Alpha-canthine specific nucleic acid aptamer and test paper detection method thereof

Abstract

The invention provides a specific nucleic acid aptamer of alpha-camarine and a test paper detection method of the specific nucleic acid aptamer. The nucleic acid aptamer has a nucleic acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2. The invention also provides a lateral flow chromatography detection test strip for alpha-calasodine. The lateral flow chromatography detection test strip comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, the conjugate pad is sprayed with a gold-labeled probe containing the aptamer and gold nanoparticles; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is fixedly provided with a first capture molecule, the first capture molecule is streptavidin, the quality control line is fixedly provided with a second capture molecule, and the second capture molecule is a conjugate of a streptavidin-biotinylated aptamer. According to the nucleic acid aptamer, the gold-labeled probe and the test strip disclosed by the invention, the detection efficiency of the alpha-camarin detection method is greatly improved, and the on-site instant demand is met.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to α-chaconine specific aptamer and its detection test paper and detection method. BACKGROUND

[0002] α-chaconine (CHA) is a major solanum alkaloid toxin naturally occurring in potato and other solanaceae plants. Its content increases significantly during potato germination, greening or improper storage. If it enters the human body through food, it may cause nausea, vomiting and other poisoning reactions, posing a potential threat to food safety. Therefore, it is of great significance to establish an efficient CHA detection method to protect agricultural product quality and human health.

[0003] Currently, a variety of analytical methods have been applied to the detection and quantitative analysis of CHA. Early methods (such as gravimetric method and colorimetric method) cannot distinguish different solanum alkaloid components and have poor specificity, and have been basically eliminated, only found in some earlier toxicology research and clinical case reports. The current mainstream detection technologies include chromatography, enzyme-linked immunosorbent assay (ELISA), biosensor method, etc.

[0004] Chromatography, combined with different detection instruments (such as HPLC-MS and GC-MS), can realize high-specificity qualitative and quantitative analysis of CHA, and is the gold standard for laboratory detection of CHA. However, chromatography requires professional instruments and operators, and the detection process is often time-consuming and costly, which is difficult to meet the needs of on-site rapid detection.

[0005] ELISA is low in cost, fast in speed and high in sensitivity, but it depends on the development of specific antibodies and has a high false positive rate and poor accuracy. Biosensor method is simple to operate and portable, suitable for various express occasions, but the biological recognition element is easily affected by the environment and has outstanding stability problems.

[0006] In addition, with the continuous development of analytical techniques, some new detection methods have shown unique advantages in CHA detection, meeting the specific needs of different application scenarios. Capillary electrophoresis is suitable for precise analysis of trace samples due to its ultra-high separation efficiency and extremely low sample consumption; near-infrared spectroscopy has been applied to potato and its product processing production line due to its non-destructive and rapid (about 30 seconds for single detection) characteristics, and can realize real-time online monitoring of CHA content. Matrix-assisted laser desorption / ionization can realize the detection of CHA in complex samples.

[0007] provides spatial distribution information of CHA in plant tissues. These emerging technologies and traditional detection methods complement each other's advantages and together build a complete CHA detection system from the laboratory to practical production applications. SUMMARY

[0008] In order to solve the problems in the prior art, the application uses a nuclease-assisted SELEX technology to screen a high-affinity CHA-specific aptamer, and systematically explores the thermodynamic changes, conformational changes and binding site analysis of the aptamer and CHA binding process. Based on the screened nucleic acid aptamer, a rapid visual detection method for CHA is established. The method overcomes the shortcomings of traditional detection methods, such as long time consumption, high cost, complicated operation and difficulty in instant detection, and not only provides new insights into the molecular recognition mechanism of nucleic acid aptamer recognizing alkaloid toxins, but also provides an efficient on-site detection method and idea for food and environmental safety detection.

[0009] In one aspect, the application provides a nucleic acid aptamer specifically binding to alpha-chaconine (CHA), wherein the nucleic acid aptamer has a nucleic acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 2.

[0010] Further, the nucleic acid aptamer is a single-stranded DNA aptamer with a length of 55 nt or 25 nt.

[0011] Further, when the nucleic acid aptamer SEQ ID NO. 1 binds to CHA, it enters its own binding pocket through van der Waals force and hydrophobic force, forms a total of 9 hydrogen bonds with T15, G17, T18 and G24 base sites, the binding energy is -9.9 kcal / mol, and the affinity constant (Kd) is 0.333 μM.

[0012] Further, the nucleic acid aptamer SEQ ID NO. 2 is obtained by truncating the nucleic acid aptamer SEQ ID NO. 1 at the 3' end 30T site, and when it binds to CHA, it forms a total of 5 hydrogen bonds with G16, A17 and T21 base sites through van der Waals force and hydrophobic force, the binding energy is -9.8 kcal / mol, and the affinity constant (Kd) is 0.326 μM.

[0013] Further, during the binding process of the nucleic acid aptamer and CHA, the enthalpy change ΔH < 0 and the entropy change ΔS < 0, the binding process is a hydrogen bond force-driven exothermic reaction, and a complex with lower heat content and higher conformational stability is formed; and after binding, the circular dichroism spectrum of the aptamer is characterized by an increase in absorbance at 285 nm and a decrease in absorbance at 240 nm, and the secondary structure content is increased.

[0014] In another aspect, the application provides a gold label probe for detecting alpha-solanine, which is a conjugate of a thiolated complementary strand of the above-mentioned aptamer and gold nanoparticles (AuNPs); the thiolated complementary strand of the above-mentioned aptamer is a thiol (-SH) modified complementary single-stranded DNA, which is a single-stranded DNA complementary to at least a part of the above-mentioned aptamer.

[0015] Further, the sequence of the complementary single-stranded DNA is shown in any one of SEQ ID NO. 3-7, preferably shown in SEQ ID NO. 6.

[0016] Further, the particle size of the gold nanoparticles is 5-50 nm.

[0017] Further, the preparation method of the gold label probe comprises:

[0018] S1) mixing TCEP with the thiolated above-mentioned aptamer and incubating to activate the thiolated above-mentioned aptamer;

[0019] S2) mixing the activated thiolated above-mentioned aptamer with gold nanoparticles, standing to allow sufficient binding, and gradually increasing the ionic concentration of the solution;

[0020] S3) purifying to obtain the gold label probe.

[0021] Further, the method for gradually increasing the ionic concentration of the solution is adding 20 μL of 0.5×PBS (containing 68 mM NaCl, 1.3 mM KCl, 4.0 mM Na2HPO4, and 1.0 mM KH2PO4) every 2 h for a total of 3 times.

[0022] In another aspect, the application provides a lateral flow chromatographic test strip for detecting alpha-solanine, which comprises a conjugate pad, a nitrocellulose membrane (NC membrane), and a water absorption pad; the conjugate pad is sprayed with the above-mentioned gold label probe; the nitrocellulose membrane is provided with a test line (T line) and a quality control line (C line), the test line is fixed with a first capture molecule, which is streptavidin, and the quality control line is fixed with a second capture molecule, which is a complex of streptavidin-biotinylated aptamer.

[0023] The complex of streptavidin-biotinylated aptamer is streptavidin and biotin-modified above-mentioned aptamer.

[0024] Further, the binding pad is pretreated with a treatment liquid, the treatment liquid comprising 1% glucose, 6% BSA, 0.5% Tween-20, 0.1M Tris-HCl, and the pH being 8.0; the coating concentration of streptavidin on the NC membrane is 4mg / mL, and the coating concentration of biotinylated aptamer is 10nM.

[0025] Further, the coating concentration of streptavidin on the detection line is 4mg / mL.

[0026] Further, the concentration of streptavidin-biotinylated aptamer complex on the quality control line is 10nM.

[0027] In still another aspect, the present application provides a method for detecting alpha-solanine, comprising the following steps:

[0028] S01) sample pretreatment: mixing the sample to be detected with a buffer solution of biotinylated aptamer and incubating;

[0029] S02) test strip detection: dropping the mixture of step S01) to the sample adding area of the lateral flow chromatographic test strip, and chromatographing for 15min at room temperature;

[0030] S03) result interpretation: if the quality control line (C line) appears a red band, and the detection line (T line) has no band or the band is significantly lighter, it is determined as positive; if the C line and the T line both appear a red band, it is determined as negative; if the C line has no band, it is determined as invalid test strip.

[0031] In the application, the biotinylated aptamer is a nucleic acid aptamer modified by biotin.

[0032] In still another aspect, the present application provides the application of the above-mentioned lateral flow chromatographic test strip for alpha-solanine in the rapid on-site detection of alpha-solanine in potatoes or products thereof.

[0033] Advantages

[0034] The CHA-specific aptamer (especially Apt-55) obtained by the lambda-exonuclease-assisted SELEX technology in the present research has outstanding molecular recognition advantages. It not only has a binding energy as low as -9.9 kcal / mol with CHA, forms nine stable hydrogen bonds through specific base sites such as T15 and G17, but also exhibits a high affinity (Kd value) of 0.333 μM, and the binding process is a hydrogen bond-driven exothermic reaction that can form a complex with lower heat content and more stable conformation. The secondary structure content of the aptamer is significantly increased after binding with CHA, as verified by circular dichroism, further strengthening the binding stability. Compared with traditional antibodies or ordinary nucleic acid aptamers, the aptamer has stronger molecular recognition specificity, higher affinity, and better anti-interference ability, which solves the core pain point of "insufficient recognition element performance leading to low detection precision and poor stability" in existing detection technologies from the source. The Apt-LFA detection system constructed based on the above high-optimization aptamer realizes a visual detection limit of 0.5 ng / mL and a visual judgment threshold of 1 ng / mL under optimal experimental conditions, which significantly improves the sensitivity compared with traditional mainstream detection technologies such as HPLC (high-performance liquid chromatography) and ELISA (enzyme-linked immunosorbent assay), and can accurately capture low-concentration α-chaconine (CHA) residues, meeting the stringent requirements of the food and environmental safety field for trace toxin detection and filling the technical gap of existing rapid detection methods in low-concentration CHA recognition.

[0035] The detection efficiency is greatly improved, and the aptamer meets the on-site demand. The detection system does not require complex instruments and professional operation training throughout the process, and the whole process from sample incubation to result interpretation can be completed in 15 minutes. Compared with chromatography (which takes several hours to several tens of hours) and traditional biosensor methods (which rely on specific detection instruments), the detection time is greatly shortened, and the detection system can be directly applied to non-laboratory scenes such as potato planting bases, processing production lines, and food supervision sites, solving the pain point of the disconnection between "laboratory precise detection" and "on-site rapid screening" in existing technologies.

[0036] The result accuracy is high, and it is highly consistent with the authoritative method. In actual potato and its product sample detection, the consistency of the test strip detection result with LC-MS / MS (liquid chromatography-tandem mass spectrometry, referring to NY / T3951-2021 standard) is more than 95%, effectively avoiding the defects of high false positive rate of ELISA method and insufficient accuracy of traditional chromatography, ensuring that the detection data can be used as a reliable basis for food safety evaluation, and providing a high-efficiency connection scheme of "rapid screening + precise verification" for CHA residue detection.

[0037] Specificity highlights, effective evasion of interference misjudgment: relying on the precise molecular recognition ability of high-quality aptamer to CHA, the detection system can specifically distinguish a- solanine, β- carajao base and other structural similar solanine components, as well as caffeine, theophylline, nicotine and other common alkaloid small molecules, with a cross reaction rate of less than 5%, solving the technical problems that the existing detection methods are easily interfered by similar components in the matrix and cannot accurately qualify CHA, especially suitable for the specific detection of CHA in complex potato matrix.

[0038] Stability and economy are taken into account, and it is convenient for large-scale application: on the one hand, high-quality aptamer itself has good chemical stability and is not easily affected by temperature, pH and other environmental factors, so that the test strip based thereon can be stored at 4℃ in the dark for 30 days without significant attenuation of detection performance (the color clarity of T line and C line is ≤10% different from that of freshly prepared test strip), overcoming the problem of poor stability of biological recognition elements in biological sensor method; on the other hand, the aptamer can be batch screened and prepared through SELEX technology, compared with traditional antibodies (long preparation period, high cost and easy to be limited by immunogenicity), the production cycle is shortened, the material cost is reduced, and the synthesis cost can be further optimized by truncation (such as Apt-25) in the future, providing economic feasibility for large-scale production and grassroots promotion of the detection system, and having outstanding application advantages in CHA detection in resource-limited areas. The construction of rapid detection system provides a super-optimal molecular recognition basis. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 A schematic diagram for screening aptamer molecules with high affinity to CHA by SELEX technology.

[0040] Figure 2 Secondary structures of initial aptamer and trimmed aptamer.

[0041] Figure 3 Affinity of initial aptamer and trimmed aptamer to CHA: (A) Apt-55 aptamer analysis result; (B) Apt-25 aptamer analysis result; (C) dissociation constant analysis of Apt-55 aptamer; (D) dissociation constant analysis of Apt-25 aptamer.

[0042] Figure 4 Isothermal titration calorimetry of aptamer and CHA binding: (A) blank control; (B) aptamer binding CHA.

[0043] Figure 5 Circular dichroism spectrum of aptamer and CHA binding.

[0044] Figure 6Molecular docking of CHA with aptamer: (A) Molecular docking results of CHA with Apt-55: the green part represents CHA, and the blue part shows the interaction sites of Apt-55 with CHA. The hydrogen bond formation sites are DT-15, DG-17, DT-18 and DG-24. (B) Molecular docking results of CHA with Apt-25: the yellow part represents CHA, and the red part shows the interaction sites of Apt-25 with CHA. The hydrogen bond formation sites are DG-16, DA-17 and DT-24.

[0045] Figure 7 Schematic diagram of Apt-LFA CHA detection based on competitive reaction.

[0046] Figure 8 Characterization of AuNPs and gold-labeled probe: (A) TEM image of synthesized AuNPs; (B) UV spectrum of AuNPs and synthesized gold-labeled probe; (C) Mey's experiment.

[0047] Figure 9 Optimization of test strip conditions: (A) Optimization of SHcDNA length (B) Optimization of PBS concentration (C) Optimization of binding pad treatment solution (D) Optimization of streptavidin concentration (E) Optimization of biotinylated aptamer concentration (F) Optimization of binding time.

[0048] Figure 10 (A) Specific detection results of Apt-LFA: 1 is α-solanine, 2 is β-solanine, 3 is caffeine, 4 is theophylline, and 5 is nicotine. (B) Sensitivity detection results of Apt-LFA. (C) Stability detection results of Apt-LFA. DETAILED DESCRIPTION

[0049] The content of the present application will be described in detail by specific examples. It should be noted that these examples are only used to illustrate the present application and not used to limit the scope of the present application. In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased in the market. The features and properties of the present application will be described in detail in the following combined with the embodiments.

[0050] In this study, a rapid detection system of CHA was established by integrating molecular recognition technology and detection method innovation. The main research contents are as follows: 1. Creation of high-affinity aptamer and analysis of molecular recognition mechanism; 2. Construction of lateral flow chromatographic detection technology for rapid detection of CHA based on high-affinity aptamer

[0051] Preparation of high affinity aptamer and analysis of molecular recognition mechanism

[0052] 1. The nucleic acid aptamer of CHA was obtained by SELEX screening technology, as shown in the following figure: Figure 1 The complete experimental flow chart of SELEX is shown in the following figure. The flow chart starts from the construction of the initial random single-stranded DNA library. The whole screening process goes through ten rounds of strict iteration. Each round of cycle includes three key steps: first, the library is incubated with the target CHA. Hind III restriction enzyme is used to specifically cut the unbound sequences to reduce non-specific interference; then the aptamer sequence bound to CHA is amplified by PCR; finally, it is purified by exonuclease. After several rounds of enrichment, the high-affinity aptamer sequence is finally determined by high-throughput sequencing. The whole process realizes the efficient screening of nucleic acid aptamer with high specificity and high affinity from massive random sequences by combining enzyme digestion selection and PCR amplification technology, which lays a molecular recognition foundation for the subsequent development of CHA rapid detection method.

[0053] The nucleic acid aptamer of CHA was obtained by SELEX screening technology, and the specific steps are as follows:

[0054] (1) Pretreatment of the initial library: take 1 OD of random initial library and dissolve it in 110 μL of ddH2O. Then heat the solution at 95°C for 5 min, and immediately transfer it to ice for cooling for 15 min for standby.

[0055] (2) Double-stranded complex preparation: mix the initial library with the complementary fixed sequence according to the equimolar ratio, and place it in the PCR instrument for annealing. The program is set as follows: 95°C denaturation for 10 min, then gradient cooling to 25°C at a rate of 0.1°C / s, to obtain dsDNA complex;

[0056] (3) Target incubation: add target CHA to the prepared double-stranded complex system, and incubate at 37°C for 60 min, so that the dsDNA specifically bound to CHA is dissociated from the double-stranded complex to form ssDNA-CHA complex;

[0057] (4) Endonuclease digestion: add Hind III restriction endonuclease (10 U / μL) to the system after CHA incubation, and react at 37°C for 30 min. This step can specifically cut the un-dissociated double-stranded complex. The cut dsDNA cannot be amplified by PCR in the next step;

[0058] (5) PCR amplification: ssDNA-CHA complex obtained in step (4) was used as a template for PCR amplification with a primer concentration of 500 nM. The reaction was first incubated at 37 °C for 30 min, then heated at 80 °C for 15 min to denature and inactivate Hind III restriction endonuclease, and finally cooled at 4 °C for 10 min to stabilize the product;

[0059] (6) Single-stranded DNA preparation: The amplification product in step (5) was recovered and purified, and lambda endonuclease (5 U / μL) was added for 37 °C reaction for 60 min to digest the complementary strand, obtaining a ssDNA secondary library;

[0060] (7) Multiple rounds of screening: The starting Library library in (1) was replaced with the secondary library obtained in (6), and steps (2) to (6) were repeated for multiple rounds of screening;

[0061] (8) Sequencing analysis: After the tenth round of screening, the latest secondary library obtained was subjected to high-throughput sequencing analysis to obtain candidate CHA aptamers for subsequent verification.

[0062] After ten rounds of SELEX screening, the final enriched library was subjected to PCR amplification, screening and sequencing to determine the sequence of the CHA aptamer to be tested.

[0063] As shown in Figure 2 , one 55 nt long aptamer Apt-55 was obtained after 10 rounds of SELEX screening. In order to further improve the affinity and specificity of the aptamer, a 25 nt short-chain aptamer Apt-25 was designed by 3' end truncation (cut at 30T site), and the sequence is shown in Table 1.

[0064] Table 1

[0065] Colloidal gold colorimetric

[0066] The affinity between the two and CHA was characterized by colloidal gold colorimetry. 5 μL of CHA with different concentrations was incubated with 5 μL of 1 μM aptamer solution (10 mM PBS, pH 7.4) at room temperature for 40 min, then 100 μL of AuNPs was added to the solution and incubated for another 40 min, followed by the addition of 9 μL of 0.5 M NaCl to induce aggregation for 15 min. Finally, the absorbance of the solution at 626 nm (characteristic peak of aggregated AuNPs) and 525 nm (characteristic peak of monomeric AuNPs) was measured using a UV spectrophotometer, and the binding capacity of Apt-55 and Apt-25 to CHA was quantitatively analyzed by calculating A626 / A525. As the concentration of aptamer increased, the affinity for CHA also gradually increased, and the binding capacity was positively correlated with the concentration of aptamer. The affinity constant (Kd) was analyzed and fitted in GraphPad Prism 8.

[0067] The results are shown in Figure 3 The Kd value of Apt-55 was 0.333 μM, and the Kd value of Apt-25 was 0.326 μM. The affinity of the trimmed aptamer was slightly improved, indicating that the removed part of the sequence may be involved in maintaining the stability of the complex after the aptamer binds to CHA, while the active center that plays a key role in recognizing the CHA target is retained in the 25 nt core sequence. In addition, the Kd values of both are in the micromolar range, and the Kd values of aptamers with good affinity are generally in the micromolar range, and the lower the affinity constant, the higher the affinity. Therefore, the results show that the original aptamer Apt-55 and the trimmed aptamer Apt-25 have strong affinity for the target CHA, and the affinities of the two are similar.

[0068] Isothermal Titration Calorimetry (ITC)

[0069] Isothermal titration calorimetry is an effective method for determining the affinity and thermodynamic parameters of the interaction between aptamer and target molecule. The aptamer is added dropwise to CHA, and the heat release is recorded over time.

[0070] The results are shown in Figure 4 As the concentration of aptamer increased, the binding between aptamer and CHA gradually tended to saturation, and the heat change tended to be stable. In contrast, no heat change was observed in the blank control group. The test data was based on Independent model fitting, and the corresponding stoichiometric ratio n, enthalpy change ΔH, entropy change ΔS, etc. were obtained, as shown in Table 2.

[0071] The results show that the aptamer Apt-55 has a high affinity for CHA. ΔH < 0 and ΔS < 0, indicating that the binding process is driven by hydrogen bonding. The exothermic binding process suggests that the aptamer and CHA form a complex with lower thermality, resulting in a more stable spatial structure.

[0072] Table 2

[0073]

[0074]

[0075] Circular Dichroism (CD)

[0076] Circular dichroism spectroscopy can be used to characterize conformational changes after aptamers bind to CHA. Type B DNA typically shows a positive peak at CD280 nm and a negative peak at CD250 nm.

[0077] The results are as follows Figure 5 As shown, the binding of the aptamer to CHA increases CD 285nm and decreases CD 240nm, indicating that the binding forms more secondary structures and makes the aptamer structure more stable. The binding of the aptamer to CHA may change the base pair stacking and helicity of the aptamer, as well as form additional secondary structures, such as heterozygous G-tetramers.

[0078] Combined with dynamics research

[0079] Molecular docking simulations were performed on Apt-55 and Apt-25 with the CHA molecule to predict and analyze binding pockets and binding sites. When CHA binds to the aptamer, it selectively interacts with specific base sites rather than binding to all base sites of the aptamer.

[0080] The results are as follows Figure 6 As shown in Figure A, the binding energy of the lowest energy conformation of Apt-55, as simulated by molecular docking, is -9.9 kcal / mol. CHA enters the binding pocket of the aptamer through van der Waals forces and hydrophobic interactions, forming a total of 9 hydrogen bonds with the T15, G17, T18, and G24 base sites of Apt-55, with bond lengths of [missing information]. and like Figure 6 As shown in Figure B, the results of 100 molecular docking simulations indicate that the binding energy between the lowest energy conformation of Apt-25 and CHA is -9.8 kcal / mol. CHA enters the binding pocket of Apt-25 through van der Waals forces and hydrophobic interactions. CHA forms five hydrogen bonds with the G16, A17, and T21 base sites of Apt-25, with bond lengths of [missing information]. and

[0081] The above results demonstrate that the screened nucleic acid aptamer has excellent affinity and target specificity for binding to CHA and can generate a complex with a stable spatial structure, and Apt-55 shows more prominent analytical performance, and thus is used for subsequent construction of a rapid detection method.

[0082] Example 2 Construction of a CHA rapid detection method based on nucleic acid aptamer

[0083] 1. Preparation of AuNPs. Before preparing colloidal gold nanoparticles (AuNPs), all glassware (Erlenmeyer flasks, beakers, etc.) and magnetic stirring rods were immersed in chromic acid solution (a mixture of potassium dichromate and concentrated sulfuric acid) overnight, then washed with ddH2O and dried for use. AuNPs were synthesized by the trisodium citrate method, and the specific steps were as follows: 99 mL of filtered ultrapure water was added to 1.0 mL of 1.0% chloroauric acid (HAuCl4·3H2O) solution, and heated to boiling using a constant temperature metal bath, about 5-8 min, with constant stirring using a magnetic stirring rod at a speed of 500 rpm. Then, while maintaining boiling, 2.0 mL of 1.0% trisodium citrate solution was rapidly added, and heating was continued, and the solution was observed to change from light yellow to gray black and finally to red, about 2-3 min, and the solution was maintained at boiling until the color was stable wine red, and heating was continued for 10 min before stopping. Continue stirring at room temperature for 10 min, then cool to room temperature in the dark, and store the synthesized AuNPs at 4°C in the dark for use. The size and morphology of the synthesized gold nanoparticles were observed using transmission electron microscopy, and the synthesized AuNPs were characterized using a UV-visible spectrometer scanning the range of 400-800 nm.

[0084] 2. Coupling of AuNPs with SHcDNA. 5 μL of 1M TCEP (Tris(2-carboxyethyl)phosphine, pH 7.0) was mixed with 200 μL of 10 μM thiol (-SH) modified Apt-55 complementary strand (SHcDNA) and incubated at room temperature in the dark for 2 h to activate the SHcDNA. The activated SHcDNA was then mixed with 800 μL of prepared AuNPs and incubated overnight (16 h) at 4°C to allow for complete binding of SHcDNA and AuNPs. Gradual salting was performed to increase the ionic strength of the solution, with 20 μL of 0.5×PBS (containing 68 mM NaCl, 1.3 mM KCl, 4.0 mM Na2HPO4, and 1.0 mM KH2PO4) added every 2 h for a total of 3 times. Finally, the synthesized gold-labeled probe was purified. The solution was stored at 4°C in the dark for 24 hours to ensure complete binding. To remove excess SHcDNA and unbound AuNPs, the solution was centrifuged at 12,000 rpm for 15 minutes, the supernatant was removed, and the precipitate was resuspended in 200 μL of 0.5×PBS (containing 0.01% Tween-20 to prevent non-specific adsorption). Centrifugation was repeated three times to ensure complete removal of unbound components. The prepared gold-labeled probe was stored at 4°C in the dark and can be ultrasonically dispersed before use.

[0085] 3. Characterization of AuNPs and gold-labeled probes. These are used as chromogenic signal molecules in Apt-LFA. For example... Figure 8 As shown in Figure A, the morphology and size of the synthesized AuNPs were examined using scanning transmission electron microscopy (TEM). The results showed that the prepared AuNPs were uniformly monodisperse, with regular spherical particles, uniform particle size distribution, and an average particle size of 20 nm. To investigate whether the AuNPs were successfully conjugated with SHcDNA, the synthesized gold-labeled probe was scanned using a UV-Vis spectrophotometer. Figure 8 As shown in Figure B, bare AuNPs exhibit a characteristic surface plasmon resonance (SPR) absorption peak at 526 nm, while the maximum absorption peak of the SHcDNA-modified AuNPs-SHcDNA complex redshifts to 531 nm, a redshift of 5 nm. This redshift phenomenon originates from the successful assembly of DNA molecules on the AuNPs surface, leading to changes in local refractive index and electron cloud distribution. This indicates that SHcDNA has been successfully coupled to the AuNPs surface, resulting in an increase in the AuNP particle size. Furthermore, the stability of the AuNPs-SHcDNA probe was evaluated using the Mey stabilization assay. When AuNPs are modified with ssDNA, the AuNPs surface acquires a strong negative charge, thereby enhancing the electrostatic repulsion between gold nanoparticles and preventing salt solution-induced particle aggregation. Figure 8As shown in FIG. C, after adding 1 M NaCl, the bare AuNPs solution will form large particle aggregates, resulting in aggregation, and the solution color changes from red to purple. In contrast, the AuNPs-SHcDNA solution remains stable after adding NaCl solution, with no obvious color change, indicating that the SHcDNA is successfully coupled to the surface of AuNPs.

[0086] 4. Preparation of aptamer-based lateral flow test strip. The principle of CHA rapid detection based on aptamer-based lateral flow assay (Apt-LFA) developed in this study is shown in FIG. Figure 7 The test strip consists of three key parts: (1) the conjugate pad area: AuNPs-labeled SHcDNA probe is sprayed; (2) the NC membrane detection line (T line): streptavidin is immobilized as a capture molecule; (3) the NC membrane control line (C line): 1 mg / mL streptavidin 10 μL, 6 μL of 100 μM biotinylated aptamer, 0.1 x PBS to 20 μL, 37°C incubation for 30 min, to obtain streptavidin-biotinylated aptamer complex, and streptavidin-biotinylated aptamer complex is immobilized as a capture molecule. Biotinylated aptamer is directly synthesized by Shanghai Generay Biotech Co., Ltd.

[0087] AuNPs-SHcDNA is sprayed on the conjugate pad of the test strip, and streptavidin is immobilized on the T line of the NC membrane, and streptavidin-biotinylated aptamer complex is immobilized on the C line.

[0088] Before detection, the sample is mixed with biotinylated aptamer, and the target in the sample can be fully combined with the biotinylated aptamer, and then added dropwise to the sample pad (i.e., the sample addition area) for detection.

[0089] In the absence of CHA in the sample, the biotinylated aptamer Bio-Apt in the sample buffer preferentially binds to AuNPs-SHcDNA on the binding pad, forming a Bio-Apt-AuNPs-SHcDNA ternary complex. This complex is captured by streptavidin on the T line of the NC membrane during subsequent chromatography, forming a visible red band. Simultaneously, unreacted free AuNPs-SHcDNA is captured by the C line, forming a second red band, resulting in red bands on both the T and C lines. Conversely, when CHA is present in the sample buffer, CHA competitively binds to Apt-55 in the buffer, inhibiting the hybridization of Apt-55 with AuNPs-SHcDNA and hindering the formation of the ternary complex, causing a significant weakening or complete disappearance of the T line band. Regardless of the presence of CHA, free AuNPs-SHcDNA is captured by the C line, producing a red band, which serves as the quality control band for normal test strip function. In summary, the interpretation criteria for the test results are as follows: a positive sample will show a red band on line C, and no band or a significantly lighter band on line T. A negative sample will show red bands on both line C and line T; if there is no band on line C, the test strip is considered invalid. This detection system achieves specific identification of CHA through a competitive binding principle, and the entire detection process can be completed within 15 minutes without the need for complex instruments and equipment.

[0090] 4. Optimization of experimental conditions.

[0091] (1) Optimization of SHcDNA length. The effect of different lengths of thiol-complementary DNA (SHcDNA) on the detection performance of the test strip was investigated. As shown in Table 3, SHcDNA lengths of 15nt, 25nt, 35nt, 45nt, and 55nt were selected and coupled with 20nm AuNPs. The prepared gold-labeled probe solution was uniformly sprayed onto the glass fiber conjugate pad and dried in a 37℃ constant temperature drying oven for 2 hours to ensure that the probe was stably fixed on the conjugate pad. Subsequently, the sample pad, conjugate pad, nitrocellulose membrane (pre-coated with the detection line and control line), and absorbent pad were assembled into a complete test strip according to the standard procedure. To determine the optimal SHcDNA length, all test strips were tested using negative samples without CHA, and the band color development of the detection line (T line) was visually compared and analyzed. Figure 9 As shown in Figure A, when the length of SHcDNA is 25nt, the C and T lines are the clearest and darkest, therefore, SHcDNA with a length of 25nt is selected.

[0092] Table 3 SHcDNA Sequences

[0093]

[0094]

[0095] (2) PBS buffer concentration optimization. The effect of PBS buffer concentration on the performance of gold-labeled probes was investigated. The phosphate groups in PBS buffer can help the stable binding of SHcDNA and AuNPs through electrostatic interaction, so the system sets 0.1x (low ionic strength), 0.5x (medium ionic strength) and 1x (high ionic strength) concentrations of PBS buffer to participate in the incubation of gold-labeled probes. After probe purification, according to the above same steps, the combination pad is sprayed and the test strip is assembled. The negative sample and positive sample (containing 50 ng / mL α-kaolin alkaloid standard) are detected by the prepared test strip, and the maximum concentration of PBS buffer with clear C and T line bands is selected. As shown in Figure 9 B, when the concentration of PBS solution is 0.5x, the band color is clear, indicating that the concentration of buffer is conducive to the binding of AuNP-SHcDNA probe, so 0.5x PBS solution is selected.

[0096] (3) Optimization of combination pad treatment solution. In this study, the formula of the combination pad treatment solution was systematically optimized to solve the problem of non-specific background signal of lateral flow chromatography test strip. Based on the principle that surfactants can improve the chromatography flow rate and protein blocking agents can reduce non-specific adsorption, different components of the treatment solution were designed for comparative study. In order to reduce non-specific background signal, in the absence of target molecules, the combination pad was treated with 5 different components of the treatment solution, and the treatment solution with the cleanest background and fastest flow rate was selected for test strip detection. The components of the 5 treatment solutions are as follows: treatment solution 1: 2% glucose, 1% Triton X-100, 1% BSA, 0.5M boric acid, pH 8.0; treatment solution 2: 2% glucose, 1% Triton X-100, 3% BSA, 0.5M boric acid, pH 8.0; treatment solution 3: 1% glucose, 3% BSA, 0.5% Tween-20, 0.1M Tris-HCl, pH 8.0; treatment solution 4: 1% glucose, 6% BSA, 0.5% Tween-20, 0.1M Tris-HCl, pH 8.0; treatment solution 5: 1% Triton X-100, 1% BSA, 2% glucose, 0.05M boric acid, pH 8.0. In order to accelerate the sample migration rate and the clean background color, as shown in Figure 9 C, treatment solution 4 (1% glucose, 6% BSA, 0.5% Tween-20, 0.1M Tris-HCl, pH 8.0) is selected.

[0097] (4) The optimal coating concentration of streptavidin and biotinylated aptamer on nitrocellulose membrane (NC membrane) was systematically optimized in this study. Based on the high affinity binding characteristics between streptavidin and biotinylated aptamer, different concentrations of biotinylated aptamer were set at 1 nM, 10 nM, and 100 nM, respectively, and then incubated with CHA. The assembled test strips were used for detection to explore their effects on the color performance of the test line (T line). The concentration of the C line and T line with uniform color distribution was selected as the optimal concentration of biotinylated aptamer. Figure 9 D, E, the final concentration of streptavidin was selected as 4 mg / mL and the concentration of biotinylated aptamer was selected as 10 nM.

[0098] (5) Optimization of the binding time. The effect of incubation time of aptamer and CHA on the detection performance was systematically investigated in this study. Based on the binding kinetics characteristics of nucleic acid aptamer and target molecules, different incubation times were set at 5 min, 15 min, 30 min, 45 min, and 60 min, respectively. The assembled test strips were then detected at different times, and all reactions were performed at room temperature. Each time was set in triplicate, and the shortest incubation time with the deepest color of the C line and T line was selected as the optimal binding time. As shown in Figure 9 F, the shortest incubation time with the deepest color of the C line and T line was selected as the optimal binding time. As shown in

[0099] 5. Sensitivity, specificity, and stability of the test strip

[0100] In order to effectively evaluate the specificity of Apt-LFA, α-solanine and β-solanine, which belong to the same solanine, as well as caffeine, theophylline, nicotine and other alkaloid small molecules were used for test strip detection. The results are shown in Figure 10 A, only when the target CHA was added, the test strip showed a positive result, and other alkaloids showed a negative result, indicating that the constructed aptamer-based lateral flow chromatography test strip could distinguish common structurally similar alkaloid molecules and had good specificity.

[0101] The sensitivity of Apt-LFA was evaluated. With the increase of CHA concentration, the T line band color gradually weakened until disappeared after CHA combined with Apt-55, and the C line was not affected, showing a clear red band, and the test strip showed a positive result. The visual detection limit refers to the minimum CHA concentration when the T line signal intensity is significantly lower than the positive result, and the visual threshold value refers to the minimum CHA concentration when the T line signal completely disappears. Therefore, by Figure 10The visual detection limit and visual threshold were 0.5 mg / mL and 1 mg / mL, respectively. As shown in Table 4, the detection limit of the Apt-LFA developed in this study was higher than that of the reported CHA detection methods, including HPLC and ELISA. The stability of the Apt-LFA was evaluated by storing the test strips at 4°C for 1-30 days, during which time the Apt-LFA was tested for detecting negative samples, and the color of the C and T lines of the test strips showed no significant changes, indicating that the performance of the test strips was not affected. The lateral flow chromatographic test strip developed for detecting CHA had good stability, which indicated that the test strip had great potential for on-site rapid detection applications. Figure 10 C. As shown in Table 4, the detection limit of the Apt-LFA developed in this study was higher than that of the reported CHA detection methods, including HPLC and ELISA. The stability of the Apt-LFA was evaluated by storing the test strips at 4°C for 1-30 days, during which time the Apt-LFA was tested for detecting negative samples, and the color of the C and T lines of the test strips showed no significant changes, indicating that the performance of the test strips was not affected. The lateral flow chromatographic test strip developed for detecting CHA had good stability, which indicated that the test strip had great potential for on-site rapid detection applications.

[0102] Table 4 Comparison of sensitivity of Apt-LFA with other methods

[0103]

[0104] 6. Actual sample detection.

[0105] To verify the practicality of the developed Apt-LFA, three spiked potato CHA samples were analyzed. As shown in Table 5, according to the standard method NY / T 3951-2021, the concentrations of CHA in the three spiked samples were 60 ng / mL, 6 ng / mL, and 1 ng / mL, respectively, as measured by LC-MS / MS. At the same time, the above three samples were detected using the constructed test strips, and the results of the test strips were compared with those of LC-MS / MS. As shown in Table 5, the results obtained by the two methods were consistent, confirming that the Apt-LFA had the ability to accurately detect CHA on-site in actual agricultural production and life.

[0106] Table 5 Results of actual sample detection

[0107]

[0108] Note: a T line without band, b positive result is T line without band.

Claims

1. A nucleic acid aptamer that specifically binds to α-carboline, characterized in that, The nucleic acid aptamer has the nucleic acid sequence described in SEQ ID NO.1 or SEQ ID NO.

2.

2. A gold-labeled probe for detecting α-carboxine, characterized in that, The gold-labeled probe is a conjugate of the complementary strand of the nucleic acid aptamer of claim 1 with thiolized form and gold nanoparticles; the complementary strand of the thiolized nucleic acid aptamer is a thiol-modified complementary single-stranded DNA, and the complementary single-stranded DNA is a single-stranded DNA that is complementary to at least a portion of the nucleic acid aptamer.

3. The gold-labeled probe according to claim 2, characterized in that, The sequence of the complementary single-stranded DNA is shown in any one of SEQ ID NO. 3-7, preferably as shown in SEQ ID NO.

6.

4. The gold-labeled probe according to claim 2, characterized in that, The gold nanoparticles have a particle size of 5nm-50nm.

5. The gold-labeled probe according to claim 2, characterized in that, The method for preparing the gold-labeled probe includes: S1) Mix TCEP with the thiolated nucleic acid aptamer of claim 1 and incubate to activate the thiolated nucleic acid aptamer; S2) The activated thiolized nucleic acid aptamers were mixed with gold nanoparticles, and after standing to allow them to fully combine, the ion concentration of the solution was gradually increased. The gold-labeled probe was obtained after purification (S3).

6. The gold-labeled probe according to claim 5, characterized in that, The method to gradually increase the ion concentration in the solution is to add 20 μL of 0.5×PBS every 2 hours for a total of 3 times.

7. A test strip for α-carbocyanine side-flow chromatography, characterized in that, It includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad arranged sequentially; the conjugate pad is coated with the gold-labeled probe according to any one of claims 2-6; the nitrocellulose membrane is provided with a detection line and a control line, the detection line is fixed with a first capture molecule, the first capture molecule being streptavidin, and the control line is fixed with a second capture molecule, the second capture molecule being a complex of streptavidin-biotinylated aptamer; The streptavidin-biotinylated aptamer complex is a conjugate of streptavidin and biotin-modified nucleic acid aptamer as described in claim 1.

8. The α-carbocyanine side-flow chromatography test strip according to claim 7, characterized in that, The conjugate pad is pretreated with a treatment solution consisting of 1% glucose, 6% BSA, 0.5% Tween-20, and 0.1M Tris-HCl, with a pH of 8.

0. The streptavidin coating concentration on the NC membrane is 4 mg / mL, and the biotinylated aptamer coating concentration is 10 nM.

9. A method for detecting α-carboline, comprising the following steps: S01) Sample pretreatment: Mix the sample to be tested with the buffer solution of the biotinylated aptamer and incubate; S02) Test strip detection: Add the mixture from step S01) to the sample pad of the α-carbohydrate side-flow chromatography test strip as described in claim 7 or 8, and perform chromatography at room temperature for 15 min; S03) Result interpretation: If a red band appears on the control line and no band or a significantly lighter band appears on the test line, the result is considered positive; if both the control line and the test line show red bands, the result is considered negative. If the control line has no band, the test strip is considered invalid. Wherein, the biotinylated aptamer is the nucleic acid aptamer as described in claim 1 that is modified with biotin.

10. The application of the α-carbohydrate side-flow layer test strip according to claim 7 or 8 in the rapid on-site detection of α-carbohydrate in potatoes or their products.

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