Bifunctional bivalent aptamer staphylococcus aureus enterotoxin B fluorescent proportional biosensor

Abstract

The invention discloses a bifunctional bivalent aptamer staphylococcus aureus enterotoxin B (SEB) fluorescent proportional biosensor. The 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 a bifunctional divalent aptamer fluorescence proportional biosensor; (4) an SEB bifunctional biosensor sequence; (5) carrying out condition optimization on the bifunctional bivalent aptamer SEB fluorescent proportional biosensor; and (6) SEB detection. The design principle is based on the competitive binding effect between an SEB target and a small molecule nucleic acid dye thioflavin T (Thioflavin T, ThT) and an aptamer, so that the fluorescence of SEB and ThT is excited to different degrees, the ratio of the fluorescence signals of SEB and ThT and the concentration of the target are in a linear relation, and finally the sensitive, rapid and low-cost proportional fluorescence detection of SEB is realized.

Description

Technical Field

[0001] The invention belongs to the field of biosensors, in particular to a bifunctional bivalent aptamer Staphylococcus aureus enterotoxin B fluorescence ratio biosensor. Background Art

[0002] Staphylococcus aureus is a common foodborne bacterial pathogen. It secretes a class of small, heat-stable toxins (22-28 kDa) extracellularly, known as Staphylococcal enterotoxins (SEs). Among these SEs, only a subset (SEA-SEI, SER, SES, and SET) are known to cause food poisoning. Consumption of improperly processed food contaminated with these SEs can lead to acute gastroenteritis. Among these, Staphylococcal enterotoxin B (SEB), the most heat-stable SE, is the most common cause of dairy product contamination; as little as 100 ng of SEB can cause symptoms. Therefore, sensitive, rapid, and low-cost SEB 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, capable of binding to specific metal ions, proteins, lipids, and even cells. They represent a highly specialized class of affinity tools. 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 then designed into the middle of the random sequence in the library to facilitate the screening process and the acquisition of highly specific aptamers. The SEB aptamer, a commonly used and recognized DNA aptamer proposed in 2012, is a DNA aptamer. Upon binding to the aptamer, the SEB molecule significantly stimulates its autofluorescence, generating a fluorescent signal. However, the affinity of the SEB DNA aptamer requires further improvement. Therefore, performing advanced evolutionary screening on the SEB DNA aptamer after rough tailoring can further optimize 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. Aptamers, which can generate output signals through pattern conversion in response to biological components that specifically recognize specific substances, offer advantages such as simple operation, rapid analysis, and stable properties. However, some aptamer biosensors require modification of the signal output molecule, which increases costs and affects the folding dynamics of the aptamer. Furthermore, to improve signal stability and detection sensitivity and reduce systematic errors caused by environmental and instrumental factors, fluorescence ratiometric aptamer sensing strategies are urgently needed. Thioflavin T (ThT), a small molecule fluorescent dye, can be embedded in specific nucleic acid conformations, such as G-quadruplexes and hairpin structures, resulting in high fluorescence quantum yields.

[0006] The present invention introduces an aptamer core sequence and proposes an advanced evolutionary screening scheme. During this screening process, the complex matrix environment of milk was simultaneously created, and aptamers with good recognition performance and excellent matrix adaptability were obtained after only eight rounds of screening. Furthermore, by integrating tailoring technology with bioinformatics, a computer simulation-assisted aptamer tailoring strategy was proposed. This strategy optimizes the tailoring of SEB DNA aptamers through "structural analysis," "molecular docking," and "tailoring optimization," further improving their affinity and luminescence intensity. Furthermore, by introducing the fluorescent small molecule ThT, a fluorescent ratiometric SEB biosensor based on a bifunctional, bivalent aptamer was successfully constructed, ultimately achieving rapid, low-cost, and sensitive ratiometric fluorescence detection of SEB. Summary of the Invention

[0007] Based on this, the present invention proposes an advanced evolutionary screening scheme and an aptamer tailoring strategy assisted by computer simulation, and successfully constructs a fluorescence ratiometric SEB biosensor based on a bifunctional bivalent aptamer.

[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 B aptamer SEB-org (SEQ ID NO.1) is initially 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 a complex matrix of real samples 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] SEB-org: 5'-CACTGGTCGTTGTTGTCTGTTGTCTGTTATGTTGTTTCGT-3', as shown in SEQ ID NO.1;

[0011] The advanced evolution screening scheme involves introducing the truncated SEB aptamer sequences obtained through advanced structural analysis, "docking simulation scoring," and "initial trimming" as core sequences into the in vitro screening of random sequences in the library, thereby reducing the time cost of aptamer screening and improving the binding ability and matrix adaptability of the aptamers.

[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 SEB and the aptamer to obtain a simulation score;

[0013] The “initial tailoring” refers to modifying the SEB aptamer using truncation and cleavage strategies based on molecular docking results and special secondary structures such as hairpins. The affinity of the tailored aptamer to SEB 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] SEB-CJ-2: 5′-CACTGGTCGT-3′, as shown in SEQ ID NO. 2;

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

[0016] SEB-CJ-4: 5'-TGTTGTCTGT-3', as shown in SEQ ID NO. 4;

[0017] SEB-CJ-5: 5′-TGTCTGTTATGTTGTTTCGT-3′, as shown in SEQ ID NO. 5;

[0018] SEB-CJ-6: 5′-CACTGGTCGTCACTGGTCGT-3′, as shown in SEQ ID NO. 6;

[0019] SEB-WK-7: 5′-N15-TGTTGTCTGT-N15 -3′, as shown in SEQ ID NO. 7;

[0020] SEB-SX-8: 5′-GAGACGGCACAAGAATGTTGTCTGTAGCCCAGCCATTATG-3′, as shown in SEQ ID NO. 8;

[0021] SEB-SX-9: 5′-CCCGGTCGCCAGAGTTGTTGTCTGTGTATCGGATTTTCGT-3′, as shown in SEQ ID NO. 9;

[0022] SEB-SX-10: 5'-GGGCGGGAGGTGCGGTGTTGTCTGTGGTCTGGCCAGTCGG-3', as shown in SEQ IDNO.10;

[0023] Specifically, molecular docking was used to predict the binding site of the aptamer SEB-org (SEQ ID NO.1) with SEB. Based on the predicted results, the aptamer SEB-org (SEQ ID NO.1) was "roughly tailored," with the principle of preserving the stem-loop structure as much as possible. The aptamer SEB-org (SEQ ID NO.1) contains only a small, unstable stem-loop structure with a long single-stranded segment at its 3' end. Therefore, in the first round of tailoring, the original 40nt aptamer was trimmed into two nucleic acid strands of 10nt and 30nt, namely SEB-CJ-2 (SEQ ID NO.2) and SEB-CJ-3 (SEQ ID NO.3). Then, the second round of tailoring was conducted on SEB-CJ-2 (SEQ ID NO.2) and SEB-CJ-3 (SEQ ID NO.3): SEB-CJ-6 (SEQ ID NO.6) was obtained by tandem research of SEB-CJ-2 (SEQ ID NO.2); SEB-CJ-4 (SEQ ID NO.4) (10nt) and SEB-CJ-5 (SEQ ID NO.5) (20nt) were obtained by tailoring the linear structure of SEB-CJ-3 (SEQ ID NO.3). After qPCR analysis, the tailored aptamer SEB-CJ-4 (SEQ ID NO.4) was determined to be the optimal core sequence. Subsequently, a total length of 78nt was constructed, and the library capacity was 10 14 A linear ssDNA library was prepared, and a random sequence of 40 nt SEB-WK-7 (SEQ ID NO. 7) was designed. The 10 nt core sequence of the aptamer SEB-CJ-4 (SEQ ID NO. 4) obtained after trimming was placed in the middle of the corresponding SEB-WK-7 (SEQ ID NO. 7) sequence. After eight rounds of evolutionary screening, including two rounds of counter-screening and three rounds of matrix evolutionary screening, 20 aptamers targeting SEB were analyzed and obtained. These aptamers were then divided into six families, with SEB-specific aptamers classified. Based on homology analysis and secondary structure data, three families of aptamers, SEB-SX-8 (SEQ ID NO. 8), SEB-SX-9 (SEQ ID NO. 9), and SEB-SX-10 (SEQ ID NO. 10), were obtained. The affinities of these three aptamers were then verified using qPCR and fluorescence spectrophotometry, and the aptamer SEB-SX-10 (SEQ ID NO. 10) with relatively outstanding performance was preliminarily identified.

[0024] The SEB aptamers screened by the advanced evolution were tailored and optimized with the assistance of computer simulation.

[0025] On the other hand, this application proposes a rational tailoring strategy for aptamers assisted by computer simulation.

[0026] 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 SEB aptamer SEA-SX-10 (SEQ ID NO. 10) is tailored and optimized. The functional properties of the tailored aptamer are then verified by fluorescence and affinity characterization methods.

[0027] The computer simulation-assisted rational tailoring strategy for aptamers refers to improving the binding ability of SEB's DNA aptamer through three steps: "molecular docking", "structural analysis" and "tailoring and modification", while also exploring the dual functions of fluorescence and recognition of the tailored aptamer;

[0028] 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 SEB and the aptamer to identify potential receptor-ligand binding sites;

[0029] 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;

[0030] The "tailoring and modification" mentioned above refers to optimizing and modifying the SEB aptamer using truncation, bivalent or multivalent strategies based on the molecular docking results and special secondary structure. The affinity of the tailored aptamer to SEB 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:

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

[0032] SEB-CJ-12: 5′-TCTGTGGTCTGGCCAGTCGG-3′, as shown in SEQ ID NO. 12.

[0033] SEB-CJ-13: 5′-GGGCGGGAGGTGCGG-3′, as shown in SEQ ID NO. 13;

[0034] SEB-CJ-14: 5′-GGGAGGTGCGGTGTTGTCTGTGGTCTGG-3′, as shown in SEQ ID NO. 14;

[0035] SEB-CJ-19: 5'-GGGAGGTGCGGTGTTGTCTGTGGTCTGGTTTTTTTTGGGAGGTGCGGTGTTGTCTGTGGTCTGG-3', such as SEQ ID NO. 19;

[0036] SEB-CJ-21: 5'-GGTCTGGTTTTTTTTGGGAGGTGCGG-3', such as SEQ ID NO. 21.

[0037] Specifically, the binding sites of the aptamer SEB-SX-10 (SEQ ID NO.10) and SEB were predicted through molecular docking, and the structure of SEB-SX-10 (SEQ ID NO.6) was clarified through structural analysis. Based on the results, the aptamer SEB-SX-10 (SEQ ID NO.10) was tailored and modified. The sequence was first split in the middle to produce SEB-CJ-11 (SEQ ID NO.11) and SEB-CJ-12 (SEQ ID NO.12). Subsequently, SEB-CJ-11 (SEQ ID NO.11) was truncated from G1-G15 and G5-G32, maintaining the specific structure of G4, to produce SEB-CJ-13 (SEQ ID NO.13) and SEB-CJ-14 (SEQ ID NO.14), respectively. After performance evaluation, SEB-CJ-14 (SEQ ID NO. 14) was selected for engineering bivalent aptamer design and construction. The bivalent spacer sequence was optimized. Computer software was used to predict the sequence conformation under different spacer base sequences of 2nt, 4nt, 6nt, 8nt, and 10nt. The optimal spacer sequence under different metal ion conditions was experimentally verified, resulting in the bivalent aptamer SEB-CJ-19 (SEQ ID NO. 19) and its truncated sequence SEB-CJ-21 (SEQ ID NO. 21). Subsequently, the fluorescent small molecule ThT was introduced, and the target toxin SEB was added to explore the fluorescence and recognition dual functions of the truncated sequence.

[0038] Application of the tailored SEB aptamer in SEB detection.

[0039] Finally, this application developed a ratiometric SEB biosensor based on a bifunctional bivalent aptamer, which is characterized by (1) the construction of a bifunctional bivalent aptamer; (2) a construction strategy for a fluorescence ratiometric biosensor; (3) optimization of reaction conditions for the fluorescence ratiometric SEB biosensor; and (4) detection of SEB.

[0040] The construction of the bifunctional bivalent aptamer is based on the principle of increasing the number of binding sites to improve the binding capacity of the aptamer. This experiment studied the structure and performance of the bivalent aptamer with different lengths of connecting chains.

[0041] The 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 SEB aptamer significantly stimulates SEB fluorescence upon binding to the target. The present invention incorporates an embedded small molecule fluorescent dye, ThT, which competes with SEB for aptamer binding sites, thereby affecting the fluorescence intensity of both ThT and SEB. SEB is then quantitatively detected ratiometrically using the ratio of the two fluorescence signals.

[0042] The sequence of the label-free ratiometric SEB luminescent aptamer biosensor is: SEB-CJ-21: 5'-GGTCTGGTTTTTTTTGGGAGGTGCGG-3', as shown in SEQ ID NO. 21;

[0043] The optimization of reaction conditions of the label-free ratiometric SEB biosensor includes the concentration of ThT in the sensor solution, the metal ion conditions and the pH;

[0044] The concentration of ThT fluorescent dye in the label-free ratio SEB biosensor solution is in the range of 0.5 to 50 μmol·L -1；

[0045] Preferably, the concentration of ThT fluorescent dye in the label-free ratiometric SEB biosensor solution is 10 μmol·L -1 ;

[0046] The buffer solution of the label-free ratio SEB 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 );

[0047] Preferably, the buffer solution of the label-free ratiometric SEA biosensor is 10 mmol·L -1 Tris-HCl (pH 7.5) Example conditions are: B9;

[0048] The buffer solution of the label-free ratio SEB biosensor is 10 mmol·L -1 The pH range of Tris-HCl is 5.5 to 9.5;

[0049] Preferably, the buffer solution of the label-free ratiometric SEB biosensor is 10 mmol·L -1 The pH of Tris-HCl is 7.5;

[0050] The detection of SEB is based on the competition between SEB and ThT for the binding sites of the SEB aptamer, which causes the fluorescence excitation intensity of the two to change. The ratio of the SEB fluorescence value to the ThT fluorescence value under specific excitation conditions shows a gradient change with the SEB concentration in the solution, thereby realizing the detection of SEB;

[0051] Specific bifunctional bivalent aptamer construction:

[0052] First, the linker sequence between the bivalent aptamers was optimized, and the sequence conformations at different intervals (0nt, 2nt, 4nt, 6nt, 8nt, 10nt) were predicted using QGRS Mapper. At the same time, the bivalent aptamers under the optimal spacer sequence were further explored using Hemin experiments. The optimal spacer sequence of B1, B2, B3, B4, B5, B6, B7, B8, and B9 under different metal ion conditions was verified: K was used in the experiment. + 、Na +The G4 conformation is regulated by metal ions. After forming a DNA enzyme with hemin, it catalyzes TMB. The optimal linker sequence and corresponding metal ion conditions are determined by the absorbance change after catalysis. Finally, the successful construction of the bivalent aptamer is characterized by exploring the binding site between the aptamer and the target toxin. Molecular docking simulation software such as OpenBabel, HDOCK, DiscoveryStudio, and PyMOL were used to explore the binding site between the aptamer and the target, and the amino acid binding site of SEB was identified.

[0053] Specific steps for establishing a standard curve:

[0054] Different concentrations of SEB were added to the solution containing 1 μmol·L -1 10mmol·L of aptamer solution -1 The mixture was incubated in Tris-HCl (pH 7.5) buffer solution for 15 min, and then 10 μmol·L -1 ThT, so that the final concentration of SEB is 0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 μg·mL -1 The fluorescence intensity of SEB and ThT in the solution was measured at 632 and 487 nm using a fluorescence spectrophotometer. The ratio of the fluorescence peaks of SEB and ThT was found to be proportional to the concentration of SEB at 0.00001 μg mL. -1 ~1 μg·mL -1 There is a good linear relationship within the range (R 2 =0.998), the linear equation is Y=0.54-0.33X.

[0055] On the other hand, the selectivity of the SEB biosensor towards SEB was demonstrated.

[0056] Staphylococcus aureus enterotoxin A (SEA), Staphylococcus aureus enterotoxin C (SEC), lactoferrin and casein were used instead of SEB. The experiment was carried out using a label-free ratiometric SEB biosensor to detect the fluorescence ratio of SEB and ThT at the characteristic wavelength.

[0057] On the other hand, the SEB biosensor detects actual samples containing SEB, and the specific operation is as follows:

[0058] Commercially available pure milk was diluted 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. -1The spike recovery experiment was performed on a SEB solution. The detection signal output was the fluorescence intensity ratio of the SEB and ThT molecules, with fluorescence excitation wavelengths of 632nm and 487nm, respectively. The fluorescence value ratio of SEB and ThT was then substituted into the standard curve to calculate the SEB content in the sample to be tested, achieving quantitative detection of SEB.

[0059] Preferably, commercially available pure milk is diluted 1 / 5 with B9 binding buffer to obtain 79 μL of actual sample. Then, the final concentration of 0, 0.01 or 0.1 μg·mL is added to the 79 μL actual sample. -1 A spike recovery experiment was performed on a SEB solution. The detection signal output was the fluorescence intensity ratio of the SEB and ThT molecules, with fluorescence excitation wavelengths of 632nm and 487nm, respectively. The fluorescence intensity ratio of SEB and ThT was then substituted into the standard curve to calculate the SEB content in the sample, achieving quantitative detection of SEB.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] 1. The present invention proposes an advanced evolutionary screening scheme, which achieves the efficient and low-cost acquisition of Staphylococcus aureus enterotoxin B aptamers; proposes a computer-assisted tailoring strategy, explores the fluorescence and recognition dual functions of Staphylococcus aureus enterotoxin B aptamers, and designs and constructs bivalent bifunctional aptamers; through truncation, bivalence and other tailoring optimization methods, the detection sensitivity and signal stability of Staphylococcus aureus enterotoxin B aptamer sequence biosensors are improved, broadening the application range of the sensor.

[0062] 3. The present invention proposes a universal ratiometric sensing method, which introduces embedded small molecule nucleic acid dyes to achieve stable ratiometric sensing detection through the principle of binding site competition.

[0063] 5. The Staphylococcus aureus enterotoxin B biosensor proposed in the present invention can realize low-cost, rapid, stable and sensitive ratio detection of Staphylococcus aureus enterotoxin B, and has certain versatility and industrialization potential.

[0064] 6. The Staphylococcus aureus enterotoxin B biosensor proposed in the present invention has a good linear relationship (R 2 =0.991), the linear regression equation is Y=0.89-0.25X, and the detection limit is as low as 0.126ng·mL -1 . BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1Results of SEB-org advanced evolutionary screening. (A) SEB-org secondary structure; (B) SEB-org tertiary structure; (C) SEB-org overall tailoring scheme; (D) affinity comparison between SEB-CJ-4 truncated sequence and SEB-org; ​​(E) affinity comparison between SEB-CJ-5 truncated sequence and SEB-org; ​​(F) SEB-WK-7 advanced evolutionary screening round monitoring results; (G) homology analysis results of the 8th round of evolutionary screening sequences.

[0066] Figure 2 Affinity evaluation of aptamers selected for evolutionary selection of Staphylococcus aureus enterotoxin B. (A) Quantitative PCR analysis of affinity of the selected sequences; (B) SEB-org fluorescence analysis of K d (C) is the SEB-SX-10 fluorescence method for determining K d Value result.

[0067] Figure 3 Molecular docking results of SEB-SX-10 and Staphylococcus aureus enterotoxin B. (A) SEB-SX-10 secondary structure; (B) SEB-SX-10 and Staphylococcus aureus enterotoxin B molecular docking demonstration; (C) SEB-SX-10 G4 structure verification.

[0068] Figure 4 The molecular docking results of SEB-CJ-14 and Staphylococcus aureus enterotoxin B. (A) is the secondary structure of SEB-CJ-14; (B) is the Ct value of SEB-CJ-13 and SEB-CJ-14; (C) is the K value of SEB-SX-10. d Value results; (D) is the K of SEB-CJ-14 d (E) is the structure prediction of each bivalent aptamer under different connection chains.

[0069] Figure 5 This demonstrates the feasibility of constructing a fluorescent ratiometric biosensor for Staphylococcus aureus enterotoxin B. (A) 2D structure of the SEB-CJ-21 / ThT complex; (B) 2D structure of the SEB-CJ-21 / Staphylococcus aureus enterotoxin B complex; (C) Fluorescence spectra of SEB-CJ-21 binding to S. aureus enterotoxin B and ThT.

[0070] Figure 6 Performance evaluation of the fluorescence ratiometric Staphylococcus aureus enterotoxin B biosensor. (A) Fluorescence spectra of dual signals at different Staphylococcus aureus enterotoxin B concentrations; (B) Standard curve for the sensor's detection of Staphylococcus aureus enterotoxin B.

[0071] Figure 7 Specificity evaluation of a fluorescent ratiometric Staphylococcus aureus enterotoxin B biosensor.

[0072] Figure 8 Optimization of reaction conditions for a fluorescent ratiometric Staphylococcus aureus enterotoxin B biosensor. (A) ThT concentration; (B) ionic conditions; (C) pH value.

[0073] Figure 9 Schematic diagram of the fluorescent ratiometric Staphylococcus aureus enterotoxin B biosensor based on a bifunctional bivalent aptamer. DETAILED DESCRIPTION

[0074] 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.

[0075] Example 1. Advanced Evolution Screening of Staphylococcus aureus Enterotoxin B Aptamers

[0076] 1. Obtaining the core sequence of Staphylococcus aureus enterotoxin B aptamer

[0077] The recognition ability and matrix adaptability of the reported Staphylococcus aureus enterotoxin B DNA aptamers need to be improved. In order to improve the performance of the aptamer, the original Staphylococcus aureus enterotoxin B 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.

[0078] The nucleotide sequences involved in the examples are shown in Table 1.

[0079] Table 1 Summary of nucleic acid sequences

[0080]

[0081]

[0082] The secondary structure of SEB-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 SEB-org (SEQ ID NO. 1) and Staphylococcus aureus enterotoxin B was performed using the HDOCK website to obtain an HDOCK score.

[0083] like Figure 1 As shown in A and B, the high-level structure of SEB-org (SEQ ID NO.1) only contains an unstable small "stem-loop" structure with a long single strand at its 3' end. Figure 1 C aptamer overall tailoring scheme, the tailoring experiment was divided into two rounds, first the original aptamer SEB-org (SEQ ID NO.1) with a full length of 40nt was tailored into two nucleic acid chains of 10nt and 30nt, respectively named SEB-CJ-2 (SEQ ID NO.2) and SEB-CJ-3 (SEQ ID NO.3). In the second round of tailoring, SEB-CJ-2 (SEQ ID NO.2) and SEB-CJ-3 (SEQ ID NO.3) were tailored separately: SEB-CJ-2 (SEQ ID NO.2) was tandemly studied to obtain SEB-CJ-6 (SEQ ID NO.6), which was then Figure 1 C shows that the HDOCK score of SEB-CJ-6 (SEQ ID NO.6) is not improved compared with SEB-org (SEQ ID NO.1); the linear structure of SEB-CJ-3 (SEQ ID NO.3) was trimmed to obtain two aptamers, SEB-CJ-4 (SEQ ID NO.4) (10nt) and SEB-CJ-5 (SEQ ID NO.5) (20nt). SEB-CJ-4 (SEQ ID NO.4) has an effect of improving affinity compared with the original sequence SEB-org (SEQ ID NO.1) ( Figure 1 D), while the affinity of SEB-CJ-5 (SEQ ID NO.5) was not significantly improved compared with SEB-org (SEQ ID NO.1), and the Ct value did not change significantly ( Figure 1 E).

[0084] The aptamer sequences used:

[0085] SEB-org: 5'-CACTGGTCGTTGTTGTCTGTTGTCTGTTATGTTGTTTCGT-3', such as SEQ ID NO.1

[0086] SEB-CJ-2: 5′-CACTGGTCGT-3′, as shown in SEQ ID NO. 2;

[0087] SEB-CJ-3: 5′-TGTTGTCTGTTGTCTGTTATGTTGTTTCGT-3′, as shown in SEQ ID NO. 3;

[0088] SEB-CJ-4: 5'-TGTTGTCTGT-3', as shown in SEQ ID NO. 4;

[0089] SEB-CJ-5: 5′-TGTCTGTTATGTTGTTTCGT-3′, as shown in SEQ ID NO. 5;

[0090] SEB-CJ-6: 5′-CACTGGTCGTCACTGGTCGT-3′, as shown in SEQ ID NO. 6;

[0091] 2. Evolution and Screening of Staphylococcus aureus Enterotoxin A Aptamers

[0092] 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 SEB-WK-7 (SEQ ID NO.7) library, making the screening more controllable and easier to obtain aptamers with good performance efficiently.

[0093] The truncated aptamer of SEB-CJ-4 (SEQ ID NO. 4) 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. The full length of the sequence was 78 nt and the library capacity was about 10 14 A random library was prepared. 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 and 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 SEB aptamer sequences were divided into six families using homology analysis, secondary structure prediction, and minimum free energy. The performance of the candidate sequences in each family was analyzed.

[0094] 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 F), thus we can know the progress of evolutionary screening and determine the final number of evolutionary screening rounds to be round 8. The library of round 8 was amplified by PCR and then sequenced. The first 20 sequences obtained by sequencing were analyzed and a homology tree was made based on the results ( Figure 1G) for homology analysis. Based on the above homology analysis and secondary structure prediction, the 20 sequences can be divided into six major families. Based on the abundance of representative sequences in each of the six major aptamer families and the homology evaluation between the sequences in each group, three candidate aptamers were finally selected: SEB-SX-8 (SEQ ID NO. 8), SEB-SX-9 (SEQ ID NO. 9), and SEB-SX-10 (SEQ ID NO. 10).

[0095] The aptamer sequences used:

[0096] SEB-CJ-4: 5'-TGTTGTCTGT-3', as shown in SEQ ID NO. 4;

[0097] SEB-WK-7: 5′-N15-TGTTGTCTGT-N15 -3′, as shown in SEQ ID NO. 7;

[0098] SEB-SX-8: 5′-GAGACGGCACAAGAATGTTGTCTGTAGCCCAGCCATTATG-3′, as shown in SEQ ID NO. 8;

[0099] SEB-SX-9: 5′-CCCGGTCGCCAGAGTTGTTGTCTGTGTATCGGATTTTCGT-3′, as shown in SEQ ID NO. 9;

[0100] SEB-SX-10: 5'-GGGCGGGAGGTGCGGTGTTGTCTGTGGTCTGGCCAGTCGG-3', as shown in SEQ IDNO.10;

[0101] 3. Affinity Verification of Staphylococcus aureus Enterotoxin B Aptamers

[0102] Subsequently, the affinity of the three aptamers SEB-SX-8 (SEQ ID NO. 8), SEB-SX-9 (SEQ ID NO. 9), and SEB-SX-10 (SEQ ID NO. 10) obtained after evolutionary screening was verified using qPCR and fluorescence titration methods.

[0103] qPCR assay principle: Use a qPCR instrument to measure the Ct values ​​of the bound aptamers SEB-SX-8 (SEQ ID NO. 8), SEB-SX-9 (SEQ ID NO. 9), and SEB-SX-10 (SEQ ID NO. 10). A lower average Ct value indicates a higher affinity for the aptamer binding to the target protein.

[0104] The specific experimental steps of the qPCR method are as follows:

[0105] First, the qPCR template was obtained by the magnetic bead immobilization target method: the enterotoxin was diluted, 10 μL of 2.5 mg / mL SEB was taken into 240 μL PBST, mixed thoroughly, and stored in a -20°C refrigerator for later use; then, the carboxyl magnetic beads (MB-COOH) were activated, 20 μL of 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 SEB protein, and 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-SEB protein complex, and magnetic separation and washing were performed to remove unbound proteins. The complex was washed three times with 100 μL PBST. The washed magnetic bead-SEB 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.

[0106] 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.

[0107] The specific experimental steps of fluorescence titration are as follows:

[0108] To the solution containing 1 μmol·L -1 Staphylococcus aureus enterotoxin B aptamer buffer solution 10 mmol·L -1Staphylococcus aureus enterotoxin B 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 concentration of Staphylococcus aureus enterotoxin B to obtain the affinity constant.

[0109] like Figure 2 As shown in A, the qPCR experimental results show that among the three sequences SEB-SX-8 (SEQ ID NO.8), SEB-SX-9 (SEQ ID NO.9), and SEB-SX-10 (SEQ ID NO.10), SEB-SX-10 (SEQ ID NO.10) has a lower average Ct value, that is, it has a stronger binding ability with SEB, with a Ct value of 14.81. Therefore, the SEB-SX-10 (SEQ ID NO.10) aptamer was selected for the experiment, and its K value was specifically determined using a fluorescence spectrophotometer. d The value was compared with that of the original aptamer SEB-org (SEQ ID NO. 1). Figure 2 As shown in B and C, the K of SEB-SX-10 (SEQ ID NO.10) aptamer d The value is 43.71nM, which is higher than the binding affinity of the original aptamer.

[0110] The aptamer sequences used in the experiment are as follows:

[0111] SEB-org: 5'-CACTGGTCGTTGTTGTCTGTTGTCTGTTATGTTGTTTCGT-3', as shown in SEQ ID NO.1;

[0112] SEB-SX-8: 5′-GAGACGGCACAAGAATGTTGTCTGTAGCCCAGCCATTATG-3′, as shown in SEQ ID NO. 8;

[0113] SEB-SX-9: 5′-CCCGGTCGCCAGAGTTGTTGTCTGTGTATCGGATTTTCGT-3′, as shown in SEQ ID NO. 9;

[0114] SEB-SX-10: 5'-GGGCGGGAGGTGCGGTGTTGTCTGTGGTCTGGCCAGTCGG-3', as shown in SEQ IDNO.10;

[0115] Example 2. Computer simulation-assisted evolutionary tailoring of Staphylococcus aureus enterotoxin B aptamers

[0116] 1. Molecular Docking of Staphylococcus aureus Enterotoxin B Aptamer

[0117] To mitigate the effects of redundant sequences in the Staphylococcus aureus enterotoxin B aptamer sequences obtained after advanced evolution screening on aptamer performance, the Staphylococcus aureus enterotoxin B aptamer was tailored using computer simulation, and 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.

[0118] The secondary structure of SEB-SX-10 (SEQ ID NO. 10) was predicted using the mfold website. RNA Composer 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 SEB-SX-10 (SEQ ID NO. 10) and Staphylococcus aureus enterotoxin B 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 SEB-SX-10 (SEQ ID NO. 10), QGRS Mapper was used to predict the G4 structure and analyze whether a G4 structure existed.

[0119] like Figure 3 As can be seen from A, the high-level structure of SEB-SX-10 (SEQ ID NO.10) mainly contains a stem-loop structure. In the aptamer-target interaction model, the aptamer can bind to the binding site of Staphylococcus aureus enterotoxin B ( Figure 3 B) At the same time, Figure 3 As shown in C, there is a G4 structure at nucleotides G5-G32. Combined with the HDOCK score, it is speculated that the G4 structure at G5-G32 is the main binding domain.

[0120] The aptamer sequences used:

[0121] SEB-SX-10: 5'-GGGCGGGAGGTGCGGTGTTGTCTGTGGTCTGGCCAGTCGG-3', as shown in SEQ ID NO. 10.

[0122] 2. Tailoring of Staphylococcus aureus Enterotoxin B Aptamers

[0123] In order to further clarify the potential binding domain of Staphylococcus aureus enterotoxin B 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.

[0124] Since SEB-SX-10 (SEQ ID NO.10) has a G4 structure, the G4 structure was retained as much as possible during cutting. First, the sequence of SEB-SX-10 (SEQ ID NO.10) was split from the middle to obtain SEB-CJ-11 (SEQ ID NO.11) and SEB-CJ-12 (SEQ ID NO.12), and then SEB-CJ-11 (SEQ ID NO.11) was cut from G1-G15 and G5-G32, maintaining the special G4 structure, and obtaining SEB-CJ-13 (SEQ ID NO.13) and SEB-CJ-14 (SEQ ID NO.14), respectively. Molecular docking was used to simulate the interaction between the aptamer and Staphylococcus aureus enterotoxin B, and the specific method was the same as above. Subsequently, the Ct values ​​of the SEB-CJ-13 (SEQ ID NO.13) and SEB-CJ-14 (SEQ ID NO.14) 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 B, 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 B. At the same time, it was compared with the K of the SEB-SX-10 (SEQ ID NO. 10) aptamer. d The results were compared with those obtained by qPCR. Figure 4 As shown in C and D, SEB-CJ-14 (SEQ ID NO.14) aptamer K d The affinity value of SEB-SX-10 (SEQ ID NO. 10) was reduced from 43.71 nM to 43.29 nM, and an aptamer with excellent affinity for Staphylococcus aureus enterotoxin B was obtained.

[0125] Subsequently, SEB-CJ-14 (SEQ ID NO. 14) was selected for the design and construction of an engineered bivalent aptamer. Under the spacer sequences of 0ntT (SEB-CJ-15), 2ntT (SEB-CJ-16), 4ntT (SEB-CJ-17), 6ntT (SEB-CJ-18), 8ntT (SEB-CJ-19), and 10ntT (SEB-CJ-20), 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 Figure E (where the yellow-labeled bases form G4), the 8ntT spacer sequence in the bivalent aptamer forms a new G4 structure, which expands the G4 central loop compared to the one without the spacer sequence, and then separates the two G4 structures starting from the 10ntT. SEB-CJ-19 (SEQ ID NO. 19), which formed the new G4 structure, was subsequently truncated to obtain the new bivalent aptamer SEB-CJ-21 (SEQ ID NO. 21).

[0126] The aptamer sequences used in the experiment are as follows:

[0127] SEB-SX-10: 5'-GGGCGGGAGGTGCGGTGTTGTCTGTGGTCTGGCCAGTCGG-3', as shown in SEQ IDNO.10;

[0128] SEB-CJ-11: 5′-GGGCGGGAGGTGCGGTGTTG-3′, as shown in SEQ ID NO. 11;

[0129] SEB-CJ-12: 5′-TCTGTGGTCTGGCCAGTCGG-3′, as shown in SEQ ID NO. 12.

[0130] SEB-CJ-13: 5′-GGGCGGGAGGTGCGG-3′, as shown in SEQ ID NO. 13;

[0131] SEB-CJ-14: 5′-GGGAGGTGCGGTGTTGTCTGTGGTCTGG-3′, as shown in SEQ ID NO. 14;

[0132] SEB-CJ-15: 5'-GGGAGGTGCGGTGTTGTCTGTGGTCTGGGGGAGGTGCGGTGTTGTCTGTGGTCTGG-3', such as SEQ ID NO.15;

[0133] SEB-CJ-16: 5'-GGGAGGTGCGGTGTTGTCTGTGGTCTGGTTGGGAGGTGCGGTGTTGTCTGTGGTCTGG-3', such as SEQ ID NO. 16;

[0134] SEB-CJ-17: 5'-GGGAGGTGCGGTGTTGTCTGTGGTCTGGTTTTGGGAGGTGCGGTGTTGTCTGTGGTCTGG-3', such as SEQ ID NO. 17;

[0135] SEB-CJ-18: 5'-GGGAGGTGCGGTGTTGTCTGTGGTCTGGTTTTTTGGGAGGTGCGGTGTTGTCTGTGGTCTGG-3', such as SEQ ID NO. 18;

[0136] SEB-CJ-19: 5'-GGGAGGTGCGGTGTTGTCTGTGGTCTGGTTTTTTTTGGGAGGTGCGGTGTTGTCTGTGGTCTGG-3', such as SEQ ID NO. 19;

[0137] SEB-CJ-20: 5'-GGGAGGTGCGGTGTTGTCTGTGGTCTGGTTTTTTTTTTGGGAGGTGCGGTGTTGTCTGTGGTCTGG-3', such as SEQ ID NO. 20;

[0138] SEB-CJ-21: 5'-GGTCTGGTTTTTTTTGGGAGGTGCGG-3', such as SEQ ID NO. 21.

[0139] Example 3. Feasibility Verification of Staphylococcus aureus Enterotoxin B Fluorescence Ratio Biosensor

[0140] 1. Construction strategy of fluorescence ratiometric biosensor

[0141] To construct a fluorescent ratiometric biosensor, SEB-SX-10 (SEQ ID NO. 10), obtained through advanced evolutionary screening, was tailored and modified using a computer simulation-guided tailoring strategy. This improved the affinity of the aptamer, resulting in the bifunctional bivalent aptamer SEB-CJ-21 (SEQ ID NO. 21). The biosensor construction strategy is based on the competition of fluorescent molecules for the binding sites of the luminescent aptamer. The modified S. aureus enterotoxin B luminescent aptamer significantly stimulates the fluorescence of S. aureus enterotoxin B upon binding to the target. The embedded small molecule fluorescent dye introduced into the system competes with S. aureus enterotoxin B for the same binding site on the aptamer, generating a dynamic equilibrium effect based on competition in the solution. Based on this principle, changes in the S. aureus enterotoxin B content in the solution alter the fluorescence intensity of ThT and S. aureus enterotoxin B. The ratio of these two fluorescence signals allows ratiometric detection of the S. aureus enterotoxin B content in the solution.

[0142] 2. Feasibility Verification of Fluorescence Ratiometric Biosensor

[0143] In order to verify the feasibility of the ratiometric sensor construction strategy, we performed molecular docking prediction on SEB-CJ-21 (SEQ ID NO. 21) and ThT. Figure 5 As shown in Figures A and B, SEB-CJ-21 (SEQ ID NO. 21) shares some of the same binding sites with ThT and Staphylococcus aureus enterotoxin B. Therefore, computer prediction and simulation indicate that the dual-signal combination of ThT and Staphylococcus aureus enterotoxin B can achieve fluorescence ratiometric sensing by competing for the same binding site of SEB-CJ-21 (SEQ ID NO. 21).

[0144] Then, 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 B significantly changes the luminescence intensity of ThT and itself, proving the feasibility of the construction strategy of the fluorescence ratio biosensor.

[0145] Example 4. Detection Performance Evaluation of Fluorescence Ratiometric Staphylococcus aureus Enterotoxin B Biosensor

[0146] 1. Sensitivity Evaluation of the Fluorescence Ratiometric Staphylococcus aureus Enterotoxin B Biosensor

[0147] SEB-CJ-21 (SEQ ID NO. 21) was used to detect the known concentrations of Staphylococcus aureus enterotoxin B. A standard curve was prepared based on the change in the fluorescence ratio of Staphylococcus aureus enterotoxin B and ThT in the solution. Different concentrations of Staphylococcus aureus enterotoxin B 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 10 μmol·L -1 ThT, and the final concentrations of Staphylococcus aureus enterotoxin B were 0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, and 10 μg mL -1 Finally, the fluorescence intensity of the solution system of Staphylococcus aureus enterotoxin B and ThT was measured at 632nm and 487nm using a fluorescence spectrophotometer, and a standard curve was drawn based on the fluorescence ratio of the two.

[0148] like Figure 6 As shown, the fluorescence ratiometric Staphylococcus aureus enterotoxin B biosensor was -1 ~1 μg·mL -1 There is a good linear relationship within the range (R 2=0.998), the linear regression equation is Y=0.54-0.33X, and the detection limit of the real sample is as low as 0.126ng·mL -1 .

[0149] 2. Specificity Evaluation of the Fluorescence Ratiometric Staphylococcus aureus Enterotoxin B Biosensor

[0150] Staphylococcus aureus enterotoxin A (SEA), Staphylococcus aureus enterotoxin C (SEC), lactoferrin and casein were used to evaluate the specificity of the Staphylococcus aureus enterotoxin B biosensor. -1 Staphylococcus aureus enterotoxin B or SEA, 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 10 μmol·L -1 ThT was measured immediately after oscillation and mixing at room temperature. Finally, the fluorescence intensity of S. aureus enterotoxin B and ThT in the solution was measured at 632 nm 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 B content.

[0151] like Figure 7 As shown, the addition of SEA, SEC, lactoferrin, and casein did not significantly affect the detection results of Staphylococcus aureus enterotoxin B, indicating that the Staphylococcus aureus enterotoxin B aptamer has good selectivity.

[0152] Example 5. Optimization of detection conditions for a fluorescence ratiometric Staphylococcus aureus enterotoxin B biosensor

[0153] First, the ThT concentration of the Staphylococcus aureus enterotoxin B sensor system was optimized. -1 Staphylococcus aureus enterotoxin B 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 shaking at room temperature. Finally, the fluorescence intensity of Staphylococcus aureus enterotoxin B and ThT in the solution system was measured at 632 nm and 487 nm using a fluorescence spectrophotometer. Figure 8As shown in A, the final concentration of ThT was 10 μmol·L -1 It has a higher detection signal-to-noise ratio and lower detection cost.

[0154] Secondly, the metal ion conditions of the Staphylococcus aureus enterotoxin B sensor system were optimized. -1 Staphylococcus aureus enterotoxin B and 10 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 B and ThT in the solution system at 632nm and 487nm was measured using a fluorescence spectrophotometer. Figure 8As shown in B, when the buffer solution is 10mmol·L -1 Na in Tris-HCl (pH 7.5) + 0mmol·L -1 , K + 140mmol·L -1 A high-intensity fluorescence signal will appear when the B9 ion solution is added to the detection system.

[0155] Finally, the pH of the Staphylococcus aureus enterotoxin B sensor system was optimized. -1 Staphylococcus aureus enterotoxin B and 10 μmol·L -1 ThT was added to a concentration of 1 μmol·L -1 10mmol·L of aptamer solution -1 The pH values ​​of the buffer solutions in Tris-HCl 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 B and ThT at 632 nm 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.

[0156] Example 6. Detection of Staphylococcus aureus Enterotoxin B in Real Samples Using a Fluorescence Ratiometric Biosensor

[0157] In the real sample spike recovery experiment, commercially available pure milk was diluted 1 / 5 with B9 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 Staphylococcal enterotoxin B solution. The fluorescence intensity ratio of the signal output molecules, Staphylococcal enterotoxin B and ThT, was detected. The fluorescence excitation wavelengths were 632 nm and 487 nm, respectively. The fluorescence intensity ratio of Staphylococcal enterotoxin B and ThT was then substituted into the standard curve to calculate the content of Staphylococcal enterotoxin B in the test sample, achieving quantitative detection of Staphylococcal enterotoxin B.

[0158] As shown in Table 1, the recovery rate of dairy products spiked with Staphylococcus aureus enterotoxin B biosensor was ideal, indicating that the biosensor can achieve quantitative detection of Staphylococcus aureus enterotoxin B in real samples.

[0159] Table 1 Experimental study on the addition of Staphylococcus aureus enterotoxin B in dairy products based on fluorescence ratiometric biosensor

[0160]

Claims

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

4.

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

3. A Staphylococcus aureus enterotoxin B nucleic acid aptamer obtained by advanced evolution screening, characterized in that: The aptamer sequence is shown in any one of SEQ ID NOs. 8 to 10, SEQ ID NOs. 13 to 14, and SEQ ID NO.

21.

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

5. A fluorescence ratio-type Staphylococcus aureus enterotoxin B luminescent aptamer biosensor, characterized in that: (1) Construction strategy of fluorescence ratiometric biosensor; (2) Sequence of fluorescence ratiometric Staphylococcus aureus enterotoxin B biosensor; (3) Optimization of fluorescence ratiometric Staphylococcus aureus enterotoxin B biosensor conditions; (4) Detection of Staphylococcus aureus enterotoxin B; The construction strategy of the fluorescence ratio 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 fluorescent ratiometric Staphylococcus aureus enterotoxin B biosensor is shown in SEQ ID NO.

21.

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 B; Staphylococcal enterotoxin A, Staphylococcal enterotoxin C, lactoferrin and casein were used instead of Staphylococcal enterotoxin B. The fluorescence ratio of Staphylococcal enterotoxin B and thioflavin T at the characteristic wavelength was detected using a fluorescence ratio-type Staphylococcal enterotoxin B biosensor.

8. The method for quantitatively detecting Staphylococcus aureus enterotoxin B using a biosensor according to any one of claims 5 to 7, wherein: Establishment of standard curve: Different concentrations of Staphylococcus aureus enterotoxin B 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 10 μmol·L -1 Thioflavin T was added to make the final concentrations of Staphylococcus aureus enterotoxin B 0, 0.00001, 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 B solution to detect the fluorescence intensity ratio of the signal output molecules Staphylococcal enterotoxin B and thioflavin T, with the fluorescence excitation wavelengths being 632 and 487 nm, respectively. The fluorescence value ratio of Staphylococcal enterotoxin B and thioflavin T was then substituted into the standard curve to calculate the content of Staphylococcal enterotoxin B 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 B detection method or a food safety detection kit.

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