Ternary material Pd / Pt / Co NPs and preparation method and application thereof

Abstract

The embodiment of the invention discloses a ternary material Pd / Pt / Co NPs as well as a preparation method and application thereof. According to the present invention, Pd / Pt / Co NPs is adopted as a signal label to construct the enhanced colorimetric-chemiluminescence-based bidirectional lateral flow chromatography test strip (ddLFIA) for quantitative detection of Myo; one end is used for colorimetric mode detection, and 3, 3 '-diaminobenzidine (DAB) is catalyzed to generate an oxidation product through peroxidase-like activity (POD) of Pd / Pt / Co NPs, so that the color of a T line is enhanced, a colorimetric signal is amplified, and the detection sensitivity is improved. At the other end, Pd / Pt / Co NPs is utilized to catalyze a luminol luminescence system, and detection in a chemiluminescence mode is carried out. According to the strategy, a more efficient multi-channel detection mode is provided, system errors and background signal interference are effectively corrected through combination of the two modes, and the accuracy of a test result is improved.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and more particularly to a ternary material Pd / Pt / Co NPs and a preparation method and application thereof. Background Art

[0002] Acute myocardial infarction (AMI) is a cardiovascular emergency characterized by acute myocardial ischemia and necrosis due to complete occlusion of a coronary artery, which can lead to cardiac dysfunction, heart failure, and even death. AMI patients typically experience a sudden onset, rapid disease progression, a poor prognosis, and may also be accompanied by various complications. Therefore, early screening and diagnosis of AMI are crucial for cost savings and improved clinical outcomes. Myoglobin (Myo) is an early biomarker of myocardial injury, and its rapid and highly sensitive detection is crucial for the diagnosis of AMI. Therefore, developing a rapid, sensitive, reliable, and accurate method for Myo detection is crucial for the diagnosis of AMI. Current Myo detection methods primarily include enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and surface plasmon resonance-based detection. However, these methods often suffer from complex equipment, long test cycles, high costs, and the need for specialized personnel, limiting their application in clinical field monitoring. In contrast, lateral flow immunoassay technology has become a research focus due to its rapid response, portability, and low cost.

[0003] However, most LFIA technologies rely on colloidal gold test strips, which lack sensitivity and are difficult to meet the detection needs of low-concentration biomarkers. Furthermore, qualitative analysis can only be performed by visual interpretation. The signal is not enhanced by secondary methods such as enzyme-linked amplification, but relies solely on the physical aggregation of markers (such as colloidal gold) for color development, resulting in a nonlinear relationship between signal intensity and analyte concentration. Although some LFIA methods based on fluorescence, surface-enhanced Raman scattering (SERS), and electrochemistry can achieve quantitative detection, most of these methods rely on a single signal output and are easily affected by serum color and light intensity, resulting in poor accuracy, low reliability, and an increased risk of misjudgment.

[0004] Noble metal nanoparticles (such as Pd, Pt, Au, etc.) are widely used in the construction of electrochemical sensors due to their excellent catalytic activity, conductivity and chemical stability. Among them, palladium (Pd) and platinum (Pt) bimetallic nanocomposites can significantly enhance the catalytic activity of nanoenzymes due to their synergistic effect, and have demonstrated high sensitivity in the detection of biomarkers such as glucose and dopamine. However, due to their limited catalytic activity towards hydrogen peroxide, the peroxidase-like activity of platinum-based nanodendritic structures is even inferior to that of HRP, that is, they cannot amplify the signal through enzyme-catalyzed reactions, resulting in difficulties in detecting low-concentration targets. Summary of the Invention

[0005] To solve the above problems, the present invention uses Pd / Pt / Co NPs as signal tags to construct a bidirectional lateral flow chromatography test strip (ddLFIA) based on enhanced colorimetry-chemiluminescence for quantitative detection of Myo. One end is used for colorimetric mode detection. Through the POD activity of Pd / Pt / Co NPs, DAB is catalyzed to produce oxidation products, which enhances the color of the T line, thereby amplifying the colorimetric signal and improving the detection sensitivity. The other end uses Pd / Pt / Co NPs to catalyze the luminol luminescence system for detection in chemiluminescence mode. This strategy provides a more efficient multi-channel detection method, and the combination of dual modes effectively corrects system errors and background signal interference, thereby improving the accuracy of the test results.

[0006] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of an embodiment of the present invention, the present invention provides a method for preparing a ternary material Pd / Pt / Co NPs, the method comprising:

[0008] (1) Poloxamer, K2PtCl4, K2PdCl4 and ascorbic acid react in the presence of water until the solution gradually changes from yellow to black, and then centrifuge, wash and disperse in water to obtain Pd / PtNPs;

[0009] (2) reacting the Pd / PtNPs, CoCl26H2O and NH3BH3 in the presence of water until a large amount of bubbles are generated, centrifuging, and dispersing in a PBS buffer solution to obtain the Pd / Pt / CoNPs.

[0010] Furthermore, in step (1),

[0011] The molar ratio of K2PtCl4, K2PdCl4 and ascorbic acid is 108:77:500, and the mass ratio of poloxamer to K2PdCl4 is 1.8-2.2:1;

[0012] The reaction conditions are as follows: stirring in a magnetic stirrer at 28-32°C for 12 h at a speed of 600 rpm;

[0013] The volume mass ratio of the water to K2PdCl4 is 15 mL:25.2 mg.

[0014] Furthermore, in step (2),

[0015] The mass ratio of CoCl2·6H2O, NH3BH3 and K2PdCl4 is 30:60:25.2;

[0016] The volume mass ratio of the PBS buffer solution to K2PdCl4 is 15 mL:25.2 mg.

[0017] According to a second aspect of the embodiments of the present invention, the present invention provides a ternary material Pd / Pt / Co NPs, which is prepared by the method described in any one of the above items.

[0018] According to a third aspect of the embodiments of the present invention, the present invention provides a use of the ternary material Pd / Pt / Co NPs as described above in lateral flow immunochromatographic analysis.

[0019] According to a fourth aspect of an embodiment of the present invention, the present invention provides a Myo detection method using a bidirectional lateral flow chromatography test strip based on the ternary material Pd / Pt / Co NPs described above, wherein the bidirectional lateral flow chromatography test strip comprises, from left to right, a first absorbent pad, a first NC membrane, a sample pad, a second NC membrane, and a second absorbent pad, the method comprising the following steps:

[0020] (1) After adjusting the pH value of the Pd / Pt / Co NPs, the Pd / Pt / Co NPs were coupled with the anti-Myo monoclonal antibody Ab1 in the presence of a PBS buffer solution, and then blocked, washed, and resuspended in a PBS buffer solution to obtain Pd / Pt / Co-Ab1;

[0021] (2) After the sample to be tested is mixed with Pd / Pt / Co-Ab1, the resulting sample solution is transferred to the sample pad;

[0022] (3) DAB and H2O2 were added dropwise to the T line of the first NC membrane to obtain the gray value of the T line. The Myo content in the test sample was obtained based on the linear relationship between the gray value difference ΔG and the Myo concentration C, where ΔG = G-G0, G and G0 are the gray values ​​of the test sample and the negative control sample, respectively;

[0023] (4) Luminol and H2O2 were added dropwise to the T line of the second NC film, and the chemiluminescence intensity of the T line area was obtained using a chemiluminescence analyzer under dark conditions. The difference in chemiluminescence intensity ΔI was used to determine the chemiluminescence intensity. CL The linear relationship between ΔI and Myo concentration C was used to obtain the Myo content in the sample to be tested, where ΔI CL =I CL -I CL0 , I CL , I CL0 are the chemiluminescence intensities of the test sample and the negative control sample, respectively.

[0024] Furthermore, in step (1),

[0025] After mixing 10 μL of 0.2 mol / L potassium carbonate with 50 μL of Pd / Pt / Co NPs, 2.5 μL of 1 mg / mL anti-Myo monoclonal antibody Ab1 and 1 mL of PBS buffer solution were added, and then gently shaken at room temperature for 120 minutes. Subsequently, 100 μL of 1% BSA solution was added and incubated at 800 rpm for 60 minutes at 25°C. Finally, the mixture was centrifuged at 8000 rpm for 8 minutes at room temperature for several washes, and the precipitate was directly resuspended in 100 μL of PBS buffer solution.

[0026] Furthermore, the T line of the first NC membrane / the second NC membrane is coated with Myo cAb, and the coating amount of Myo cAb on each centimeter width of the T line is 350-400 ng, and the C line is coated with goat anti-mouse IgG, and the coating amount of goat anti-mouse IgG on each centimeter width of the C line is 350-400 ng;

[0027] The two-way lateral flow chromatography test strip adopts RB45 model glass fiber.

[0028] Further,

[0029] In step (2), the amount of Pd / Pt / Co-Ab1 used is 20 μL;

[0030] In step (3), the DAB was prepared with anhydrous ethanol as solvent at a concentration of 40 mmol / L and a volume of 50 μL, and the H2O2 was prepared with HAc-NaAc buffer solution at pH 4.0 as solvent at a concentration of 20 mmol / L and a volume of 50 μL; after the dropwise addition of DAB and H2O2, the reaction time was 5 min;

[0031] In step (4), the luminol is prepared by diluting 30% H2O2 with distilled water at a concentration of 4 mmol / L and a volume of 50 μL of a pH NaOH aqueous solution and a concentration of 10 mmol / L and a volume of 50 μL of H2O2.

[0032] Furthermore, in step (3), the linear relationship is: ΔG = 17.1690LogC - 11.6274, R 2 =0.9945, where ΔG is expressed in au and C is expressed in ng / mL;

[0033] In step (4), the linear relationship is: ΔI CL =10.6940C+237.44943, R 2 =0.9985, where ΔI CL The unit of AU is au, and the unit of C is ng / mL.

[0034] The embodiments of the present invention have the following advantages:

[0035] The present invention first prepared the ternary material Pd / Pt / Co NPs and explored its POD activity and catalytic mechanism. Subsequently, using Pd / Pt / Co NPs as signal labels, a ddLFIA test strip based on a signal amplification strategy was developed, which can simultaneously complete the detection of Myo in both colorimetric and chemiluminescence modes. The constructed method has good anti-interference, repeatability and accuracy for the detection of Myo. On the basis of dual-mode detection, the color change of DAB-H2O2 is enhanced by Pd / Pt / Co NPs. This strategy reduces the detection limit of ddLFIA by 10 times, and to a certain extent overcomes the problem of insufficient sensitivity of traditional test strips. In addition, the design of the bidirectional structure of ddLFIA realizes dual-mode simultaneous detection, greatly saving detection time and improving detection efficiency. In summary, the developed sensing platform can quickly and easily detect Myo, and the dual-signal sensing strategy provides a flexible and universal method for designing LFIA test strips for multiplex detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0037] Figure 1 Structural and compositional analysis of the Pd / Pt / Co NPs nanozyme provided by the present invention, (A) Schematic diagram of the preparation of Pd / Pt / Co NPs nanozyme; (B) Actual images of Pd / Pt NPs and Pd / Pt / Co NPs nanozymes; (C, D) TEM images of Pd / Pt / Co NPs; (E) SEM image; (F) STEM image; (G) elemental distribution map; (H) EDS spectrum.

[0038] Figure 2 High-resolution XPS spectra of Pd / PtNPs and Pd / Pt / Co NPs, (A) overall spectrum of Pd / PtNPs and corresponding to that in Pd / PtNPs; (B) XPS spectra of Pd 3d and (C) Pt 4f; (D) schematic diagram of electron migration in Pd / Pt / Co NPs; (E) overall spectrum of Pd / PtNPs and corresponding to that in Pd / Pt NPs; (F) XPS spectra of Pd 3d, (G) Pt4f and (H) Co 2p.

[0039] Figure 3(A) UV-visible absorption spectra of various chromogenic substrates catalyzed by Pd / Pt / Co NPs after the addition of H2O2, inset: color changes of different substrates; (B) chemiluminescence spectrum measurement of Pd / Pt / Co NPs; (C) activity comparison of Pd / Pt / Co NPs and Pd / Pt NPs; (D) chemiluminescence kinetic curves of Pd / Pt / Co NPs and Pd / Pt NPs in different systems; (E) reaction images of Pd / Pt / Co NPs and Pd / Pt NPs in different luminescence systems.

[0040] Figure 4 Steady-state kinetic analysis of Pd / Pt / Co NPs: Michaelis-Menten model corresponding to TMB (A) and H2O2 (C), Lineweaver-Burk model corresponding to TMB (B) and H2O2 (D).

[0041] Figure 5 (A) Schematic depiction of the catalytic process of Pd / Pt / Co NPs in the TMB system; (B) Changes in the relative activity of Pd / Pt / Co NPs in the TMB-H2O system after the addition of free radical scavengers; (C) Fluorescence spectra of TA and (D) HE in different reaction systems. Experimental conditions: Pd / Pt / Co NPs, H2O2 (5 mmol / L), Trp, IPA, and BQ (0.2 mol / L), HE and TA (1 mg / mL), and 0.1 mol / L acetate buffer (pH 4.0).

[0042] Figure 6 (A) Comparison of UV absorption spectra of Pd / Pt / Co NPs, Ab1 and Pd / Pt / Co-Ab1; (B) Zeta potential changes of Pd / Pt / Co NPs, Ab1 and Pd / Pt / Co-Ab1.

[0043] Figure 7 Schematic diagram of the composition of ddLFIA labeled with Pd / Pt / Co NPs.

[0044] Figure 8 This is the effect of K2CO3 addition on the gray value of T line.

[0045] Figure 9 Figures 1 and 2 show the effect of (A) Pd / Pt / Co NPs dosage on the grayscale value of the T line; (B) the effect of antibody dosage on the grayscale value of the T line; and (C) the effect of Pd / Pt / Co-Ab1 probe volume on the grayscale value of the T line. (Insets show images of the test strips under each condition; top: C line; bottom: T line).

[0046] Figure 10Effects of (A) pH, (B) luminol concentration, and (C) H2O2 concentration on the POD activity of Pd / Pt / Co NPs.

[0047] Figure 11 Effects of (A) pH; (B) different reaction substrates; and (C) reaction time on the grayscale value of the T line (the insets are actual images of the test strips under each condition, top: C line; bottom: T line).

[0048] Figure 12 ddLFIA images of different Myo concentrations (A) and images after enhanced colorimetry (B); T-line grayscale value difference response graph of ddLFIA at different Myo concentrations (C) and corresponding linear calibration graph (D); chemiluminescence intensity response graph of different Myo concentrations (E) and corresponding linear calibration graph (F) using the chemiluminescence method (n = 3).

[0049] Figure 13 The anti-interference performance of the ddLFIA test strips in the colorimetric mode (A) and the chemiluminescence mode (D); the reproducibility of the ddLFIA test strips in the colorimetric mode (B) and the chemiluminescence mode (E); and the stability of the ddLFIA test strips in the colorimetric mode (C) and the chemiluminescence mode (F) (n=3) (p<0.05). DETAILED DESCRIPTION

[0050] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0051] Example 1 Synthesis, Characterization and POD Activity Study of Pd / Pt / Co NPs

[0052] (1) Synthesis of Pd / Pt / Co NPs

[0053] Pd / Pt / Co NPs nanozymes were prepared by a one-pot method using Pt / Pd NPs as precursors and NH3BH3 as a reducing agent. Figure 1 -A). The specific contents are as follows:

[0054] 50 mg of Pluronic F127 was sonicated in a 10 mL solution of 9 mL of K₂PtCl₄ (12 mmol / L) and 1 mL of K₂PdCl₄ (77 mmol / L). 5 mL of ascorbic acid (100 mmol / L) was added as a reducing agent. The mixture was sonicated continuously for 30 min in a 25°C water bath and then stirred in a magnetic stirrer at 30°C for 12 h at 600 rpm, during which time the solution gradually turned from yellow to black. After the reaction, the solid phase was collected by centrifugation and washed with acetone and distilled water for five consecutive cycles (8000 rpm for 8 min each). The solid phase was then dispersed in 15 mL of water to obtain Pd / Pt NPs for further use. Next, 5 mL of the prepared Pd / Pt NPs was dispersed in 25 mL of distilled water. 10 mg of CoCl₂·6H₂O was added and sonicated at room temperature to homogenize the mixture. 20 mg of NH₃BH₃ was then added until abundant bubbles formed, concluding the reaction. After the reaction was completed, the mixture was centrifuged at 12000 rpm for 10 min at room temperature, the supernatant was discarded, and the precipitate was retained and redispersed in 5 mL of PBS to obtain Pd / Pt / Co NPs, which were then stored at 4°C.

[0055] (2) Characterization of Pd / Pt / Co NPs

[0056] from Figure 1 As can be seen in Figure 2-B, both Pd / PtNPs and Pd / Pt / CoNPs appear black due to the reduction of Co by NH3BH3. 2+ H2 will be generated during the preparation process, so bubbles will be generated during the preparation process. TEM image ( Figure 1 -C) shows that Pd / Pt / Co NPs have an irregular spherical structure with protrusions on the surface. Figure 1 -D shows the morphology of single Pd / Pt / Co NPs with an average particle size of about 40-50 nm, high surface area and good dispersion. Figure 1 -E shows that the particle size of Pd / Pt / Co NPs is relatively uniform and the shape is consistent with TEM. In the element distribution map ( Figure 1 -F, G) can be found that Pt is most distributed in the outermost layer, a small amount of Co is distributed on its surface, and Pd is mainly distributed in the inner layer. The element distribution of Pd / Pt / Co NPs was analyzed by energy dispersive spectroscopy (EDS) line scanning method. The results showed that the element contents of Pd, Pt and Co were 65.15%, 21.87% and 14.04% respectively ( Figure 1 -H). This is consistent with the results of the elemental distribution map, further indicating that Pd / Pt / Co NPs were successfully prepared.

[0057] In order to study the effect of elemental interactions on peroxidase activity, XPS spectroscopy was used to analyze the element valence in Pd / PtNPs and Pd / Pt / CoNPs. Figure 2 As shown in -A, E, Pd / PtNPs are composed of Pd, Pt, C and O, while Pd / Pt / Co NPs contain Pd, Pt, C, O and Co. According to the spectrum of Pd element, the presence of Co slightly increases the Pd 3d 3 / 2 and Pd3d 5 / 2 The binding energy of Figure 2 -B) increased to 340.6eV and 335.3eV ( Figure 2 -F). Similarly, the presence of Co will 5 / 2 and Pt 4f 7 / 2 The binding energies of the orbitals are 74.2eV and 70.9eV ( Figure 2 -C) increased to 74.5eV and 71.2eV ( Figure 2 -G). It is well known that in bulk alloys, electrons migrate from metals on the left side of the periodic table to electron-rich metals on the right side. However, at the surface, the electron migration is reversed. The binding energy of Pd and Pt increases, indicating that electrons automatically migrate from Pt / PtNPs to Co, producing electron-deficient Pt / PtNPs ( Figure 2 -D). Existing studies have shown that electron-deficient catalysts exhibit enhanced interactions with substrates, thereby improving catalytic activity. Therefore, it is speculated that Co doping improves the catalytic activity of Pt / PtNPs. In addition, for Pd / Pt / Co NPs, the characteristic peaks of Co 2p3 / 2 and Co 2p1 / 2 are 796.8eV and 781.0eV, respectively ( Figure 2 -H), and Co 2+ The positions of the characteristic peaks are basically consistent. The above results show that the Co element is successfully doped on Pt / PtNPs.

[0058] (3) Study on the catalytic activity of Pd / Pt / Co NPs

[0059] In order to explore the nanozyme activity of Pd / Pt / Co NPs, this study was verified using 3,3',5,5'-tetramethylbenzidine (TMB), DAB and o-phenylenediamine (OPD) as reaction substrates.

[0060] To a 96-well plate, 160 μL of pH 4.0 HAc-NaAc buffer solution, 10 μL of Pd / Pt / Co NPs, and 10 μL of 2 mmol / L H₂O₂ were added and mixed thoroughly. Immediately, 10 μL of TMB, DAB, or OPD (all at 40 mmol / L) was added and mixed using a vortex mixer. After a 1-minute reaction, UV-visible absorption spectra were recorded at 652 nm.

[0061] like Figure 3 -A, in the presence of H2O2, the color of their oxidation products changed and the absorption spectrum also changed, which indicates that Pd / Pt / Co NPs have strong POD activity. For example, the oxidation product of TMB catalyzed by Pd / Pt / Co NPs produces a significant absorption peak at 652nm, and the characteristic absorption peaks of DAB and OPD oxidation products are located at 470nm and 450nm, respectively. Figure 3 As shown in Figure 1-B, the signal generated by the luminol chemical system catalyzed by Pd / Pt / Co NPs is concentrated at 425 nm, which is consistent with the emission wavelength of luminol, indicating that Pd / Pt / Co NPs have excellent enzyme catalytic activity.

[0062] To verify whether Co doping improves the catalytic activity of Pd / PtNPs, the POD and OXD activities of Pd / PtNPs and Pd / Pt / Co NPs were compared under the same conditions.

[0063] To a 96-well plate, 160 μL of pH 4.0 HAc-NaAc buffer solution, 10 μL of Pd / Pt NPs or Pd / Pt / Co NPs, and either 10 μL of 2 mmol / L H₂O₂ (with or without) were added, mixed thoroughly, and then 10 μL of TMB (40 mmol / L) was immediately added and mixed using a vortex mixer. After a 1-minute reaction, UV-visible absorption spectra were recorded at 652 nm.

[0064] Based on the above research, the possible reaction mechanism of Pd / Pt / Co NPs in the luminol luminescence reaction system was elucidated by combining equations 1 to 6. Under alkaline conditions, luminol loses a proton to form LH - (Equation 1). Pd / Pt / CoNPs catalyze H2O2 to generate ·OH and ·O 2- (Formula 2). Subsequently, LH - and HO 2- Reacts with OH to form L - (Formula 4). Then, ·O 2- With L ·-The reaction generates an unstable excited state 3-aminophthalate anion (3-APA*) (Equation 5), which emits a strong light state at 440 nm when it returns to the ground (Equation 6).

[0065] Luminol+OH-→LH - +H2O (1)

[0066]

[0067] H2O2+OH - →HO 2- +H2O (3)

[0068] LH - + OH+HO 2- →·O 2- +L ·- +H2O (4)

[0069] L ·- +·O 2- →3-APA* (5)

[0070] 3-APA*→3-APA+hυ (6)

[0071] like Figure 3 -C, regardless of the presence of H2O2, the absorbance intensity of TMB catalyzed by Pd / Pt / Co NPs was stronger than that of Pd / PtNPs, and its chemiluminescence intensity for luminol system was also higher than that of Pd / Pt NPs ( Figure 3 -D). From Figure 3 As can be seen from Figure 3-E, the blue color produced by the reaction of Pd / Pt / Co NPs with TMB is deeper, and the blue light produced after the reaction with the luminol system is also higher than that of Pd / Pt NPs. The above results show that Co doping effectively improves the catalytic activity of Pd / Pt NPs.

[0072] In order to further explore the POD activity of Pd / Pt / Co NPs, steady-state kinetic analysis was performed. 10 μL Pd / Pt / Co NPs, 90 μL pH 4.0 HAc-NaAc buffer solution, 50 μL H2O2, and 50 μL TMB were added to a 96-well plate in sequence. The characteristic absorption peak intensity at 652 nm was monitored in real time using a UV-visible spectrometer, and the absorbance (A) at different reaction time points (t) was recorded. 652nm The initial reaction rate (V) is calculated by ΔA 652nm / t calculation, and each concentration was measured 3 times. Specifically, the TMB concentration was fixed at 20mmol / L, and the absorbance was measured at H2O2 concentrations of 10, 20, 40, 60, 80, 100, 200, 300, 400, and 500mmol / L, or the H2O2 concentration was fixed at 50mmol / L, and the absorbance at different TMB concentrations (1, 2, 4, 6, 8, 15, 20, 30, and 50mmol / L) was measured. The kinetic analysis used the Michaelis-Menten model, and the specific parameters were calculated using the double reciprocal Lineweaver-Burk equation: 1 / V=K m / V m [S]+1 / V m Where V is the initial reaction rate, V max is the maximum reaction rate, [S] is the substrate concentration (mmol / L), K m is the Michaelis constant (mmol / L), which reflects the affinity between the enzyme and the substrate.

[0073] like Figure 4 As shown in Figure 2, under standard reaction conditions (pH 4.0, 25°C), the corresponding TMB ( Figure 4 -A) and H2O2( Figure 4 -C) steady-state kinetic curves, and K was calculated according to the Michaelis-Menten equation m and V max Value. Figure 4 -B and 4-D show that the reciprocal of TMB and H2O2 concentrations (1 / C) showed good linear fit with the corresponding 1 / v, respectively.

[0074] The K of Pd / Pt / Co NPs, Pd-Pt, and natural horseradish peroxidase (HRP) m and V max The results are compared in Table 1.

[0075] Table 1 K of Pd / Pt / Co NPs and other substances m and V max Compare

[0076]

[0077] Note: For the preparation method of Pd-Pt, please refer to Liu S., Li Z., Li C., et al. A "three-in-one" multifunctional palladium / platinum nanoparticles-driven multimodal lateralflow immunoassays for point-of-care testing of Staphylococcus aureus[J]. Sensors and Actuators B: Chemical, 2024, 413: 135877.

[0078] As shown in Table 1, Pd / Pt / Co NPs exhibited a lower K for H2O2 than HRP. m The K value for TMB is m However, it is worth noting that, whether H2O2 (when measuring H2O2, the concentrations of Pd / Pt / Co NPs and TMB were fixed) or TMB (when measuring TMB, the concentrations of Pd / Pt / Co NPs and H2O2 were fixed), trimetallic Pd / Pt / Co NPs showed better catalytic performance for both substrates compared with Pd / Pt NPs, not only K m The value is lower and it shows a larger V max , which fully demonstrates the important role of cobalt doping in improving the catalytic activity of nanozyme POD.

[0079] The present invention reveals the catalytic mechanism of Pd / Pt / Co NPs POD activity through systematic free radical capture experiments.

[0080] Free Radical Verification: To a 96-well plate, 160 μL of pH 4.0 HAc-NaAc buffer, 10 μL of Pd / Pt NPs or Pd / Pt / Co NPs, and 10 μL of 2 mmol / L H₂O₂ were added sequentially. Immediately thereafter, 10 μL of TMB (40 mmol / L) and 10 μL of a scavenger (1 mg / mL) were added and mixed using a vortex mixer. After a 1-minute reaction, UV-visible absorption spectra were recorded at 652 nm.

[0081] like Figure 5 As shown in Figure 2A, in the catalytic system with the presence of H2O2, reactive oxygen species (ROS) such as hydroxyl radicals (·OH), superoxide anions (·O 2- ) and singlet oxygen ( 1 O2) may participate in the reaction process. Generally speaking, ·OH, ·O 2- and1 O2 is easily generated from H2O2 in the nanozyme catalytic reaction, and then reacts with the reaction substrate DAB to produce brown oxidation products. In order to clarify the active species that dominate this reaction, the experiment was designed to use a specific scavenger system: isopropyl alcohol (IPA, ·OH scavenger), p-benzoquinone (BQ, ·O 2- scavenger) and tryptophan (Trp, 1 O2 scavenger). Figure 5 -B, the relative activity of the system was significantly reduced to 52.8±2.7% (p<0.05) after the addition of BQ, while the activity of the IPA and Trp treatment groups remained at 102.56±6.7% and 89.62±4.8% of the original level, respectively. This difference indicates that 2- It is the key free radical in the catalytic process. To further verify this conclusion, the fluorescence probe method was used for auxiliary characterization: when terephthalic acid (TA) was used as the ·OH probe, the fluorescence changes of each reaction system at 360nm were not obvious ( Figure 5 -C), and dihydroethidium (HE) as ·O 2- When the probe was added, the fluorescence of Pd / Pt / Co NPs was significantly enhanced at 610 nm ( Figure 5 -D). These results indicate that Pd / Pt / Co NPs can generate ·O by catalyzing the decomposition of H2O2 2- Dominates the TMB oxidation process.

[0082] Example 2 Preparation and Characterization of Pd / Pt / Co-Ab1

[0083] (1) Preparation of Pd / Pt / Co-Ab1

[0084] The pH of the Pd / Pt / Co NPs prepared in Example 1 was adjusted with potassium carbonate (aqueous solution, 0.2 mol / L). 2.5 μL of 1 mg / mL anti-Myo monoclonal antibody Ab1 (CAT-6006, Zhengzhou Saitukang Biotechnology Co., Ltd.) was added dropwise to 100 μL of pH-adjusted Pd / Pt / Co NPs, and 1 mL of pH 7.2-7.4 PBS buffer solution was added. The mixture was then gently shaken at room temperature for 120 minutes to form a Pd / Pt / Co NPs-labeled Ab1 complex (Pd / Pt / Co-Ab1). Excess binding sites on the Pd / Pt / Co NPs were then blocked with 100 μL of 1% BSA solution and incubated at 800 rpm for 60 minutes at 25°C. Finally, the mixture was washed several times by centrifugation at 8000 rpm for 8 minutes at room temperature, and the precipitate was directly resuspended in 100 μL of pH 7.2-7.4 PBS buffer solution.

[0085] (2) Characterization of Pd / Pt / Co-Ab1

[0086] In this study, UV-visible spectroscopy and Zeta potential characterization techniques were used to verify whether the Pd / Pt / Co-Ab1 immunoprobe was successfully prepared. Figure 6 -A shows that pure Ab1 has a typical absorption peak at 280nm, while Pd / Pt / Co NPs have no obvious absorption in this wavelength range. When Pd / Pt / Co NPs are coupled with Ab1, an absorption peak also appears at the same position, indicating that Pd / Pt / Co-Ab1 is successfully prepared. Zeta potential test ( Figure 6 -B) found that the surface potential of Ab1 was -5.32 mV, while the surface potential of Pd / Pt / Co NPs was +1.72 mV. After coupling, the Zeta potential of the Pd / Pt / Co-Ab1 probe changed to -3.64 mV, a significant change that once again verified the successful coupling of Ab1 with Pd / Pt / Co NPs.

[0087] Example 3 Detection of Myo

[0088] (1) Preparation of ddLFIA test strips

[0089] The ddLFIA test strip consists of six parts: a PVC base, a sample pad, two NC membranes, and two absorbent pads. The sample pad is located in the middle of the test strip, and an NC membrane and an absorbent pad are placed on both sides of the sample pad. That is, the ddLFIA test strip from left to right consists of: the first absorbent pad, the first NC membrane (C line, T line), the sample pad, the second NC membrane (T line, C line), and the second absorbent pad ( Figure 7 ).

[0090] The preparation method of ddLFIA test strips is as follows:

[0091] First, a 2.5 × 10 cm glass fiber (RB45, Shanghai Jieyi Biotechnology Co., Ltd.) was soaked in PBS (10 mmol / L, pH 7.4) containing 0.5% PVP and 1% Tween-20, completely wetted, and then dried overnight at 37°C. Separately, 0.25 mg / mL Myo cAb (CAT-6005, Zhengzhou Saitukang Biotechnology Co., Ltd.) and 0.25 mg / mL goat anti-mouse IgG (CAT-9023, Zhengzhou Saitukang Biotechnology Co., Ltd.) were uniformly coated onto the NC membrane at a constant speed, forming the T and C lines, respectively, with a 5 mm interval between them. The coating amount of Myo cAb per centimeter of T line and goat anti-mouse IgG per centimeter of C line was 370 ng. The membrane was then dried overnight in an oven set at 37°C. Finally, all the processed sample pads, NC membranes and absorbent papers were placed on the PVC base in the order of absorbent pad, NC membrane, sample pad, NC membrane and absorbent pad, with adjacent parts overlapping by 2 mm. After assembly, they were cut into 5 mm wide test strips and stored in sealed bags to prevent the NC membrane from getting wet.

[0092] (2) Myo detection principle

[0093] In this study, Myo was used as the analyte and a novel dual-signal bidirectional LFIA was established based on competitive immunoassay. Figure 7As shown, when Myo appears, it first undergoes an antigen-antibody reaction with Pd / Pt / Co-Ab1 to form a Myo-Pd / Pt / Co-Ab1 complex. As the liquid moves, the Myo-Pd / Pt / Co-Ab1 complex is captured by the Myo cAb coated on the T line, forming a "sandwich complex," resulting in a black band on the T line. Unbound Pd / Pt / Co-Ab1 is then captured by the IgG on the C line, also appearing black on the C line. When a DAB-H2O2 solution is added to the T line, the T line changes from black to reddish-brown due to DAB oxidation by H2O2 under the catalytic activity of Pd / Pt / Co. In colorimetric mode, the T line's colorimetric signal can be further quantified using Image J software, not only by visual semiquantitative detection. Notably, the use of Pd / Pt / Co NPs to catalyze the DAB color reaction significantly enhances signal intensity and widens the color variation of the T line, thereby improving the sensitivity and detection range of this method. In addition, to achieve a dual-mode strategy in a single test, the other side of the test strip can also use the Pd / Pt / Co NPs catalyzed luminol system to achieve chemiluminescence mode detection. Simply add luminol solution and H2O2 solution to the T line, and then use a chemiluminescence analyzer to read the signal to achieve quantitative analysis in chemiluminescence mode. In short, if both the T line and the C line are colored, it indicates that the sample contains Myo and the test result is positive. Conversely, if only the C line is colored, it is negative. If the C line does not color, the test strip is considered invalid.

[0094] (3) Detection method

[0095] A series of Myo standard solutions with concentrations of 1 to 1000 ng / mL were prepared using pH 7.2 to 7.4 PBS buffer solution. 100 μL of Myo standard solutions of different concentrations were premixed with 20 μL of Pd / Pt / Co-Ab1 prepared in Example (1) for 5 min to allow Myo to bind to Pd / Pt / Co-Ab1 to form a "Myo-Pd / Pt / Co-Ab1" complex, which was then transferred to the sample pad of the test strip. The sample solution flowed forward along the NC membrane under capillary action. After the immune reaction was completed, 50 μL of DAB (40 mmol / L, prepared with anhydrous ethanol as solvent) and 50 μL of H2O2 (40 mmol / L, prepared with 30% H2O2 diluted with pH 4.0 HAc-NaAc buffer solution as solvent) were added dropwise to the left T line. After a few minutes, a 4K high-definition camera was used to capture images of the ddLFIA test strip, and Image J was used to extract the grayscale value of the T line area. On the other side, 50 μL of H2O2 (10 mmol / L, prepared by diluting 30% hydrogen peroxide with distilled water) and 50 μL of luminol (4 mmol / L, prepared by using a NaOH aqueous solution with a pH of 14 as a solvent) were added dropwise to the T line, and the chemiluminescence intensity was measured using a chemiluminescence analyzer in the dark.

[0096] (4) Optimization of experimental conditions

[0097] To achieve good analytical results, this study optimized several key parameters at a Myo concentration of 10 μg / mL. Because the electrostatic interaction between antigen and antibody is significantly affected by pH, K2CO3 was added to adjust the pH of the system to achieve optimal conditions. Figure 8 The results show that when 10 μL K2CO3 is added, the T line color is the darkest and the gray value is the smallest, so adding 10 μL K2CO3 is the optimal condition.

[0098] As the volume of Pd / Pt / Co NPs increases, the color of the T line gradually increases ( Figure 9 -A). When the volume of Pd / Pt / Co NPs is 50 μL, the T line is the darkest. When the volume is greater than 50 μL, the stability of the excessive Pd / Pt / Co NPs decreases and aggregation occurs, which in turn affects their mobility on the NC membrane. Therefore, 50 μL is the optimal volume of Pd / Pt / Co NPs. Figure 9As shown in Figure 2-B, the grayscale value of the T line gradually decreases with the increase of Ab1 volume and then tends to be stable. When the volume of Ab1 is 2.5μL (dosage is 2.5μg), the T line color is the darkest and the grayscale value is the smallest. Therefore, the optimal amount of Ab1 is 2.5μg. Similarly, when the volume of the probe Pd / Pt / Co-Ab1 is 20μL, the grayscale value drops to the lowest. Therefore, in order to avoid the loss of antibodies, the optimal amount of Pd / Pt / Co-Ab1 probe is 20μL ( Figure 9 -C).

[0099] At the same time, in order to ensure that the constructed chemiluminescence achieves the best detection performance, the key factors affecting the experimental results were deeply explored. Considering that pH value has a significant effect on enzyme activity, the catalytic activity of Pd / Pt / Co NPs on the luminol-H2O2 system (luminol is prepared by NaOH solution of different pH values, with a volume of 50μL, and H2O2 is prepared by diluting 30% H2O2 with distilled water, with a volume of 50μL) under different pH (10-14) conditions was explored. Figure 10 As shown in Figure 1-A, the catalytic activity of Pd / Pt / Co NPs reached its highest when the pH value of luminol was 14. Therefore, pH 14 was the optimal condition for subsequent chemiluminescence analysis. Figure 10 As shown in Figure 1-B, the chemiluminescence intensity gradually increases with increasing luminol concentration. The chemiluminescence intensity reaches its peak at 4 mmol / L, making 4 mmol / L the optimal luminol concentration. Similarly, the chemiluminescence intensity in this system reaches its peak at 10 mmol / L H2O2, making 10 mmol / L the optimal H2O2 concentration.

[0100] The specific effects of the pH of the HAc-NaAc buffer solution, the type of reaction substrate, and the reaction time on the experimental results in the colorimetric reaction system (reaction substrate - H2O2, the concentration of the reaction substrate is 40mmol / L, prepared from anhydrous ethanol, the volume is 50μL; the concentration of H2O2 is 20mmol / L, prepared from 30% H2O2 diluted with HAc-NaAc buffer solution, the volume is 50μL) were further explored. It can be clearly seen that with the increase of pH, the absorbance also shows a trend of gradual increase ( Figure 11 -A). When pH reaches 4, the absorbance reaches its maximum. Therefore, in subsequent experiments, pH 4.0 was used as the optimal colorimetric condition for the reaction. At the same time, in order to obtain the best color enhancement effect, different substrates were used for comparison. The results are as follows Figure 11When using 3-amino-9-ethylcarbazole (AEC) and TMB, the grayscale value was low, followed by TMB and OPD. 4-chloro-1-naphthol (4-CN) showed almost no change, indicating that ACE and DAB had a strong enhancement effect. However, as can be seen from the actual image, AEC itself is yellow, and after enhancement, it has a high background, making it unsuitable for enhancing signal intensity. Finally, the effect of reaction time was examined. After the addition of the colorimetric reagent, the reaction became more complete with time, and the grayscale value change reached its maximum after 5 minutes. Therefore, using the DAB-H2O2 system, a reaction time of 5 minutes was the optimal condition.

[0101] (5) Detection of Myo

[0102] Under the optimized experimental conditions, ddLFIA test strips were used to detect Myo in gradient concentrations. The colorimetric analysis results showed that ( Figure 12 -A), as the concentration of Myo standard solution increased from 1 ng / mL to 1000 ng / mL, the black color of the T line of the ddLFIA test strip gradually deepened. The addition of DAB-H2O2 significantly enhanced the signal intensity of the T line, and the detection limit observed by the naked eye decreased from 10 ng / mL to 1 ng / mL ( Figure 12 -B). Figure 12 -C, in the concentration range of 10 to 1000 ng / mL, the gray value difference ΔG (ΔG = G-G0, G and G0 are the gray values ​​of the test sample and the negative control sample, respectively) increases with the increase of Myo concentration (C), and there is an obvious linear correlation between the two ( Figure 12 -D). The linear regression equation obtained by fitting is ΔG=17.1690LogC-11.6274(R 2 =0.9945), LOD is as low as 0.34 ng / mL (3σ / k). For the chemiluminescence mode, the difference in chemiluminescence intensity of the T line of the ddLFIA test strip ΔI CL (ΔI CL =I CL -I CL0 , I CL , I CL0 The chemiluminescence intensity of the test sample and the negative control sample also increased with the increase of Myo concentration (C). Figure 12 -E). Figure 12 -F, in the concentration range of 10 to 1000 ng / mL, the linear regression equation is ΔI CL =10.6940C+237.44943(R 2 =0.9985), and the LOD reached 4.50 ng / mL.

[0103] Example 4 Performance Test of ddLFIA Test Strips

[0104] (1) LFIA test strip performance evaluation

[0105] The analysis results show that compared with the traditional single-mode detection system, the test strip constructed by the present invention exhibits the following advantages: it has a lower detection limit and a wide detection range, which is 2 orders of magnitude wider than the Myo test strip reported in the literature; the unique dual-mode (colorimetric / chemiluminescence) design realizes the self-verification function of the test results, significantly improving the detection reliability. Referring to the requirements of China's "Guidelines for the Technical Review of Registration of Myocardial Marker Detection Reagents", the detection range of 10-1000ng / mL of this test strip completely covers the critical value range required for clinical diagnosis (myocardial injury patients>50ng / mL), and the detection limit is significantly lower than the 80ng / mL required by the guidelines. In summary, the ddLFIA test strip meets the strict requirements of Myo for actual clinical applications.

[0106] (2) Study on the anti-interference, reproducibility and stability of ddLFIA

[0107] In order to evaluate the anti-interference ability of the immunoassay strip, the ddLFIA strip was used to detect multiple potential interfering substances in serum, including BSA, creatine kinase isoenzyme (CK-MB), C-reactive protein (CRP), cardiac troponin I (cTnI), and prostate-specific antigen (PSA). Figure 13 As shown in the figure, when detecting interfering substances or Mix, since they cannot specifically bind to Pd / Pt / Co~Ab1, and will not be captured by Myo cAb on the T line, ΔG and ΔI CL There was no significant change compared with the blank group. However, when Myo was detected at 100 ng / mL, ΔG and ΔI CL Similarly, when Myo is mixed with other interfering substances, the ΔG and ΔI of the T line CL will also increase accordingly ( Figure 13 -A and Figure 13 According to statistical analysis, no significant differences were found between the interfering substances and Mix group compared with the blank group in either the colorimetric or chemiluminescence mode (p>0.05), while significant differences were found between Myo and Myo+Mix group compared with the blank group (p<0.05). In summary, the ddLFIA test strips have a certain degree of anti-interference ability.

[0108] In terms of reproducibility, seven independent batches of ddLFIA test strips were prepared under the same conditions. They were used to measure 100 ng / mL of Myo and the changes in colorimetric and chemiluminescent signals were evaluated. The results showed that the corresponding RSDs were 3.58% and 1.94%, respectively. Figure 13 -B, Figure 13-E), were all less than 5%, indicating that the ddLFIA test strips had good reproducibility.

[0109] In terms of stability, the ddLFIA test strips were stored at room temperature for a period of time, and 100 ng / mL of Myo was measured to investigate the effect of different storage times on the detection performance of the test strips. Figure 13 -C and Figure 13 As shown in Figure 3-F, the colorimetric signal of the ddLFIA test strip remained above 88.08% with an RSD of 4.54% over 28 days. The chemiluminescent signal remained above 88.93% with an RSD of 4.17%. These results indicate that the ddLFIA test strip has good stability.

[0110] Example 5 Actual sample detection

[0111] To validate the feasibility of the ddLFIA test strip in testing real serum samples, the Myo concentration in human serum was determined using the same assay procedure and compared with the results from a commercial kit. As shown in Table 2, the calculated recoveries of Myo using the ddLFIA test strip ranged from 93.53% to 102.15%, with the RSDs for three replicates less than 5%. Furthermore, the results obtained with the ddLFIA test strip were consistent with those from a commercial ELISA kit. Independent-sample t-test analysis showed no significant difference between the results obtained with the ddLFIA test strip and the ELISA kit (p>0.05), demonstrating the reliability and accuracy of the ddLFIA test strip in testing real samples.

[0112] Table 2 Detection of Myo in serum by ddLFIA and ELISA kits (n=3)

[0113]

[0114]

[0115] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing ternary material Pd / Pt / Co NPs, characterized in that: The method comprises: (1) Poloxamer, K2PtCl4, K2PdCl4 and ascorbic acid react in the presence of water until the solution gradually changes from yellow to black, and then centrifuge, wash and disperse in water to obtain Pd / PtNPs; (2) reacting the Pd / PtNPs, CoCl26H2O and NH3BH3 in the presence of water until a large amount of bubbles are generated, centrifuging, and dispersing in a PBS buffer solution to obtain the Pd / Pt / CoNPs.

2. The method for preparing the ternary material Pd / Pt / Co NPs according to claim 1, characterized in that: In step (1), The molar ratio of K2PtCl4, K2PdCl4 and ascorbic acid is 108:77:500, and the mass ratio of poloxamer to K2PdCl4 is 1.8-2.2:1; The reaction conditions are as follows: stirring in a magnetic stirrer at 28-32°C for 12 h at a speed of 600 rpm; The volume mass ratio of the water to K2PdCl4 is 15 mL:25.2 mg.

3. The method for preparing the ternary material Pd / Pt / Co NPs according to claim 1, characterized in that: In step (2), The mass ratio of CoCl2·6H2O, NH3BH3 and K2PdCl4 is 30:60:25.2; The volume mass ratio of the PBS buffer solution to K2PdCl4 is 15 mL:25.2 mg.

4. A ternary material Pd / Pt / Co NPs, characterized in that: It is made by the method according to any one of claims 1 to 3.

5. Use of the ternary material Pd / Pt / Co NPs according to claim 4 in lateral flow immunochromatographic analysis.

6. A Myo detection method using a bidirectional lateral flow chromatography test strip based on the ternary material Pd / Pt / Co NPs according to claim 4, wherein the bidirectional lateral flow chromatography test strip comprises, from left to right, a first absorbent pad, a first NC membrane, a sample pad, a second NC membrane, and a second absorbent pad, characterized in that: The method comprises the following steps: (1) After adjusting the pH value of the Pd / Pt / Co NPs, the Pd / Pt / Co NPs were coupled with the anti-Myo monoclonal antibody Ab1 in the presence of a PBS buffer solution, and then blocked, washed, and resuspended in a PBS buffer solution to obtain Pd / Pt / Co-Ab1; (2) After the sample to be tested is mixed with Pd / Pt / Co-Ab1, the resulting sample solution is transferred to the sample pad; (3) DAB and H2O2 were added dropwise to the T line of the first NC membrane to obtain the gray value of the T line. The Myo content in the test sample was obtained based on the linear relationship between the gray value difference ΔG and the Myo concentration C, where ΔG = G-G0, G and G0 are the gray values ​​of the test sample and the negative control sample, respectively; (4) Luminol and H2O2 were added dropwise to the T line of the second NC film, and the chemiluminescence intensity of the T line area was obtained using a chemiluminescence analyzer under dark conditions. The difference in chemiluminescence intensity ΔI was used to determine the chemiluminescence intensity. CL The linear relationship between ΔI and Myo concentration C was used to obtain the Myo content in the sample to be tested, where ΔI CL =I CL -I CL0 , I CL , I CL0 are the chemiluminescence intensities of the test sample and the negative control sample, respectively.

7. The lateral flow chromatography detection method according to claim 6, characterized in that In step (1), 10 μL of 0.2 mol / L potassium carbonate was mixed with 50 μL of Pd / Pt / Co NPs, and then 2.5 μL of 1 mg / mL anti-Myo monoclonal antibody Ab1 and 1 mL of PBS buffer solution were added. The mixture was then gently shaken at room temperature for 120 min, followed by the addition of 100 μL of 1% BSA solution. The mixture was incubated at 800 rpm for 60 min at 25°C. Finally, the mixture was washed several times by centrifugation at 8000 rpm for 8 min at room temperature, and the precipitate was directly resuspended in 100 μL of PBS buffer solution.

8. The lateral flow chromatography detection method according to claim 6, characterized in that The T line of the first NC membrane / the second NC membrane is coated with the capture antibody Myo cAb, and the coating amount of Myo cAb on each centimeter width of the T line is 350-400 ng, and the C line is coated with goat anti-mouse IgG, and the coating amount of goat anti-mouse IgG on each centimeter width of the C line is 350-400 ng; The two-way lateral flow chromatography test strip adopts RB45 model glass fiber.

9. The lateral flow chromatography detection method according to claim 6, characterized in that In step (2), the amount of Pd / Pt / Co-Ab1 used is 20 μL; In step (3), the DAB was prepared with anhydrous ethanol as solvent at a concentration of 40 mmol / L and a volume of 50 μL, and the H2O2 was prepared with HAc-NaAc buffer solution at pH 4.0 as solvent at a concentration of 20 mmol / L and a volume of 50 μL; after the dropwise addition of DAB and H2O2, the reaction time was 5 min; In step (4), the luminol is prepared with a NaOH aqueous solution at pH 14.0, with a concentration of 4 mmol / L and a volume of 50 μL, and the H2O2 concentration is 10 mmol / L and a volume of 50 μL, which is prepared by diluting 30% H2O2 with distilled water.

10. The lateral flow chromatography detection method according to claim 6, characterized in that In step (3), the linear relationship is: ΔG = 17.1690LogC-11.6274, R 2 =0.9945, where ΔG is expressed in au and C is expressed in ng / mL; In step (4), the linear relationship is: ΔI CL =10.6940C+237.44943, R 2 =0.9985, where ΔI CL The unit of AU is au, and the unit of C is ng / mL.

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