Method for detecting histamine based on paper-based sensor

Abstract

This invention discloses a histamine detection method based on a paper-based sensor, belonging to the field of analytical detection. The method includes the following steps: First, KSAP and PtFe nanozyme are co-incubated to form a complex; second, the complex is dropped onto a layer-modified paper surface; subsequently, histamine is added for incubation; finally, TMB and H2O2 are added sequentially for color development, and quantitative detection of histamine is achieved through image analysis. This invention utilizes the synergistic kinetic mechanism and high specificity of KSAP combined with the high catalytic activity of PtFe nanozyme, along with the low cost and portability of the paper-based platform, to achieve rapid, sensitive, and visual detection of histamine in aquatic products, showing promising application prospects in on-site food safety monitoring.

Description

Technical Field

[0001] This invention relates to a method for constructing a paper-based sensor for small molecule target detection based on the combination of kinetic co-aptamer pairs (KSAP) and PtFe nanozymes, belonging to the field of analytical detection. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Aptamers are a class of single-stranded DNA or RNA molecules that can specifically recognize and bind to their target molecules, showing broad application prospects in fields such as biosensing, diagnosis, and therapy. However, single aptamers often struggle to balance binding rate and affinity, limiting their sensitivity and response speed in practical detection.

[0004] Therefore, it is necessary to design a dynamic cooperative aptamer pair (KSAP) that utilizes the combined advantages of two aptamers with complementary dynamic properties to improve overall recognition performance. This is of great significance for designing highly sensitive and stable biosensors.

[0005] In existing technologies, single histamine aptamers (such as HIS3-T2) have been coupled with nanozymes (such as AuNPs@FeP) to achieve nM-level detection on solution platforms. However, when such sensors are transferred to paper-based platforms, the detection sensitivity typically decreases significantly due to inherent problems such as uneven sample diffusion, limited reaction time, and significant background interference. For example, the detection limit of a single HIS3-T2 aptamer on a paper-based platform (5.21 nM) is approximately 2.8 times lower than that on a solution platform (1.89 nM). Therefore, how to restore or improve detection sensitivity in the "constrained environment" of a paper-based platform is a pressing technical problem to be solved in this field.

[0006] Nanozymes are a class of nanomaterials with enzyme-like catalytic activity, and can be used as signal transduction elements in the field of biosensing. Bimetallic nanozymes, due to their high peroxidase activity, good stability, and tunable catalytic properties, can be used for colorimetric detection.

[0007] Paper-based sensors offer advantages such as low cost, ease of operation, and convenient on-site detection. By combining aptamers with nanozymes, paper-based colorimetric sensors can be constructed, enabling highly sensitive and visual detection of small molecule targets. However, currently, there are no reports on introducing two synergistic aptamers (such as the kinetically synergistic aptamer pair KSAP proposed in this invention) to compensate for the loss of platform sensitivity, especially in the detection of the small molecule target histamine on paper-based platforms.

[0008] Based on the aforementioned technical problems, this invention proposes a paper-based sensor solution that combines kinetic synergistic aptamer pairs (KSAP) with highly active PtFe nanozymes. Summary of the Invention

[0009] To address the aforementioned limitations of existing technologies, this invention provides a method for detecting small-molecule target histamine, belonging to the field of analytical detection technology. This method utilizes a designed kinetic co-aptamer pair (KSAP) signal recognition element (one with a high binding rate but weak binding force, and the other with a slow binding rate but strong affinity) coupled with a signal transduction element of a highly catalytically active PtFe bimetallic nanozyme synthesized in a one-pot process. This constructs a modified paper-based sensor, enabling sensitive, rapid, and visualized detection of the target small-molecule histamine.

[0010] Specifically, it includes the following: In a first aspect, the present invention provides a histamine detection method based on a paper-based sensor, comprising the following steps: Kinetic co-aptamer pairs (KSAP) were co-incubated with PtFe nanozymes to form aptamer-nanozyme complexes. The aptamer-nanozyme complex was dropped onto a modified paper substrate; After the test sample containing histamine is added and reacted, TMB and H2O2 are added to carry out a colorimetric reaction. The image of the paper substrate after color development is acquired, and the concentration of target histamine in the test sample is calculated based on the color intensity. The dynamic cooperative aptamer pair (KSAP) is composed of a mixture of capture aptamer L2 and stable aptamer HIS3-T2 in a set ratio; The nucleotide sequence of the capture aptamer L2 is shown in SEQ ID NO.1, and the nucleotide sequence of the stable aptamer HIS3-T2 is shown in SEQ ID NO.2.

[0011] Preferably, the kinetic synergistic aptamer pair (KSAP) is composed of a mixture of capture aptamer L2 and stabilizing aptamer HIS3-T2 in a molar ratio of 1:1.

[0012] Preferably, the PtFe nanozyme is synthesized by a one-pot method, using K2PtCl4 and FeCl3 as precursors, with a Pt to Fe molar ratio of 1:1. The reaction is carried out at 60-65°C for 2 hours under L-ascorbic acid reduction, and the product is obtained by centrifugation and washing.

[0013] Preferably, the paper base is modified by perforating the filter paper into a disc and then modifying it layer by layer with chitosan (CS) and polyethylene glycol (PEG) to form a CS / PEG layer-modified paper base.

[0014] Preferably, histamine concentration is detected by colorimetric signal detection, wherein the colorimetric signal detection includes: acquiring the RGB values ​​of the paper substrate after the reaction using a smartphone camera, and then using the Euclidean distance model D= Calculate the color intensity, where R0, G0, and B0 are the RGB values ​​of the blank group, and Ri, Gi, and Bi are the RGB values ​​of the experimental group.

[0015] Preferably, the KSAP-PtFe composite has a histamine detection limit of 2.21 nM and a linear range of 3.05-781.25 nM on a paper-based platform.

[0016] The detection limit of histamine of the KSAP-PtFe complex on the paper-based platform is more than 2.4 times lower than that of a single HIS3-T2-PtFe complex and more than 9 times lower than that of a single L2-PtFe complex; the detection limit of a single HIS3-T2-PtFe complex on the paper-based platform is 5.21 nM, and the detection limit of a single L2-PtFe complex on the paper-based platform is 19.79 nM.

[0017] The Michaelis constant of the PtFe nanozyme for H2O2 K m The maximum reaction rate was 9.43 mM. V max It is 2.66 × 10 - 6 M·s -1 The equilibrium dissociation constant of the kinetic co-adaptor for KSAP K d 3.78×10 -8 M, the rate constant k on It is 5.48×10 -5 1 / Ms, dissociation rate constant k off 2.07×10 -2 1 / s.

[0018] Preferably, the method is suitable for the rapid detection of histamine in front-end or back-end aquatic products.

[0019] In a second aspect, the present invention provides a paper-based sensor for histamine detection, comprising: Paper-based substrate modified with CS / PEG layers; An aptamer-nanozyme complex loaded on the paper substrate, the complex being formed by co-incubation of KSAP and PtFe nanozymes with a kinetic synergistic aptamer; The kinetic co-aptamer pair KSAP is composed of a capture aptamer L2 and a stable aptamer HIS3-T2 mixed in a molar ratio of 1:1. The nucleotide sequence of L2 is shown in SEQ ID NO.1, and the nucleotide sequence of HIS3-T2 is shown in SEQ ID NO.2.

[0020] The histamine detection limit of the paper-based sensor is 2.21 nM, and the molar ratio of Pt to Fe in the PtFe nanozyme is 1:1.

[0021] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) Collaborative breakthroughs on paper-based platforms On the paper-based platform, the detection limit was 2.21 nM for the KSAP-PtFe system, 5.21 nM for the HIS3-T2-PtFe system, and 19.79 nM for the L2-PtFe system. The detection limit of the KSAP system was lower than that of the HIS3-T2 and L2 systems.

[0022] (2) Characterization evidence of dynamic cooperative mechanism BLI measurements showed that the equilibrium dissociation constant of KSAP ( K d =3.78×10 -8 M) is lower than L2 (1.75×10 -7 M) and HIS3-T2 (5.95×10 -8 M), binding rate constant (k) on =5.48×10 -5 M -1 s -1 ) higher than L2 (4.68×10 -5 M -1 s -1 ) and HIS3-T2 (1.83×10 -5 M -1 s -1 (Example 1). Particle size, zeta potential, and circular dichroism spectroscopy results showed that the particle size, surface potential, and CD peaks of the KSAP-PtFe complex changed after the addition of the target.

[0023] (3) Catalytic performance of PtFe nanozymes The PtFe nanozyme used in this invention for H2O2 K m It is 9.43 mM. V max It is 2.66 × 10 -6 M·s -1 ; while AuNPs@FeP'sK m It is 500 mM. V max 1.25×10 -8 M·s -1 .

[0024] (4) Sensor selectivity and application potential Selectivity experiments showed that in the presence of histamine analogs (putrescine, tryptamine, tyramine, cadaverine, spermine, histidine), the sensor's colorimetric signal did not change significantly compared to the blank group; in a system with mixed interfering substances, the histamine detection signal was not significantly interfered with. This sensor can be used for monitoring the freshness of aquatic products. Attached Figure Description

[0025] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 : Capture the BLI curve of aptor L2.

[0027] Figure 2 BLI curve of stable aptamer HIS3-T2.

[0028] Figure 3 BLI curve of the synergistic complementary aptamer KSAP.

[0029] Figure 4 The change of absorbance over time under different Pt to Fe feed ratios.

[0030] Figure 5 TEM and EDS mapping of PtFe nanozymes; A is TEM image at different magnifications, and B is EDS mapping image.

[0031] Figure 6 Particle size distribution of PtFe nanozymes.

[0032] Figure 7 Michaelis equation curves of PtFe nanozymes on TMB.

[0033] Figure 8 : Double reciprocal curves of PtFe nanozyme against TMB.

[0034] Figure 9 Michaelis equation curves of PtFe nanozymes against H2O2.

[0035] Figure 10 Double reciprocal curves of PtFe nanozymes against H2O2.

[0036] Figure 11 : KSAP signal response diagram to the target.

[0037] Figure 12 Particle size distribution of single or complex PtFe nanozymes, aptamers, and targets after incubation.

[0038] Figure 13 Zeta potential diagrams of single or complex PtFe nanozymes, aptamers, and targets after incubation.

[0039] Figure 14 Circular dichroism chromatograms of single or complex PtFe nanozymes, aptamers, and targets after incubation.

[0040] Figure 15 : Contact angle and apparent diffusion diagram of modified paper substrate; A is the contact angle of paper substrate with different modifications, and B is the apparent diffusion diagram of paper substrate with different modifications.

[0041] Figure 16 Scanning electron microscope image of paper substrate surface.

[0042] Figure 17 Standard curve of KSAP@PtFe paper-based sensor.

[0043] Figure 18 Standard curve of HIS3-T2@PtFe paper-based sensor.

[0044] Figure 19 Standard curve of L2@PtFe paper-based sensor.

[0045] Figure 20 Selectivity and anti-interference properties of paper-based sensors. Detailed Implementation

[0046] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0047] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0048] The following investigation was conducted in a typical embodiment of the present invention: Kinetic Synergistic Adaptor Pair (KSAP) Synergy and Kinetic and Thermodynamic Studies: Two aptamers with complementary functions were selected: aptamer A (high binding rate, K d Larger) and aptamer B (slower binding rate, K d (Smaller). Binding curves of the two aptamers and their combinations (KSAP) with the target molecule were measured using BLI technology. The binding rate constant (k) was obtained by fitting the BLI curves. on ), dissociation rate constant (k off ) and equilibrium dissociation constant ( K d Compare the parameters of single aptamers and KSAP to analyze the synergistic effect.

[0049] Synthesis and characterization of PtFe nanozymes: A one-pot method was used to synthesize PtFe bimetallic nanozymes using K2PtCl4 solution and FeCl3 solution as precursors. The final product was obtained after centrifugation and washing. The Pt:Fe feed ratio was adjusted, and the optimal ratio of 1:1 was determined through catalytic activity testing. Further morphology analysis was performed using HRTEM and particle size analysis, and elemental analysis was conducted using EDS mapping.

[0050] Construction and characterization of aptamer-nanozyme composite system: First, the complex was prepared by co-incubating KSAP with PtFe nanozymes to form an aptamer-nanozyme complex. Its hydration size and zeta potential were characterized, and the structural changes of single aptamer, kinetic co-aptamer pair (KSAP), KSAP+PtFe nanozyme, KSAP+target, and KSAP+PtFe nanozyme+target were analyzed by circular dichroism spectroscopy (CD).

[0051] Construction and performance evaluation of paper-based sensors: After perforating filter paper into appropriately sized discs, the following modifications were applied: single chitosan (CS) modification, CS+PEG blend solution modification, CS / PEG layer-by-layer modification, PEG / CS layer-by-layer modification, CS / PVA blend solution modification, CS / PVA layer-by-layer modification, PVA / CS layer-by-layer modification, CS / SA (sodium alginate) layer-by-layer modification, and SA / CS layer-by-layer modification (the modification order corresponds to the order in which the components appear). The contact angles of the modified paper were tested, and the CS / PEG layer-by-layer modified paper substrate with a medium contact angle was selected to slow down diffusion efficiency and achieve uniform dispersion. SEM measurements of the paper substrate were performed, and the paper substrate was used for colorimetric analysis.

[0052] The incubated KSAP+PtFe nanozyme was added dropwise to the center of the paper substrate. After reacting with the target, TMB and H2O2 were added dropwise to change the target concentration, obtaining a standard curve and LOD. Finally, the selectivity and anti-interference ability of the paper-based sensor were evaluated.

[0053] The method for detecting histamine, a small molecule target based on a colorimetric biosensor, of the present invention can simply and quickly determine histamine content, with good selectivity and anti-interference properties and low cost, which is of great significance for monitoring the freshness of aquatic products at both the upstream and downstream stages.

[0054] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0055] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0056] Example 1: Study on the Synergy of Kinetic Synergistic Adaptor Pairs (KSAP) In this invention, two histamine nucleic acid aptamers were selected as the original aptamers: L2 and HIS3-T2.

[0057] The nucleotide sequence of the capture aptamer L2 is shown in SEQ ID NO.1, as shown below (direction 5'-3'): ACAGGGAACTGTTGGTTGCGGTTCTTCCGATCTGCTGTGTTCT.

[0058] The nucleotide sequence of the stable aptamer HIS3-T2 is shown in SEQ ID NO.2, as shown below (direction 5'-3'): TGAGCCCAAGCCCTGGTATGAATGGATTCTCGTTGGGCTGG.

[0059] The affinity and binding rate between the captured aptamer L2 and the stable aptamer HIS3-T2 were determined using a biomacromolecule interaction analyzer (BLI). The aptamer concentration was 500 nM, and the target concentration ranged from 62.5 nM to 1000 nM. The results for aptamer L2 are as follows: Figure 1 As shown, the measurement results of aptamer HIS3-T2 are as follows: Figure 2 As shown in the figure. The results indicate that the response signals of both aptamers increase with increasing concentration, suggesting that binding is concentration-dependent, and that the degree of binding and dissociation phases differs between the two groups. Therefore, the data in Table 1 are obtained by fitting their curves. It can be seen that the L2 aptamer... K dCompared to HIS3-T2, it has approximately 3 times higher affinity for the target, but lower affinity. However, it has a higher binding rate constant, enabling it to quickly capture the target. But this is accompanied by a high dissociation rate constant, making the binding relatively unstable. Conversely, aptamer HIS3-T2 has high target affinity, a low binding rate, and a low dissociation rate. Therefore, when the target dissociates from L2, it can be firmly stabilized by HIS3-T2. The two aptamers are functionally synergistic and complementary. Therefore, by combining the two aptamers (mixed in a 1:1 molar ratio), a new kinetic synergistic aptamer pair (KSAP) was constructed, which improved both affinity and binding rate constant. Figure 3 Furthermore, the dissociation rate also decreased (the high dissociation rate significantly shifted towards the low dissociation rate). This kinetic synergistic aptamer pair can, to some extent, compensate for the gap between thermodynamics and kinetics in aptamers, achieving a win-win situation of fast and tight aptamers.

[0060] Table 1. Equilibrium dissociation constants of HIS3-T2, L2 aptamers, and KSAP obtained by BLI curve fitting. K d , binding rate constant k on and dissociation rate constant k off <![CDATA[ K d (M)]]> <![CDATA[k on (1 / Ms)]]> <![CDATA[k off (1 / s)]]> L2 <![CDATA[1.75×10 -7 ]]> <![CDATA[4.68×10 -5 ]]> <![CDATA[8.18×10 -2 ]]> HIS3-T2 <![CDATA[5.95×10 -8 ]]> <![CDATA[1.83×10 -5 ]]> <![CDATA[1.09×10 -2 ]]> KSAP <![CDATA[3.78×10 -8 ]]> <![CDATA[5.48×10 -5 ]]> <![CDATA[2.07×10 -2 ]]> Example 2 Synthesis and Characterization of PtFe Nanozymes The PtFe nanozyme was prepared according to the following steps: 150 μL of 20 mM K₂PtCl₄ solution and 150 μL of 20 mM FeCl₃ solution were prepared. 8.1 mg glycine, 19.8 mg polyvinylpyrrolidone (PVP, molecular weight 58000), and 1050 μL ultrapure water were added to a 1.5 mL centrifuge tube and vortexed for 10 minutes. The mixture was preheated at 65 °C for 5 minutes, and then 150 μL of freshly prepared 1.0 mL L-ascorbic acid solution was added and vortexed for 1 minute. The reaction system was incubated at 65 °C for 2 hours. After the reaction, the product was collected by centrifugation at 10000 rpm for 15 minutes and washed three times with ultrapure water to remove unreduced FeCl₃, yielding PtFe nanozyme. The catalytic activity of the nanozyme was compared by adjusting the molar ratio of Pt to Fe (1:0.25, 1:0.5, 1:1, 1:2, 1:4, 1:8). Figure 4 Based on economic factors and enzyme activity levels, the optimal ratio of Pt:Fe was ultimately selected as 1:1.

[0061] TEM and EDS mapping of nanozymes, such as Figure 5As shown, the nanozymes are uniformly distributed and have a consistent morphology, forming clusters. Elemental analysis revealed that Pt is the most abundant element, with a small amount of Fe distributed within. The particle size was found to be relatively small, at 28.55 ± 2.86 nm. Figure 6 After the morphology meets the requirements, steady-state dynamic analysis is performed on it. Figure 7-10 The catalytic parameters of PtFe nanozymes for the substrates TMB and H2O2 were determined. Under a fixed H2O2 concentration (10 mM), the Michaelis constant of the PtFe nanozyme for TMB was measured by varying the TMB concentration. K m The maximum reaction rate (V) is 0.195 mM. max The value is 1.89 × 10 -6 M·s -1 Its Michaelis constant is 0.45 times that of natural horseradish peroxidase (HRP). Under a fixed TMB concentration (0.4 mM), the H2O2 concentration was varied, and the effect of PtFe nanozyme on H2O2 was measured. K m It is 9.43 mM, V max It is 2.66 × 10 -6 M·s⁻ -1 As shown in Table 2, compared with AuNPs@FeP, the PtFe nanozyme of the present invention exhibits a significantly higher affinity for H2O2 ( K m The maximum reaction rate was also significantly higher (9.43 mM vs. 500 mM). max 2.66×10 -6 vs. 1.25×10 -8 M·s -1 Furthermore, PtFe nanozymes have a strong effect on H2O2. K m The molecular weight (9.43 mM) is significantly lower than that of traditional Pt nanoparticles (205.6 mM), indicating that its affinity for the substrate is increased by approximately 22 times; its maximum reaction rate (V0) is also significantly lower. max It also improved by about 27 times (2.66 × 10⁻⁶) compared to Pt nanoparticles. -6 vs. 9.79×10 -8 M·s -1 The above results demonstrate that PtFe nanozymes exhibit excellent affinity and high catalytic activity for both substrates, providing ideal output performance for signal transduction.

[0062] The above results show that the PtFe nanozyme exhibits significant affinity and high catalytic activity for both substrates, demonstrating its potential as a high-performance nanozyme. Therefore, this nanozyme can provide excellent signal output for recognition transduction.

[0063] Table 2 Kinetic parameters of different nanozymes

[0064] Example 3 Construction and characterization of aptamer-nanozyme composite system The signal recognition element kinetic synergistic aptamer pair KSAP from Example 1 and the signal transduction element PtFe nanozyme from Example 2 were co-incubated to form an aptamer-nanozyme composite system. For example... Figure 11 As shown, the target exhibits a signal inhibition response mode to the aptamer-nanozyme complex, which is presumably due to target-induced aptamer conformational rearrangement. Without target binding, KSAP adsorbs onto the PtFe nanozyme surface in a relatively relaxed conformation, having little impact on catalytic activity. However, in the presence of the target, KSAP specifically recognizes and binds to it, and the target continuously achieves a distribution equilibrium among KSAPs, folding to form a stable three-dimensional structure. This leads to an increase in the space occupied by KSAP on the nanozyme surface, hindering the substrate (TMB and H2O2) from approaching the catalytic active site of the nanozyme, thus reducing catalytic efficiency. Analysis of particle size changes shows that the particle size increases after the addition of KSAP (D...). PtFe =186.80±4.94 nm → D PtFe+KSAP =200.60±3.44 nm), indicating that KSAP adsorbs onto the surface of the nanozyme in a relatively relaxed conformation; the particle size decreases after the addition of the target (D PtFe+KSAP =200.60±3.44 nm → D PtFe+ KSAP+target =194.70±4.24 n), indicating that KSAP undergoes conformational folding due to target induction, resulting in a reduction in space occupancy. This trend is consistent with the change in the unfolded to folded state of KSAP on the nanozyme surface. Figure 12 Zeta potential results are as follows: Figure 13 As shown, Z PtFe = -6.85±0.63 mv,Z PtFe+KSAP = -11.53±0.93 mv, Z PtFe+ KSAP+target = -14.73±0.75 mV, the gradually increasing negative potential indicates that after the KSAP conformation folds, its phosphate backbone is more exposed to the solution environment, increasing the surface negative charge density. Circular dichroism peak shift results ( Figure 14 This also demonstrates the rearrangement and changes in the surface structure of nanozymes after the addition of the target.

[0065] Example 4: Construction and Performance Evaluation of Paper-Based Sensors After punching holes in Whatman NO.1 filter paper into appropriately sized discs, the following modifications were performed: single chitosan (CS) modification, CS+PEG blend solution modification, CS / PEG layer-by-layer modification, PEG / CS layer-by-layer modification, CS / PVA blend solution modification, CS / PVA layer-by-layer modification, PVA / CS layer-by-layer modification, CS / SA (sodium alginate) layer-by-layer modification, SA / CS layer-by-layer modification (the modification order corresponds to the order in which the components appear), with a concentration of 0.5 wt%. Specific methods include: (1) Single chitosan (CS) modification: add CS solution and dry; (2) CS+PEG blend solution modification: add a mixed solution of CS and polyethylene glycol (PEG) and dry; (3) CS / PEG layer-by-layer modification: first add CS solution, dry, then add PEG solution and dry; (4) PEG / CS layer-by-layer modification: first add PEG solution, dry, then add CS solution and dry; (5) CS+PVA blend solution modification: add CS and polyvinyl alcohol. (6) CS / PVA layer-by-layer modification: first add CS solution, dry, then add PVA solution, dry; (7) PVA / CS layer-by-layer modification: first add PVA solution, dry, then add CS solution, dry; (8) CS / SA layer-by-layer modification: first add CS solution, dry, then add sodium alginate (SA) solution, dry; (9) SA / CS layer-by-layer modification: first add sodium alginate (SA) solution, dry, then add CS solution, dry.

[0066] In this embodiment, the viscosity of CS is 100-200 mPa·s (degree of deacetylation ≥95%), the weight-average molecular weight of PEG is 3000-5000 Da, the weight-average molecular weight of PVA is 60-70 kDa, and the viscosity of SA is 150-300 mPa·s. However, those skilled in the art will understand that the above molecular weights or viscosities can be adjusted within a certain range, as long as a substantially similar medium contact angle and uniform sample dispersion effect can be achieved. The contact angle after modification was tested (…). Figure 15 It can be seen that PEG and CS-modified paper substrates have controllable contact angles and superior apparent diffusion effects. Therefore, the CS / PEG layer-by-layer modification paper substrate with a medium contact angle was selected from the three groups to slow down the diffusion efficiency and achieve uniform dispersion. Analysis of the surface morphology of the paper substrate can yield the following results: Figure 16 As the number of modification layers increases, the surface becomes smoother and the porosity decreases.

[0067] The aptamer-nanozyme complex from Example 3 was added dropwise onto a paper substrate in two portions. After drying, different concentrations of target were added and reacted for ten minutes. Then, TMB and H2O2 were added sequentially, and after each ten-minute reaction, a photograph was taken using a smartphone in professional mode, with the camera 70 cm from the table. The image was analyzed by professional software to identify and obtain its RGB values. Subsequently, these values ​​were calculated using the following Euclidean distance model (D) formula: D = Where R0, G0, and B0 represent the mean R, G, and B values ​​of the blank group, respectively, and Ri, Gi, and Bi represent the mean R, G, and B values ​​of the experimental group, respectively. The resulting standard curve is shown below. Figure 17 The standard curve is a two-segment curve. When the histamine concentration is 3.05 nM-48.83 nM, the quantitative correlation between color intensity and histamine concentration is expressed as y=18.00x+184.54 (R0). 2 =0.9932); when the histamine concentration is 48.83 nM-781.25 nM, the quantitative correlation between color intensity and histamine concentration is expressed as y=1.08x+1010.69 (R = 0.9932); 2 =0.9989). The linear range of histamine was 3.05–781.25 nM, and the limit of detection (LOD) was 2.21 nM. To evaluate the superiority of the KSAP@PtFe paper-based sensor, HIS3-T2@PtFe and L2@PtFe were selected to construct control paper-based sensors, such as... Figure 18 As shown, the quantitative correlation between the color intensity of the HIS3-T2@PtFe paper-based sensor and histamine concentration is expressed as y = 2.67x + 763.98 (R 2 =0.9911), with a linear range of 7–448 nM and a limit of detection (LOD) of 5.21 nM. This indicates that although the HIS3-T2 aptamer exhibits high affinity, its binding rate is lower than that of KSAP. It may bind fewer targets within the same time frame compared to KSAP, hence the LOD is approximately 2.4 times that of the KSAP system (5.21 nM vs. 2.21 nM). Achieving the same binding effect may require a longer time. Meanwhile, Figure 19 As shown, the quantitative correlation between the color intensity of the L2@PtFe paper-based sensor and histamine concentration is expressed as y = 1.93x + 214.70 (R 2 =0.9949), with a linear range of 25–400 nM and a detection limit (LOD) of 19.79 nM. This indicates that the L2 aptamer has a high binding rate but low affinity, and is prone to dissociation after binding to the target within the same time. Therefore, its detection limit is about 9 times that of the KSAP system (19.79 nM vs. 2.21 nM).

[0068] The inventors did not wish to be bound by any specific theory. The limited reaction time of paper-based platforms (typically only a few minutes to a dozen minutes) places higher demands on the binding rate of the recognition element. The high binding rate L2 aptamer in KSAP ensures the capture of a sufficient amount of target in a short time, while the high affinity HIS3-T2 aptamer prevents target dissociation and loss under the fluid shear forces in the paper-based environment. This kinetic synergistic mechanism of 'fast capture and stable retention' precisely compensates for the shortcomings of paper-based platforms, such as narrow reaction time windows and susceptibility to fluid shear forces, thereby restoring detection sensitivity.

[0069] In summary, the synergistic kinetic mechanism of KSAP combined with the high catalytic activity of PtFe nanozymes results in a paper-based sensor based on KSAP-PtFe nanozymes exhibiting superior binding performance and detection efficiency.

[0070] To evaluate the selectivity and anti-interference ability of the colorimetric biosensor, six histamine analogues (putrescine, tryptophan, tyramine, cadaverine, spermine, histidine, and mixtures of the above with histamine) were selected for testing, with a histamine concentration of 200 nM. Figure 20 The results show that when each interfering substance exists alone, the sensor's colorimetric signal does not change significantly compared to the blank group; in the system where mixed interfering substances coexist, the detection signal of histamine is not significantly interfered with, and the color intensity remains stable. These results demonstrate that the paper-based sensor based on KSAP-PtFe nanozymes has good specific recognition ability for histamine and can still achieve accurate detection in complex matrices, showing good potential for practical applications.

[0071] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for histamine detection based on a paper-based sensor, characterized in that, Includes the following steps: Kinetic co-aptamer pairs (KSAP) were co-incubated with PtFe nanozymes to form aptamer-nanozyme complexes. The aptamer-nanozyme complex was dropped onto a modified paper substrate; After the test sample containing histamine is added and reacted, TMB and H2O2 are added to carry out a colorimetric reaction. The image of the paper substrate after color development is acquired, and the concentration of target histamine in the test sample is calculated based on the color intensity. The dynamic cooperative aptamer pair (KSAP) is composed of a mixture of capture aptamer L2 and stable aptamer HIS3-T2 in a set ratio; The nucleotide sequence of the capture aptamer L2 is shown in SEQ ID NO.1, and the nucleotide sequence of the stable aptamer HIS3-T2 is shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that, The kinetic synergistic aptamer pair (KSAP) is composed of a capture aptamer L2 and a stable aptamer HIS3-T2 mixed in a molar ratio of 1:

1.

3. The method according to claim 1, characterized in that, The PtFe nanozyme was synthesized in a one-pot process using K2PtCl4 and FeCl3 as precursors, with a Pt to Fe molar ratio of 1:

1. The reaction was carried out at 60-65°C for 2 hours under L-ascorbic acid reduction, and the product was obtained by centrifugation and washing.

4. The method according to claim 1, characterized in that, The paper base is modified by punching holes in the filter paper into a disc, and then modifying it layer by layer with chitosan (CS) and polyethylene glycol (PEG) to form a CS / PEG layer-modified paper base.

5. The method according to claim 1, characterized in that, Histamine concentration is detected by colorimetric signal detection, which includes: acquiring the RGB values ​​of the paper substrate after the reaction using a smartphone camera, and then using the Euclidean distance model D = ... Calculate the color intensity, where R0, G0, and B0 are the RGB values ​​of the blank group, and Ri, Gi, and Bi are the RGB values ​​of the experimental group.

6. The method according to claim 1, characterized in that, The aptamer-nanozyme complex is dropped onto a paper substrate, dried, and then incubated with the sample solution for 5-15 minutes.

7. The method according to claim 1, characterized in that, The method is applicable to the rapid detection of histamine in front-end or back-end aquatic products.

8. A paper-based sensor for histamine detection, characterized in that, include: Paper-based substrate modified with CS / PEG layers; An aptamer-nanozyme complex loaded on the paper substrate, the complex being formed by co-incubation of KSAP and PtFe nanozymes with a kinetic synergistic aptamer; The kinetic co-aptamer pair KSAP is composed of a mixture of the capture aptamer L2 and the stable aptamer HIS3-T2. The nucleotide sequence of L2 is shown in SEQ ID NO.1, and the nucleotide sequence of HIS3-T2 is shown in SEQ ID NO.

2.

9. The paper-based sensor according to claim 8, characterized in that, The dynamic co-aptamer pair KSAP is composed of a mixture of capture aptamer L2 and stabilizing aptamer HIS3-T2 in a molar ratio of 1:

1.

10. The paper-based sensor according to claim 8, characterized in that, The molar ratio of Pt to Fe in the PtFe nanozyme is 1:1.

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