free ratiometric Staphylococcus aureus enterotoxin A luminescent aptamer biosensor. Background Art

[0002] Staphylococcus aureus enterotoxin A (SEA) is one of the enterotoxins secreted extracellularly by Staphylococcus aureus and can contaminate dairy products during food processing. SEA has been shown to be closely associated with a variety of foodborne illnesses, ranging from gastroenteritis to life-threatening toxic shock. In addition, SEA is relatively stable and highly resistant to proteases, high temperatures, and extreme pH environments. Even heating to 100°C for 30 minutes cannot completely destroy it. Therefore, rapid, sensitive, and low-cost SEA detection methods are urgently needed to provide technical support for dairy product quality and safety monitoring.

[0003] Aptamers are oligonucleotide sequences (ssDNA or RNA) with specific recognition capabilities. They possess the ability to bind to specific small molecules, proteins, lipids, and even cells, making them a highly specialized type of affinity tool. Aptamers are typically obtained through systematic evolution of ligands by exponential enrichment (SELEX) or other modified SELEX strategies. To efficiently obtain high-performance target aptamers, advanced evolutionary screening strategies have been developed. After obtaining the aptamer core sequence through truncation, cleavage, and substitution, a core sequence with certain recognition properties is designed into the middle of the random sequence in the library to facilitate the screening process and the acquisition of high-performance aptamers. A commonly recognized and commonly used SEA aptamer is a DNA aptamer proposed in 2014. Upon binding to the aptamer, the SEA molecule's autofluorescence is significantly stimulated, generating a fluorescent signal. However, the excitation intensity of SEA DNA aptamers is relatively weak, and their affinity needs to be further improved. Therefore, advanced evolutionary screening of SEA DNA aptamers after rough tailoring can further optimize the aptamer performance, promote its applicability, and enhance the detection performance of aptamer sensors.

[0004] Computer simulation-assisted rational tailoring of aptamers has matured with the advancement of bioinformatics. Using computer simulation to tailor aptamers can avoid the influence of redundant sequences in sequences obtained after advanced evolutionary screening, thereby obtaining more reliable aptamers. Computer simulation can clearly define the binding site between the aptamer and the target, while tailoring technology allows for the rational redesign of the aptamer conformation through truncation, deletion, bivalent, or multivalent construction without disrupting the target binding site. Tailoring aptamers to an optimized, compact structure is crucial for maintaining or improving aptamer performance. However, aptamer sequences obtained through evolutionary screening may contain redundant bases that may not be involved in target binding or may even interfere with binding. Therefore, it is essential to focus on the rational modification of aptamers to improve their affinity, specificity, and bioavailability.

[0005] Aptamer biosensors have developed rapidly in recent years. They can generate output signals through mode conversion of biological components that specifically recognize specific substances. They have the advantages of low sample usage, simple operation, fast analysis speed, and stable properties. However, some aptamer sensors require modification of signal output molecules, which will increase costs and affect the folding dynamics of the aptamer. In addition, in order to improve signal stability and detection sensitivity and reduce systematic errors caused by environmental and instrumental factors, label-free and ratiometric aptamer sensing detection strategies are urgently needed. Thioflavin T (ThT), as a small molecule fluorescent dye, can be embedded in specific nucleic acid conformations, such as G-quadruplexes and hairpin structures, thereby generating high fluorescence quantum yields.

[0006] The present invention introduces the aptamer core sequence and proposes an advanced evolutionary screening scheme. During the screening process, a complex matrix environment of milk was simultaneously created, and aptamers with good recognition performance and good matrix adaptability were obtained after only 8 rounds of screening. In addition, the tailoring technology was integrated with bioinformatics to propose a computer simulation-assisted aptamer tailoring strategy. This strategy optimizes the tailoring of SEA DNA aptamers through "structural analysis", "molecular docking" and "tailoring optimization", further improving their affinity and luminescence intensity. In addition, by introducing the fluorescent small molecule ThT, a label-free ratiometric SEA luminescent aptamer biosensor was successfully constructed, ultimately achieving rapid, low-cost, and ultra-sensitive ratiometric fluorescence detection of pore SEA. Summary of the Invention

[0007] Based on this, the present invention proposes an advanced evolutionary screening scheme, an aptamer tailoring strategy assisted by computer simulation, and a general method for ratiometric aptamer sensing, and successfully constructs a label-free ratiometric SEA luminescent aptamer biosensor.

[0008] On the one hand, the present application proposes an advanced evolution screening scheme for aptamers.

[0009] The aptamer advanced evolution screening scheme is an efficient screening based on the aptamer core sequence. First, the Staphylococcus aureus enterotoxin A aptamer SEA-org (SEQ ID NO.1) is preliminarily trimmed through structural analysis and molecular docking simulation scoring. Then, a truncated core sequence with certain recognition ability is obtained through qPCR experimental verification. The core sequence is then designed in the middle position of the random sequence of the screening library. Screening is carried out according to the SELEX in vitro screening process, and the complex matrix of the actual sample is introduced during the screening process. Finally, multiple candidate aptamer sequences are obtained. The functional properties of the candidate aptamers are then verified by fluorescence and affinity characterization methods. The aptamer sequence is:

[0010] SEA-org: 5′-TACTTATGCATTTCCTCCCACGATCTTATTTGAGAGTGAC-3′, as shown in SEQ ID NO. 1;

[0011] The advanced evolution screening scheme refers to the in vitro screening of random sequences of the library using the truncated SEA aptamer sequences obtained through advanced structural analysis, "docking simulation scoring" and "initial trimming" as core sequences, so as to reduce the time cost of aptamer screening and improve the binding ability and matrix adaptability of the aptamer.

[0012] The “docking simulation score” refers to the use of computer software to analyze and predict the secondary and tertiary structures of the aptamer, and to perform docking simulation on the interaction between SEA and the aptamer to obtain a simulation score;

[0013] The “initial tailoring” refers to modifying the SEA aptamer using truncation and mutation strategies based on molecular docking results and special secondary structures such as hairpins. The affinity of the tailored aptamer to SEA is verified by qPCR, and the specificity and matrix adaptability of the effective sequence are characterized to determine the core sequence. The aptamer sequence is:

[0014] SEA-CJ-2: 5′-TTATTTGAGAGTGAC-3′, as shown in SEQ ID NO. 2;

[0015] SEA-CJ-3: 5′-TTTCCTCCCACGATC-3′, as shown in SEQ ID NO. 3;

[0016] SEA-CJ-4: 5′-TTTCCTCCCACGATCTTATTTGAGAGTGAC-3′, as shown in SEQ ID NO. 4;

[0017] SEA-WK-5: 5′-N12-TTATTTGAGAGTGAC-N13 -3′, as shown in SEQ ID NO. 5;

[0018] SEA-SX-6: 5'-GAGGGTGGGGGCTTATTTGAGAGTGACGGATGGTGTGGGG-3', as shown in SEQ ID NO.6;

[0019] SEA-SX-7: 5′-GCCGCCGCGCCCTTATTTGAGAGTGACTGCCCACCATCGT-3′, as shown in SEQ ID NO. 7;

[0020] SEA-SX-8: 5'-GGGTTCGGGGGCATTATTTGAGAGTGACGCTGGGTGGGTGA-3', as shown in SEQ ID NO.8;

[0021] Specifically, molecular docking was used to predict the binding site of the aptamer SEA-org (SEQ ID NO.1) with SEA. Based on the results, the aptamer SEA-org (SEQ ID NO.1) was "roughly tailored," with the principle of preserving the stem-loop structure as much as possible. The aptamer SEA-org (SEQ ID NO.1) contains only a stable "stem-loop" structure. In addition, the aptamer has a long single-stranded segment at the 5' end, away from the "stem-loop" region. It is speculated that this single-stranded segment may be a redundant sequence. Therefore, a tailored SEA-CJ-4 (SEQ ID NO.4) was designed and compared with the original aptamer. After removing the 10nt sequence at the 5' end, a second round of tailoring split the 30nt SEA-CJ-4 (SEQ ID NO.4) sequence from the middle into two 15nt nucleic acid chains, namely SEA-CJ-2 (SEQ ID NO.2) and SEA-CJ-3 (SEQ ID NO.3). After qPCR, the tailored aptamer SEA-CJ-2 (SEQ ID NO. 2) was determined to be the optimal core sequence. 14A linear ssDNA library was constructed, and a random sequence of 40 nt was designed for SEA-WK-5 (SEQ ID NO. 5). The resulting 15 nt core sequence of the aptamer SEA-CJ-2 (SEQ ID NO. 2) was clipped and inserted into the center of the corresponding SEA-WK-5 (SEQ ID NO. 5) sequence. After eight rounds of evolutionary screening, including two rounds of counter-screening and three rounds of matrix-based evolutionary screening, 20 aptamers targeting SEA were identified and grouped into five families. Based on homology analysis and secondary structure data, three families of aptamers were identified: SEA-SX-6 (SEQ ID NO. 6), SEA-SX-7 (SEQ ID NO. 7), and SEA-SX-8 (SEQ ID NO. 8). The affinities of these three aptamers were then verified using qPCR and fluorescence spectrophotometry, leading to the initial identification of SEA-SX-6 (SEQ ID NO. 6), which exhibited relatively outstanding performance.

[0022] The SEA aptamers screened by the advanced evolution are tailored and optimized with the assistance of computer simulation.

[0023] On the other hand, this application proposes a rational tailoring strategy for aptamers assisted by computer simulation.

[0024] The computer simulation-assisted rational aptamer tailoring strategy is to use computer software to perform high-level structural simulation of the aptamer sequence, followed by docking with the ligand molecule. Based on the potential binding domains and interaction sites obtained from the docking simulation results, the SEA aptamer SEA-SX-6 (SEQ ID NO. 6) is tailored and optimized. The functional properties of the tailored aptamer are then verified by fluorescence and affinity characterization methods.

[0025] The computer simulation-assisted rational tailoring strategy for aptamers refers to the dual functions of improving the binding ability and luminescence intensity of the DNA aptamer of SEA through three steps: "molecular docking", "structural analysis" and "tailoring and modification".

[0026] The “molecular docking” mentioned above refers to the use of computer software to predict the secondary and tertiary structures of the aptamer, and to perform docking simulations on the interaction between SEA and the aptamer to identify potential receptor-ligand binding sites;

[0027] The “structural analysis” mentioned above refers to the use of computer software to analyze whether the aptamer has a special secondary structure, such as a G-quadruplex;

[0028] The "tailoring and modification" mentioned above refers to optimizing and modifying the SEA aptamer using truncation, bivalent or multivalent strategies based on the molecular docking results and special secondary structure. The affinity of the tailored aptamer to SEA is verified by qPCR and fluorescence spectrophotometry. The specificity and matrix adaptability of the effective sequence are characterized to preliminarily determine the binding domain and grasp the tailoring direction. The aptamer sequence is:

[0029] SEA-CJ-9: 5′-GAGGGTGGGGGCTTATTTGA-3′, as shown in SEQ ID NO. 9;

[0030] SEA-CJ-10: 5′-GAGTGACGGATGGTGTGGGG-3′, as shown in SEQ ID NO. 10;

[0031] SEA-CJ-11: 5′-GGATGGTGTGGGG-3′, as shown in SEQ ID NO. 11;

[0032] SEA-CJ-12: 5′-ACGGATGGTGTGGGG-3′, as shown in SEQ ID NO. 12;

[0033] SEA-CJ-15: 5′-GGATGGTGTGGGGTTTTGGATGGTGTGGGG-3′, as shown in SEQ ID NO. 15;

[0034] Specifically, the binding site of the aptamer SEA-SX-6 (SEQ ID NO.6) with SEA was predicted through "molecular docking," and the structure of SEA-SX-6 (SEQ ID NO.6) was clarified through "structural analysis." Based on the results, the aptamer SEA-SX-6 (SEQ ID NO.6) was "tailored and modified." The sequence was first split in the middle to obtain SEA-CJ-9 (SEQ ID NO.9) and SEA-CJ-10 (SEQ ID NO.10). Subsequently, SEA-CJ-10 (SEQ ID NO.10) was truncated from G8-G20 and A6-G20, maintaining the specific G4 structure, to obtain SEA-CJ-11 (SEQ ID NO.11) and SEA-CJ-12 (SEQ ID NO.12), respectively. After performance evaluation, SEA-CJ-11 (SEQ ID NO. 11) was selected for engineered bivalent aptamer design and construction, and the bivalent spacer sequence was optimized. Computer software was used to predict the sequence conformations under different spacer base sequences of 0nt, 2nt, 4nt, 6nt, 8nt, and 10nt. The optimal spacer sequence under different metal ion conditions was experimentally verified, and the bivalent aptamer SEA-CJ-15 (SEQ ID NO. 15) was obtained.

[0035] Application of the tailored SEA aptamer in SEA detection.

[0036] Finally, this application developed a label-free ratiometric SEA luminescent aptamer biosensor, which is characterized by (1) the construction strategy of the label-free ratiometric biosensor; (2) the optimization of the reaction conditions of the label-free ratiometric SEA biosensor; and (3) the detection of SEA.

[0037] The construction strategy of this label-free ratiometric biosensor is based on the competitive binding of fluorescent molecules with target substances to the binding sites of luminescent aptamers. The tailored SEA luminescent aptamer significantly stimulates SEA fluorescence upon binding to the target. The present invention introduces an embedded small molecule fluorescent dye that competes with SEA for the aptamer binding sites, thereby affecting the fluorescence intensity of ThT and SEA. The ratio of the two fluorescence signals is then used to quantitatively detect SEA in a ratiometric manner.

[0038] The sequence of the label-free ratiometric SEA luminescent aptamer biosensor is: SEA-CJ-15: 5'-GGATGGTGTGGGGTTTTGGATGGTGTGGGG-3', as shown in SEQ ID NO. 15;

[0039] The reaction conditions optimization of the label-free ratiometric SEA biosensor include the concentration of ThT in the sensor solution, the metal ion conditions and the pH;

[0040] The concentration range of ThT fluorescent dye in the label-free ratiometric SEA biosensor solution is 0.5-50 μmol·L -1；

[0041] Preferably, the concentration of ThT fluorescent dye in the label-free ratiometric SEA biosensor solution is 5 μmol·L -1 ;

[0042] The buffer solution of the label-free ratiometric SEA biosensor is 10 mmol·L -1 The ionic conditions of Tris-HCl (pH 7.5) are: B1 (Na + 140mmol·L -1 )、B2(Na + 135mmol·L -1 ,K + 5mmol·L -1 )、B3(Na + 125mmol·L -1 ,K + 15mmol·L -1 )、B4(Na +115mmol·L -1 ,K + 25mmol·L -1 )、B5(Na + 100mmol·L -1 ,K + 40mmol·L -1 )、B6(Na + 80mmol·L -1 ,K + 60mmol·L -1 )、B7(Na + 70mmol·L -1 ,K + 70mmol·L -1 )、B8(Na + 20mmol·L -1 ,K + 120mmol·L -1 )、B9(Na + 0mmol·L -1 ,K + 140mmol·L -1 );

[0043] Preferably, the buffer solution of the label-free ratiometric SEA biosensor is 10 mmol·L -1 Tris-HCl (pH 7.5) Example conditions are: B2;

[0044] The buffer solution of the label-free ratiometric SEA biosensor is 10 mmol·L -1 The pH range of Tris-HCl is 5.5 to 9.5;

[0045] Preferably, the buffer solution of the label-free ratiometric SEA biosensor is 10 mmol·L -1 The pH of Tris-HCl is 7.5;

[0046] The detection of SEA is based on the competition between SEA and ThT for the binding sites of the SEA aptamer, which causes the fluorescence excitation intensity of the two to change. The ratio of the SEA fluorescence value to the ThT fluorescence value under specific excitation conditions shows a gradient change with the SEA concentration of the solution, thereby realizing the detection of SEA;

[0047] Specific steps for establishing a standard curve:

[0048] Different concentrations of SEA were added to the solution containing 1 μmol·L -1 10mmol·L of aptamer solution -1The mixture was incubated in Tris-HCl (pH 7.5) buffer solution for 15 min, and then 5 μmol·L -1 ThT, so that the final concentration of SEA is 0, 0.0001, 0.001, 0.01, 0.1, 1, 10 μg·mL -1 The fluorescence intensity of SEA and ThT in the solution was measured at 632 and 487 nm using a fluorescence spectrophotometer. The ratio of the fluorescence peaks of SEA to ThT was significantly different from that of SEA at a SEA concentration of 0.0001 μg·mL. -1 ~1 μg·mL -1 There is a good linear relationship within the range (R 2 =0.990), the linear regression equation is Y=0.99-0.31X, and the detection limit for real samples is as low as 0.144ng·mL -1 .

[0049] On the other hand, the selectivity of the SEA biosensor towards SEA was demonstrated.

[0050] Staphylococcus aureus enterotoxin B (SEB), Staphylococcus aureus enterotoxin C (SEC), lactoferrin and casein were used instead of SEA. The experiment was carried out using a label-free ratiometric SEA biosensor to detect the fluorescence ratio of SEA and ThT at the characteristic wavelength.

[0051] On the other hand, the SEA biosensor detects actual samples containing SEA, and the specific operation is as follows:

[0052] Commercially available pure milk was diluted with binding buffer to obtain 79 μL of actual sample. Then, the final concentrations of 0, 0.01, or 0.1 μg mL were added to the 79 μL actual sample. -1 The SEA solution was spiked and recovered, and the detection signal output was the fluorescence intensity ratio of the SEA and ThT molecules, with fluorescence excitation wavelengths of 632 and 487 nm, respectively. The fluorescence value ratio of SEA and ThT was then substituted into the standard curve to calculate the SEA content in the sample to be tested, achieving quantitative detection of SEA.

[0053] Preferably, commercially available pure milk is diluted 1 / 5 with B2 binding buffer to obtain 79 μL of actual sample. Then, a final concentration of 0, 0.01 or 0.1 μg·mL is added to the 79 μL actual sample. -1The SEA solution was spiked and recovered. The detection signal output was the fluorescence intensity ratio of the SEA and ThT molecules, with fluorescence excitation wavelengths of 632 and 487 nm, respectively. The fluorescence intensity ratio of SEA and ThT was then substituted into the standard curve to calculate the SEA content in the sample to be tested, achieving quantitative detection of SEA.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] 1. The present invention proposes an advanced evolutionary screening scheme to achieve efficient and low-cost acquisition of Staphylococcus aureus enterotoxin A aptamers; proposes a computer-assisted tailoring strategy to achieve dual functional improvements in the recognition ability and luminescence intensity of Staphylococcus aureus enterotoxin A aptamers; and improves the detection sensitivity and signal stability of Staphylococcus aureus enterotoxin A aptamer sequence biosensor through truncation, bivalence and other tailoring optimization methods, thereby broadening the application range of the sensor.

[0056] 3. The present invention proposes a universal ratiometric sensing method, which is to introduce embedded small molecule nucleic acid dyes into the luminescent aptamer system to achieve stable ratiometric sensing detection through the principle of binding site competition.

[0057] 5. The Staphylococcus aureus enterotoxin A biosensor proposed in the present invention can realize low-cost, rapid, stable and sensitive proportional detection of Staphylococcus aureus enterotoxin A, and has certain versatility and industrialization potential.

[0058] 6. The Staphylococcus aureus enterotoxin A biosensor proposed by the present invention is 0.0001 μg·mL -1 ~1 μg·mL -1 There is a good linear relationship within the range (R 2 =0.990), the linear regression equation is Y=0.99-0.31X, and the detection limit of the real sample is as low as 0.144ng·mL -1 . BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Results of the SEA-org advanced evolutionary screening. (A) SEA-org's secondary structure; (B) SEA-org's tertiary structure; (C) SEA-org's overall tailoring plan; (D) Affinity comparison of various truncated sequences; (E) SEA-WK-5 advanced evolutionary screening round monitoring results; (F) Homology analysis results of the 8th round of evolutionary screening sequences.

[0060] Figure 2Affinity evaluation of aptamers selected for evolutionary screening of Staphylococcus aureus enterotoxin A. (A) Quantitative PCR analysis of affinity of the selected sequences; (B) SEA-org fluorescence analysis of K d (C) is the SEA-SX-6 fluorescence method to determine K d Value result.

[0061] Figure 3 Molecular docking results of SEA-SX-6 and Staphylococcus aureus enterotoxin A. (A) The secondary structure of SEA-SX-6; (B) The molecular docking demonstration of SEA-SX-6 and Staphylococcus aureus enterotoxin A; (C) The G4 structure verification of SEA-SX-6.

[0062] Figure 4 The molecular docking results of SEA-CJ-11 and Staphylococcus aureus enterotoxin A. (A) is the secondary structure of SEA-CJ-11; (B) is the Ct value of SEA-CJ-11 and SEA-CJ-12; (C) is the K value of SEA-SX-6. d Value results; (D) is the K of SEA-CJ-11 d (E) is the structure prediction of each bivalent aptamer under different connection chains.

[0063] Figure 5 This demonstrates the feasibility of constructing a label-free ratiometric Staphylococcus aureus enterotoxin A biosensor. (A) 2D structure of the SEA-CJ-15 / ThT complex; (B) 2D structure of the SEA-CJ-15 / Staphylococcus aureus enterotoxin A complex; (C) Fluorescence spectra of SEA-CJ-15 binding to both S. aureus enterotoxin A and ThT.

[0064] Figure 6 Evaluation of the detection performance of a label-free ratiometric Staphylococcus aureus enterotoxin A biosensor. (A) Fluorescence spectra of dual signals at different Staphylococcus aureus enterotoxin A concentrations; (B) Standard curve for the sensor's detection of Staphylococcus aureus enterotoxin A.

[0065] Figure 7 Specificity evaluation of a label-free ratiometric Staphylococcus aureus enterotoxin A biosensor.

[0066] Figure 8 Optimization of reaction conditions for a label-free ratiometric Staphylococcus aureus enterotoxin A biosensor. (A) ThT concentration; (B) ionic conditions; (C) pH value.

[0067] Figure 9 Schematic diagram of the label-free ratiometric Staphylococcus aureus enterotoxin A biosensor. DETAILED DESCRIPTION

[0068] 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.

[0069] Example 1. Advanced Evolution Screening of Staphylococcus aureus Enterotoxin A Aptamers

[0070] 1. Obtaining the core sequence of Staphylococcus aureus enterotoxin A aptamer

[0071] The recognition ability and matrix adaptability of the reported Staphylococcus aureus enterotoxin A DNA aptamers need to be improved. In order to improve the performance of the aptamer, the original Staphylococcus aureus enterotoxin A aptamer was preliminarily trimmed with the principle of retaining the stem-loop structure as much as possible and removing redundant bases. The core sequence obtained after trimming was verified by qPCR.

[0072] The nucleotide sequences involved in the examples are shown in Table 1.

[0073] Table 1 Summary of nucleic acid sequences

[0074]

[0075]

[0076] The secondary structure of SEA-org (SEQ ID NO. 1) was predicted using the mfold website. Based on this, the RNA 3D structure of the aptamer was obtained using RNAComposer. The RNA 3D structure was then converted to a DNA 3D structure using BIOVIA Discovery Studio software and energy minimized. Finally, molecular docking of SEA-org (SEQ ID NO. 1) and Staphylococcus aureus enterotoxin A was performed using the HDOCK website to obtain an HDOCK score.

[0077] like Figure 1 As shown in A and B, the high-level structure of SEA-org (SEQ ID NO.1) is mainly composed of a stable "stem-loop" structure. In addition, the aptamer has a long single chain at the 5' end away from the "stem-loop" region, and it is speculated that this single chain may be a redundant sequence. Figure 1C aptamer overall trimming scheme, after removing the 5' end 10nt sequence, it was found that the HDOCK score of SEA-CJ-4 (SEQ ID NO.4) (-338.08) was similar to that of the original aptamer SEA-org (SEQ ID NO.1) (-337.49), indicating that the 5' end 10nt sequence was a redundant sequence and did not play a role in binding to the target. At the same time, it was speculated that it might be because there were still too many redundant sequences in the remaining regions. Therefore, even if this single chain was removed, it did not play a key role in improving its affinity. In order to better narrow the scope of identifying redundant sequences, the second round of trimming split the SEA-CJ-4 (SEQ ID NO.4) sequence from the middle into two nucleic acid chains, SEA-CJ-2 (SEQ ID NO.2) and SEA-CJ-3 (SEQ ID NO.3), and it was determined by qPCR method ( Figure 1 D), SEA-CJ-2 (SEQ ID NO. 2) is the best core sequence.

[0078] The aptamer sequences used:

[0079] SEA-org: 5′-TACTTATGCATTTCCTCCCACGATCTTATTTGAGAGTGAC-3′, as shown in SEQ ID NO. 1;

[0080] SEA-CJ-2: 5′-TTATTTGAGAGTGAC-3′, as shown in SEQ ID NO. 2;

[0081] SEA-CJ-3: 5′-TTTCCTCCCACGATC-3′, as shown in SEQ ID NO. 3;

[0082] SEA-CJ-4: 5′-TTTCCTCCCACGATCTTATTTGAGAGTGAC-3′, as shown in SEQ ID NO. 4;

[0083] 2. Evolution and Screening of Staphylococcus aureus Enterotoxin A Aptamers

[0084] Advanced evolutionary screening is also a method for in vitro screening to obtain specific aptamers. Compared with traditional in vitro screening, the random sequence region of the evolutionary screening library is added with a core sequence to form the SEA-WK-5 (SEQ ID NO.5) library, making the screening more controllable and easier to efficiently obtain aptamers with good performance.

[0085] The truncated aptamer of SEA-CJ-2 (SEQ ID NO. 2) obtained by preliminary cutting was used as the core sequence and designed in the middle position of the random sequence (40 nt) of the ssDNA library, so that the full length of the sequence was 80 nt and the library capacity was about 10 14 The target enterotoxin protein was then used as the target for aptamer evolutionary screening, and other common foodborne SEs were used as counter-screening toxins. Using a magnetic bead-immobilized target method, a simple procedure consisting of incubation binding, denaturation separation, PCR amplification, enzyme digestion, alcohol precipitation, and qPCR instrument characterization was performed. Pure milk was added for interference in the third, fifth, and seventh rounds of screening. Eight rounds of evolutionary screening, including two rounds of counter-screening, were performed to obtain multiple aptamer sequences. The SEA aptamer sequences were divided into five families based on homology analysis, secondary structure prediction, and minimum free energy. The performance of the candidate sequences in each family was analyzed.

[0086] The evolutionary screening of each round of aptamers was monitored by qPCR, and the Ct value obtained in each round was used as the binding efficiency of the aptamer and the target toxin in each round ( Figure 1 E), thus knowing the progress of evolutionary screening, the final number of evolutionary screening rounds was determined to be round 8. The library of round 8 was amplified by large amounts of PCR and then sequenced. The first 20 sequences obtained by sequencing were analyzed, and a homology tree was made based on the results ( Figure 1 F) for homology analysis. Based on the above homology analysis and secondary structure prediction, the 20 sequences can be divided into five major families. Based on the abundance of representative sequences in each of the five major aptamer families and the homology evaluation between each group of sequences, three candidate aptamers were finally selected: SEA-SX-6 (SEQ ID NO. 6), SEA-SX-7 (SEQ ID NO. 7), and SEA-SX-8 (SEQ ID NO. 8).

[0087] The aptamer sequences used:

[0088] SEA-CJ-2: 5′-TTATTTGAGAGTGAC-3′, as shown in SEQ ID NO. 2;

[0089] SEA-WK-5: 5′-N12-TTATTTGAGAGTGAC-N13 -3′, as shown in SEQ ID NO. 5;

[0090] SEA-SX-6: 5'-GAGGGTGGGGGCTTATTTGAGAGTGACGGATGGTGTGGGG-3', as shown in SEQ ID NO.6;

[0091] SEA-SX-7: 5′-GCCGCCGCGCCCTTATTTGAGAGTGACTGCCCACCATCGT-3′, as shown in SEQ ID NO. 7;

[0092] SEA-SX-8: 5'-GGGTTCGGGGGCATTATTTGAGAGTGACGCTGGGTGGGTGA-3', as shown in SEQ ID NO.8;

[0093] 3. Affinity Verification of Staphylococcus aureus Enterotoxin A Aptamer

[0094] Subsequently, the affinity of the three aptamers SEA-SX-6 (SEQID NO.6), SEA-SX-7 (SEQ ID NO.7), and SEA-SX-8 (SEQ ID NO.8) obtained after evolutionary screening was verified using qPCR and fluorescence titration methods.

[0095] qPCR assay principle: Use a qPCR instrument to measure the Ct values ​​of the bound aptamers SEA-SX-6 (SEQ ID NO. 6), SEA-SX-7 (SEQ ID NO. 7), and SEA-SX-8 (SEQ ID NO. 8). A lower average Ct value indicates a higher affinity for the aptamer binding to the target protein.

[0096] The specific experimental steps of the qPCR method are as follows:

[0097] First, the qPCR template was obtained by the magnetic bead immobilization target method: the enterotoxin was diluted, 10 μL of 4 mg / mL SEA was taken into 390 μL PBST (0.01 M, pH 7.3), mixed thoroughly, and placed in a -20°C refrigerator for use; then, the carboxyl magnetic beads (MB-COOH) were activated, 20 μL MB-COOH was taken into a 2 mL centrifuge tube, and the supernatant was removed by magnetic separation. The beads were magnetically separated and washed twice with 40 μL MEST, and then the supernatant was removed; 20 μL of freshly prepared EDC solution and 20 μL NHS solution was added to a centrifuge tube containing MB-COOH, vortexed to mix the magnetic beads thoroughly and suspend them, and activated at 25°C for 30 minutes; the activated magnetic beads were coupled with SEA protein, the supernatant was removed by magnetic separation, 100 μL of diluted enterotoxin protein solution was added, and the mixture was coupled at 25°C for 1 hour and then placed in a 4°C refrigerator overnight; the aptamer was then selectively bound to the magnetic bead-SEA protein complex, magnetic separation and washing were performed to remove unbound proteins, and the complex was washed three times with 100 μL PBST; the washed magnetic bead-SEA protein complex was resuspended in 100 μL The cells were thoroughly mixed in PBST and the supernatant was removed by magnetic separation. 100 μM aptamer was diluted to 0.1 μM with Hepes buffer solution. The obtained 0.1 μM aptamer (100 μL) was heated and denatured at 95°C for 5 min, immediately placed on ice, and placed for 10 min before being added to a 2 mL centrifuge tube containing magnetic beads. The cells were bound and cultured at 25°C for 1 h. After incubation, magnetic separation was performed, and the unbound ssDNA was in the supernatant. The supernatant was removed and the cells were washed 3 times with PBST and 3 times with water. Finally, 100 μL of 1× Taq enzyme buffer solution was added, heated at 95°C for 10 min, and immediately placed on ice for 10 min to dissociate the bound ssDNA and obtain the amplification template.

[0098] During the qPCR amplification process, to ensure high specificity of the amplification reaction, a two-step qPCR reaction procedure was adopted, and a melting section was added to monitor the formation of nonspecific products. The procedure included pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 15 seconds, and annealing and extension at 60°C for 30 seconds. Finally, the affinity was compared by comparing the Ct values ​​of each aptamer determined by the qPCR instrument.

[0099] The specific experimental steps of fluorescence titration are as follows:

[0100] To the solution containing 1 μmol·L -1 Staphylococcus aureus enterotoxin A aptamer buffer solution 10 mmol·L -1 Staphylococcus aureus enterotoxin A solution of different concentrations was added to Tris-HCl (pH 7.5), mixed and incubated at room temperature for 15 minutes, and the fluorescence value of the solution at 632 nm excitation was measured using a fluorescence spectrophotometer. Then, a curve was fitted based on the relationship between the fluorescence value and the Staphylococcus aureus enterotoxin A concentration to obtain the affinity constant.

[0101] like Figure 2 As shown in A, the qPCR experimental results show that among the three sequences SEA-SX-6 (SEQ ID NO.6), SEA-SX-7 (SEQ ID NO.7), and SEA-SX-8 (SEQ ID NO.8), the average Ct value of SEA-SX-6 (SEQ ID NO.6) is lower, that is, the binding ability with SEA is stronger, and the Ct value is 15.75. Therefore, the SEA-SX-6 (SEQ ID NO.6) aptamer was selected in the experiment, and its K value was specifically determined using a fluorescence spectrophotometer. d The values ​​were compared with those of the original aptamer SEA-org (SEQ ID NO. 1). Figure 2 As shown in B and C, the K of SEA-SX-6 (SEQ ID NO.6) aptamer d The value is 48.38nM, which is higher than the binding affinity of the original aptamer.

[0102] The aptamer sequences used in the experiment are as follows:

[0103] SEA-org: 5′-TACTTATGCATTTCCTCCCACGATCTTATTTGAGAGTGAC-3′, as shown in SEQ ID NO. 1;

[0104] SEA-SX-6: 5'-GAGGGTGGGGGCTTATTTGAGAGTGACGGATGGTGTGGGG-3', as shown in SEQ ID NO.6;

[0105] SEA-SX-7: 5′-GCCGCCGCGCCCTTATTTGAGAGTGACTGCCCACCATCGT-3′, as shown in SEQ ID NO. 7;

[0106] SEA-SX-8: 5'-GGGTTCGGGGGCATTATTTGAGAGTGACGCTGGGTGGGTGA-3', as shown in SEQ ID NO.8;

[0107] Example 2. Computer simulation-assisted evolutionary tailoring of Staphylococcus aureus enterotoxin A aptamers

[0108] 1. Molecular Docking of Staphylococcus aureus Enterotoxin A Aptamer

[0109] To mitigate the effects of redundant sequences in the Staphylococcus aureus enterotoxin A aptamer sequence obtained after advanced evolution screening on aptamer performance, the Staphylococcus aureus enterotoxin A aptamer was tailored using computer simulation. The tailored results were verified using qPCR and fluorescence spectrophotometry. First, molecular docking was used to predict potential binding sites between the aptamer and the target.

[0110] The secondary structure of SEA-SX-6 (SEQ ID NO. 6) was predicted using the mfold website. RNAComposer was then used to obtain the RNA 3D structure of the aptamer. The RNA 3D structure was then converted to a DNA 3D structure using BIOVIA Discovery Studio software and energy minimized. Finally, molecular docking of the binding of SEA-SX-6 (SEQ ID NO. 6) and Staphylococcus aureus enterotoxin A was performed using the HDOCK website, and the resulting aptamer-target interaction model was analyzed using BIOVIA Discovery Studio software. Furthermore, due to the high GC content of SEA-SX-6 (SEQ ID NO. 6), QGRSMapper was used to predict the G4 structure to analyze whether a G4 structure existed.

[0111] like Figure 3 As can be seen from A, the high-level structure of SEA-SX-6 (SEQ ID NO. 6) mainly contains a stem-loop structure. In the aptamer-target interaction model, it can be found that the G1-A20 bases are the binding sites between the aptamer and Staphylococcus aureus enterotoxin A ( Figure 3 B) At the same time, Figure 3 As shown in C, there is a G4 structure at nucleotides G28-G40. Combined with the HDOCK score, it is speculated that the G4 structure at G28-G40 is the main binding domain.

[0112] The aptamer sequences used:

[0113] SEA-SX-6: 5'-GAGGGTGGGGGCTTATTTGAGAGTGACGGATGGTGTGGGG-3', as shown in SEQ ID NO. 6.

[0114] 2. Tailoring of Staphylococcus aureus Enterotoxin A Aptamer

[0115] In order to further clarify the potential binding domain of Staphylococcus aureus enterotoxin A and the aptamer, the aptamer was tailored based on the molecular docking results. The secondary structure of the obtained new sequence is shown in the figure below. Figure 4 As shown in A.

[0116] Since SEA-SX-6 (SEQ ID NO.6) has a G4 structure, the G4 structure was retained as much as possible during cutting. First, the SEA-SX-6 (SEQ ID NO.6) aptamer was split from the middle to obtain SEA-CJ-9 (SEQ ID NO.9) and SEA-CJ-10 (SEQID NO.10), and then SEA-CJ-10 (SEQ ID NO.10) was cut from G8-G20 and A6-G20, maintaining the special G4 structure, and obtaining SEA-CJ-11 (SEQ ID NO.11) and SEA-CJ-12 (SEQ ID NO.12) respectively. Molecular docking was used to simulate the interaction between the aptamer and Staphylococcus aureus enterotoxin A, and the specific method was the same as above. Subsequently, the Ct values ​​of the SEA-CJ-11 (SEQ ID NO.11) and SEA-CJ-12 (SEQ ID NO.12) sequences obtained by qPCR were as follows: Figure 4 As shown in B, the affinity of each candidate aptamer was preliminarily predicted. -1 Aptamer and 0.1 μg mL -1 After incubation with Staphylococcus aureus enterotoxin A, the fluorescence value of the solution at 632 nm excitation was measured using a fluorescence spectrophotometer. The affinity constant was obtained by fitting a curve based on the relationship between the fluorescence value and the concentration of Staphylococcus aureus enterotoxin A. At the same time, it was compared with the K of the SEA-SX-6 (SEQ ID NO. 6) aptamer. d The results were compared with those obtained by qPCR. Figure 4 As shown in C and D, SEA-CJ-11 (SEQ ID NO.11) aptamer K d The affinity value of SEA-SX-6 (SEQ ID NO. 6) was reduced from 48.38 nM to 38.61 nM, and an aptamer with excellent affinity for Staphylococcus aureus enterotoxin A was obtained.

[0117] Subsequently, SEA-CJ-11 (SEQ ID NO. 11) was selected for the design and construction of an engineered bivalent aptamer. Under the spacer sequences of 0ntT (SEA-CJ-13), 2ntT (SEA-CJ-14), 4ntT (SEA-CJ-15), 6ntT (SEA-CJ-16), 8ntT (SEA-CJ-17), and 10ntT (SEA-CJ-18), the sequence structures under different spacers were predicted using QGRS Mapper. It was found that the bivalent aptamer formed different G4 structures under different spacer sequences. Figure 4As shown in E (where the yellow-marked bases constitute G4), the 4ntT spacer sequence in the bivalent aptamer forms a new G4 structure, which expands the G4 central ring compared to the one without the spacer sequence, and then separates the two G4 structures starting from 8ntT.

[0118] The aptamer sequences used in the experiment are as follows:

[0119] SEA-SX-6: 5'-GAGGGTGGGGGCTTATTTGAGAGTGACGGATGGTGTGGGG-3', as shown in SEQ ID NO.6;

[0120] EA-CJ-9: 5′-GAGGGTGGGGGCTTATTTGA-3′, as shown in SEQ ID NO. 9;

[0121] SEA-CJ-10: 5′-GAGTGACGGATGGTGTGGGG-3′, as shown in SEQ ID NO. 10;

[0122] SEA-CJ-11: 5′-GGATGGTGTGGGG-3′, as shown in SEQ ID NO. 11;

[0123] SEA-CJ-12: 5′-ACGGATGGTGTGGGG-3′, as shown in SEQ ID NO. 12;

[0124] SEA-CJ-13: 5′-GGATGGTGTGGGGGGATGGTGTGGGG-3′, as shown in SEQ ID NO. 13;

[0125] SEA-CJ-14: 5′-GGATGGTGTGGGGTTGGATGGTGTGGGG-3′, as shown in SEQ ID NO. 14;

[0126] SEA-CJ-15: 5′-GGATGGTGTGGGGTTTTGGATGGTGTGGGG-3′, as shown in SEQ ID NO. 15;

[0127] SEA-CJ-16: 5'-GGATGGTGTGGGGTTTTTTGGATGGTGTGGGG-3', as shown in SEQ ID NO. 16;

[0128] SEA-CJ-17: 5'-GGATGGTGTGGGGTTTTTTTTGGATGGTGTGGGG-3', as shown in SEQ ID NO. 17;

[0129] SEA-CJ-18: 5'-GGATGGTGTGGGGTTTTTTTTTTGGATGGTGTGGGG-3', as shown in SEQ ID NO. 18.

[0130] Example 3. Feasibility Verification of Label-free Ratio-based Staphylococcus aureus Enterotoxin A Biosensor

[0131] 1. Construction strategy of label-free ratiometric biosensor

[0132] In order to construct a label-free ratiometric biosensor, SEA-SX-6 (SEQ ID NO.6) obtained by advanced evolution screening was tailored and modified through a tailoring strategy guided by computer simulation, which improved the affinity of the aptamer and obtained the bifunctional bivalent aptamer SEA-CJ-15 (SEQ ID NO.15). The construction strategy of the biosensor is based on the competition of fluorescent molecules for the binding sites of the luminescent aptamer. After binding to the target, the modified Staphylococcus aureus enterotoxin A luminescent aptamer can significantly excite the fluorescence of Staphylococcus aureus enterotoxin A. The embedded small molecule fluorescent dye introduced into the system can compete with Staphylococcus aureus enterotoxin A for the same binding site of the aptamer and produce a dynamic equilibrium effect based on competition in the solution. Based on this principle, changes in the content of Staphylococcus aureus enterotoxin A in the solution will change the fluorescence luminescence intensity of ThT and Staphylococcus aureus enterotoxin A. The content of Staphylococcus aureus enterotoxin A in the solution is detected ratiometrically by the ratio of the two fluorescence signals ( Figure 9 ).

[0133] 2. Feasibility Verification of Label-free Ratiometric Biosensor

[0134] In order to verify the feasibility of the ratiometric sensor construction strategy, we performed molecular docking prediction on SEA-CJ-15 (SEQ ID NO.15) and ThT. Figure 5 As shown in Figures A and B, SEA-CJ-15 (SEQ ID NO. 15) shares some of the same binding sites with ThT and Staphylococcus aureus enterotoxin A. Therefore, computer predictions and simulations indicate that the dual-signal combination of ThT and Staphylococcus aureus enterotoxin A can achieve ratiometric sensing by competing for the same binding site on SEA-CJ-15 (SEQ ID NO. 15).

[0135] Subsequently, the feasibility of the proportional sensor was explored at the experimental level. Figure 5 As shown in Figure C, the addition of Staphylococcus aureus enterotoxin A significantly changes the luminescence intensity of ThT and itself, proving the feasibility of the construction strategy of the label-free ratiometric biosensor.

[0136] Example 4. Detection Performance Evaluation of a Label-free Ratio-based Staphylococcus aureus Enterotoxin A Biosensor

[0137] 1. Sensitivity Evaluation of a Label-free Ratiometric Staphylococcus aureus Enterotoxin A Biosensor

[0138] SEA-CJ-15 (SEQ ID NO. 15) was used to detect Staphylococcus aureus enterotoxin A of known concentrations, and a standard curve was prepared based on the change in the fluorescence ratio of Staphylococcus aureus enterotoxin A and ThT in the solution. Different concentrations of Staphylococcus aureus enterotoxin A were added to a solution containing 1 μmol·L -1 10mmol·L of aptamer solution -1 The cells were incubated in Tris-HCl (pH 7.5) buffer solution for 15 min, and then 5 μmol·L -1 ThT, and the final concentrations of Staphylococcus aureus enterotoxin A were 0, 0.0001, 0.001, 0.01, 0.1, 1, and 10 μg mL -1 Finally, the fluorescence intensity of Staphylococcus aureus enterotoxin A and ThT in the solution was measured at 632 and 487 nm using a fluorescence spectrophotometer, and a standard curve was drawn based on the fluorescence ratio of the two.

[0139] like Figure 6 As shown, the label-free ratiometric Staphylococcus aureus enterotoxin A biosensor was tested at 0.0001 μg·mL -1 ~1 μg·mL -1 There is a good linear relationship within the range (R 2 =0.990), the linear regression equation is Y=0.99-0.31X, and the detection limit for real samples is as low as 0.144ng·mL -1 .

[0140] 2. Specificity Evaluation of a Label-free Ratiometric Staphylococcus aureus Enterotoxin A Biosensor

[0141] Staphylococcus aureus enterotoxin B (SEB), Staphylococcus aureus enterotoxin C (SEC), lactoferrin and casein were used to evaluate the specificity of the Staphylococcus aureus enterotoxin A biosensor. -1 Staphylococcus aureus enterotoxin A or SEB, SEC, lactoferrin, and casein were added to a mixture containing 1 μmol·L -1 10mmol·L of aptamer solution -1 The cells were incubated in Tris-HCl (pH 7.5) buffer solution for 15 min, and then 5 μmol·L -1 ThT was measured immediately after oscillation and mixing at room temperature. Finally, the fluorescence intensities of S. aureus enterotoxin A and ThT in the solution were measured at 632 and 487 nm using a fluorescence spectrophotometer. The ratio of the two values ​​was inserted into the standard curve to calculate the corresponding S. aureus enterotoxin A content.

[0142] like Figure 7 As shown, the addition of SEB, SEC, lactoferrin, and casein did not significantly affect the detection results of Staphylococcus aureus enterotoxin A, indicating that the Staphylococcus aureus enterotoxin A aptamer has good selectivity.

[0143] Example 5. Optimization of detection conditions for a label-free ratiometric Staphylococcus aureus enterotoxin A biosensor

[0144] First, the ThT concentration of the Staphylococcus aureus enterotoxin A sensor system was optimized. -1 Staphylococcus aureus enterotoxin A and 0.5, 1, 5, 10 and 50 μmol·L -1 ThT was added to contain 0.1 μg mL -1 10mmol·L of aptamer solution -1 The solution was mixed in Tris-HCl (pH 7.5) buffer solution and then immediately measured after oscillation at room temperature. Finally, the fluorescence intensity of Staphylococcus aureus enterotoxin A and ThT in the solution system was measured at 632 and 487 nm using a fluorescence spectrophotometer. Figure 8 As shown in A, the final concentration of ThT was 5 μmol·L -1 It has a higher detection signal-to-noise ratio and lower detection cost.

[0145] Secondly, the metal ion conditions of the Staphylococcus aureus enterotoxin A sensor system were optimized. -1 Staphylococcus aureus enterotoxin A and 5 μmol·L -1 ThT was added to a concentration of 1 μmol·L -1 10mmol·L of aptamer solution - 1 In Tris-HCl (pH 7.5) buffer solution, the ionic conditions of the buffer solution are B1 (Na + 140mmol·L -1 )、B2(Na + 135mmol·L -1 ,K + 5mmol·L -1 )、B3(Na+ 125mmol·L -1 ,K + 15mmol·L -1 )、B4(Na + 115mmol·L -1 ,K + 25mmol·L -1 )、B5(Na + 100mmol·L -1 ,K + 40mmol·L -1 )、B6(Na + 80mmol·L -1 ,K + 60mmol·L -1 )、B7(Na + 70mmol·L -1 ,K + 70mmol·L -1 )、B8(Na + 20mmol·L -1 ,K + 120mmol·L -1 )、B9(Na + 0mmol·L -1 ,K + 140mmol·L -1 ), shaken and mixed at room temperature and then measured immediately. Finally, the fluorescence intensity of Staphylococcus aureus enterotoxin A and ThT in the solution system was measured at 632 and 487 nm using a fluorescence spectrophotometer. Figure 8 As shown in B, when the buffer solution is 10mmol·L -1 Na in Tris-HCl (pH 7.5) + 135mmol·L -1 , K + 5mmol·L -1 A high-intensity fluorescence signal will appear when the + 135mmol·L -1 , K + 5mmol·L -1 Ionic solution.

[0146] Finally, the pH of the Staphylococcus aureus enterotoxin A sensor system was optimized. -1 Staphylococcus aureus enterotoxin A and 5 μmol·L -1 ThT was added to a concentration of 1 μmol·L -1 10mmol·L of aptamer solution -1The pH values ​​of the buffer solutions were 5.5, 6.5, 7.5, 8.5, and 9.5, respectively. The samples were shaken and mixed at room temperature and then immediately measured. Finally, the fluorescence intensities of Staphylococcus aureus enterotoxin A and ThT at 632 and 487 nm were measured using a fluorescence spectrophotometer. Figure 8 As shown in C, the buffer solution is 10mmol·L -1 The highest signal-to-noise ratio was obtained when the pH of Tris-HCl was 7.5.

[0147] Example 6. Application of label-free ratiometric biosensor for detection of Staphylococcus aureus enterotoxin A in real samples

[0148] In the real sample spike recovery experiment, commercially available pure milk was diluted 1 / 5 with B2 binding buffer to obtain 79 μL of real sample. Then, the final concentrations of 0, 0.01, or 0.1 μg mL were added to the 79 μL real sample. -1 A spike recovery experiment was performed on a solution of Staphylococcus aureus enterotoxin A. The fluorescence intensity ratio of the signal output molecules, Staphylococcus enterotoxin A and ThT, was detected, with fluorescence excitation wavelengths of 632 and 487 nm, respectively. The fluorescence intensity ratio of Staphylococcus enterotoxin A to ThT was then substituted into the standard curve to calculate the content of Staphylococcus enterotoxin A in the test sample, achieving quantitative detection of Staphylococcus enterotoxin A.

[0149] As shown in Table 2, the recovery rate of dairy products spiked with Staphylococcus aureus enterotoxin A biosensor was ideal, indicating that the biosensor can achieve quantitative detection of Staphylococcus aureus enterotoxin A in real samples.

[0150] Table 2. Staphylococcus aureus enterotoxin A spike experiment in dairy products based on label-free ratiometric biosensor

[0151]

Claims

1. A high-performance nucleic acid aptamer of Staphylococcus aureus enterotoxin A obtained by tailoring, characterized in that: The aptamer sequence is shown in SEQ ID NO.

2.

2. Use of the nucleic acid aptamer sequence according to claim 1 in the development of a Staphylococcus aureus enterotoxin A detection method or a food safety detection kit.

3. A Staphylococcus aureus enterotoxin A nucleic acid aptamer obtained by advanced evolution screening, characterized in that: The aptamer sequence is shown in any one of SEQ ID NOs. 6 to 8, SEQ ID NOs. 11 to 12, and SEQ ID NO.

15.

4. Use of the nucleic acid aptamer sequence according to claim 3 in the development of a Staphylococcus aureus enterotoxin A detection method or a food safety detection kit.

5. A label-free ratiometric Staphylococcus aureus enterotoxin A luminescent aptamer biosensor, characterized in that: (1) Construction strategy of label-free ratiometric biosensor; (2) Sequence of label-free ratiometric Staphylococcus aureus enterotoxin A biosensor; (3) Optimization of label-free ratiometric Staphylococcus aureus enterotoxin A biosensor conditions; (4) Detection of Staphylococcus aureus enterotoxin A; The construction strategy of the label-free ratiometric biosensor is that the luminescent target competes with the embedded small molecule nucleic acid dye for sites, the ratio of the dual fluorescence signals is linearly related to the target concentration, and the target is quantitatively detected according to the fluorescence ratio value; The sequence of the label-free ratiometric Staphylococcus aureus enterotoxin A biosensor is shown in SEQ ID NO.

15.

6. The biosensor according to claim 5, wherein The concentration of the thioflavin T fluorescent dye in the sensor solution ranges from 0.5 to 50 μmol·L -1 .

7. The biosensor according to claim 5, characterized in that The sensor's selectivity for Staphylococcus aureus enterotoxin A; Staphylococcal enterotoxin B, Staphylococcal enterotoxin C, lactoferrin and casein were used instead of Staphylococcal enterotoxin A. The fluorescence ratio of Staphylococcal enterotoxin A and thioflavin T at the characteristic wavelength was detected using a label-free ratiometric Staphylococcal enterotoxin A biosensor.

8. The method for quantitatively detecting Staphylococcus aureus enterotoxin A using a biosensor according to any one of claims 5 to 7, wherein: Establishment of standard curve: Different concentrations of Staphylococcus aureus enterotoxin A were added to a solution containing 1 μmol·L -1 10mmol·L of aptamer solution -1 The cells were incubated in Tris-HCl buffer solution for 15 min, and then 5 μmol·L -1 Thioflavin T was added to make the final concentrations of Staphylococcus aureus enterotoxin A 0, 0.0001, 0.001, 0.01, 0.1, 1, and 10 μg mL -1 After mixing under oscillation at room temperature, the fluorescence was immediately measured; the fluorescence intensity of the solution system at 632 and 487 nm was measured using a fluorescence spectrophotometer.

9. The method for detecting a real sample containing Staphylococcus aureus enterotoxin A using a sensor according to any one of claims 5 to 7, characterized in that: The specific operations are as follows: Commercially available pure milk was diluted 5-fold with binding buffer to obtain 79 μL of actual sample; then, 0, 0.01, or 0.1 μg mL-1 of the final concentration of 0 μg mL-1 was added to the 79 μL actual sample. -1 A spiked recovery experiment was performed on a Staphylococcal enterotoxin A solution, and the fluorescence intensity ratio of the signal output molecules Staphylococcal enterotoxin A and thioflavin T was detected, with the fluorescence excitation wavelengths being 632 and 487 nm, respectively. The fluorescence value ratio of Staphylococcal enterotoxin A and thioflavin T was then substituted into the standard curve to calculate the content of Staphylococcal enterotoxin A in the sample to be tested, thereby realizing the quantitative detection of Staphylococcal enterotoxin A.

10. Use of the biosensor according to any one of claims 5 to 7 or the method according to claim 8 or 9 in the development of a Staphylococcus aureus enterotoxin A detection method or a food safety detection kit.

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