Multi-mode lateral flow immunodetection system based on PdPtRu nanoprobe and application
By preparing PdPtRu nanoprobes, the problems of signal instability and insufficient sensitivity in traditional lateral flow immunochromatography were solved, and multimodal output of colorimetric, photothermal and catalytic signals was realized, which improved the sensitivity and stability of detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-11
- Publication Date
- 2026-03-31
AI Technical Summary
Traditional lateral flow immunochromatography uses gold nanoparticles as a colorimetric signal, which has problems such as limited sensitivity, unstable signal, susceptibility to background interference, and poor quantitative resolution.
Using PdPtRu nanoprobes and hexadecyltrimethylammonium chloride as a structure directing agent, a branched metal structure nanoprobe with surface protrusions and internal nanopores was prepared to achieve multimodal output of colorimetric, photothermal and catalytic signals.
It improves detection sensitivity, signal stability and applicability, and enhances detection sensitivity and multimodal signal output capability.
Smart Images
Figure CN121762826A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of lateral flow immunoassay technology, and more specifically, to a multimodal lateral flow immunoassay system and its application based on PdPtRu nanoprobes. Background Technology
[0002] The widespread transmission of pathogens in environmental media and the food supply chain poses a serious and escalating threat to global public health, ecological security, and critical infrastructure resources. The location of the first reported positive case is often not the primary source of contamination, but rather the place where it is first detected. This situation is exacerbated by the time lag between the onset of contamination and case confirmation. Given the inherent time lag of traditional detection methods, by the time the initial point of contamination is identified, pathogens may have already spread widely through water, airborne vectors, biological carriers, or human movement, exceeding the speed of public health interventions and undermining timely prevention and control strategies. To address this urgent need, lateral flow immunoassay (LFIA), with its ease of operation, low cost, and portability, has emerged as one of the most promising analytical platforms, enabling rapid, point-of-care diagnosis in a variety of environments.
[0003] However, traditional lateral flow immunochromatography typically uses gold nanoparticles as a colorimetric signal, which is accompanied by problems such as limited sensitivity, unstable signal, susceptibility to background interference, and poor quantitative resolution.
[0004] Therefore, the purpose of this invention is to establish a multimodal lateral flow immunoassay system based on PdPtRu nanoprobes, which has important practical significance. Summary of the Invention
[0005] In view of this, the present invention proposes a multimodal lateral flow immunoassay system and its application based on PdPtRu nanoprobes, aiming to solve at least one of the problems mentioned in the background art.
[0006] This invention proposes a multimodal lateral flow immunoassay system based on PdPtRu nanoprobes and its applications, including: Lateral flow immunoassay strips, colorimetric solution, signal acquisition module, and PdPtRu nanoprobes; The lateral flow immunoassay strip includes: a nitrocellulose membrane, a sample pad, and an absorbent pad; The acquisition module is used to acquire the absorbance and temperature signals of the lateral flow immunoassay strip after testing.
[0007] Preferably, the preparation method of the PdPtRu nanoprobe is as follows: Hexadecyltrimethylammonium chloride was added to water and heated and stirred until dissolved. After cooling, NaOH solution was added to obtain a hexadecyltrimethylammonium chloride solution. H2PdCl4 solution, H2PtCl6 solution and RuCl3·3H2O solution were added sequentially to the hexadecyltrimethylammonium chloride solution and stirred to disperse. After standing, ascorbic acid solution was added and stirred to mix. After the mixture was finished, centrifugation was performed, the precipitate was collected, and the precipitate was post-treated to obtain PdPtRu nanoparticle (PP NPs) solution. The PdPtRu nanoparticle solution was mixed with potassium carbonate solution, and then anti-Salmonella typhimurium antibody was added and stirred. Subsequently, gelatin solution was added and incubated to obtain PdPtRu nanoprobes.
[0008] Preferably, the concentrations of the H2PdCl4 solution, H2PtCl6 solution, and RuCl3·3H2O are 10 mmol / L; The volume ratio of the H2PtCl6 solution, H2PtCl6 solution, and RuCl3·3H2O is 1:1:1.
[0009] Preferably, the post-processing is as follows: The precipitate was washed with an ethanol / deionized water mixture and then added to water to prepare a 0.021 mg / mL PdPtRu nanoparticle solution.
[0010] Preferably, the potassium carbonate solution has a mass concentration of 1%, and the volume ratio of the PdPtRu nanoparticle solution to the potassium carbonate solution is 50:1.
[0011] Preferably, the method for preparing the lateral flow immunoassay strip is as follows: The sample pad was sealed with bovine serum albumin (BSA) solution and then dried. Anti-mouse IgG solution was used as the control line antibody, and capture antibody was used as the detection line antibody. After being diluted with phosphate-buffered saline, the solutions were spotted onto nitrocellulose membranes to form the control line (C line) and the detection line (T line). The dried sample pad, the spotted NC membrane, and the absorbent pad are overlapped and fixed onto the backing card, and then cut into 3 mm wide test strips to obtain lateral flow immunoassay strips.
[0012] Preferably, the colorimetric solution comprises: sodium acetate-acetic acid (NaAc-HAc) buffer (pH 6.0), H2O2 solution, and TMB solution.
[0013] The present invention also provides a method for detecting the concentration of pathogenic bacteria in a sample using the multimodal lateral flow immunoassay system based on PdPtRu nanoprobes, wherein the detection method includes: colorimetric detection, enzyme-catalyzed detection, and photothermal detection.
[0014] Preferably, the colorimetric detection method is as follows: the sample solution is mixed with the PdPtRu nanoprobe and added to the sample pad of the lateral flow immunoassay strip, incubated for 20 minutes, the absorbance signal of the T line color in the lateral flow immunoassay strip is detected using a signal acquisition module, and the absorbance signal is substituted into the colorimetric detection standard curve to obtain the concentration of pathogenic bacteria in the sample; The enzyme-catalyzed detection method is as follows: the sample solution is mixed with the PdPtRu nanoprobe and added to the sample pad of the lateral flow immunoassay strip. After incubation for 20 minutes, the lateral flow immunoassay strip is immersed in the colorimetric solution. Then, the absorbance signal of the T line color in the lateral flow immunoassay strip is detected using a signal acquisition module. The absorbance signal is substituted into the enzyme-catalyzed detection standard curve to obtain the concentration of pathogenic bacteria in the sample. The photothermal detection method is as follows: the sample solution is mixed with the PdPtRu nanoprobe and added to the sample pad of the lateral flow immunoassay strip. After incubation for 20 minutes, the T line of the lateral flow immunoassay strip is irradiated with a laser. After the incubation, the temperature signal of the T line is detected by the signal acquisition module. The temperature signal is then substituted into the photothermal detection standard curve to obtain the concentration of pathogenic bacteria in the sample.
[0015] Preferably, the pathogenic bacterium is Salmonella typhimurium.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention designs a structurally tunable trimetallic palladium-platinum-ruthenium (PdPtRu) nanoprobe, using hexadecyltrimethylammonium chloride (CTAC) as a structure directing agent to guide the aggregation and alignment of metal precursors along their hydrophobic chains, thereby achieving in-situ reduction and forming a branched metal structure with surface protrusions and internal nanopores. This geometric feature generates a localized electromagnetic field concentration on the nanoprobe surface, amplifying the photothermal and catalytic responses. This enables multimodal output of colorimetric, photothermal, and catalytic signals in lateral flow immunochromatography (LFIA), thereby improving the detection sensitivity, signal stability, and applicability of this invention. Attached Figure Description
[0017] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 The image shows the characterization and testing results of the PPR NPs alloy provided in this embodiment of the invention. Figure 2 The figure shows the evaluation and theoretical analysis results of the photothermal performance of PPR NPs provided in the embodiments of the present invention; Figure 3 The figure shows the results of the study and analysis of the catalytic activity of PPR NPs provided in the embodiments of the present invention; Figure 4 The images provided in this embodiment of the invention are photographic images of PPR NPs-multiLFIA in colorimetric and photothermal modes. Figure 5 This is a signal response diagram of PPR NPs-multiLFIA in colorimetric and photothermal modes provided in an embodiment of the present invention; Figure 6 The image shows the test strip detection results of PPR NPs-multiLFIA and AuNPs-LFIA in catalytic mode provided in the embodiments of the present invention. Figure 7 This is a signal response diagram of PPR NPs-multiLFIA in catalytic mode provided in an embodiment of the present invention; Figure 8 The results show the specificity evaluation of PPR NPs-multiLFIA in colorimetric, catalytic, and photothermal modes provided in the embodiments of the present invention.
[0018] Figure 9 These are photographic images of fruit sample detection using PPR NPs-multiLFIA in colorimetric, catalytic, and photothermal modes, as provided in this embodiment of the invention. Figure 10 This is a data analysis diagram of fruit sample detection using PPR NPs-multiLFIA in colorimetric, catalytic, and photothermal modes, as provided in this embodiment of the invention. Figure 11 These are photographic images of lettuce samples detected by PPR NPs-multiLFIA in colorimetric, catalytic, and photothermal modes, as provided in this embodiment of the invention. Figure 12 This is a data analysis diagram of lettuce sample detection using PPR NPs-multiLFIA in colorimetric mode, catalytic mode, and photothermal mode, as provided in this embodiment of the invention. Figure 13 This is a comparison chart of the recovery rates of two concentrations in lettuce and fruit samples provided in this embodiment of the invention with those of the standard method. Detailed Implementation
[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.
[0020] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0024] This invention proposes a multimodal lateral flow immunoassay system based on PdPtRu nanoprobes, comprising: Lateral flow immunoassay strips, colorimetric solution, and signal acquisition module; The lateral flow immunoassay strip includes: a PdPtRu nanoprobe, a nitrocellulose membrane, a sample pad, and an absorbent pad; The acquisition module is used to acquire the absorbance and temperature signals of the lateral flow immunoassay strip after testing.
[0025] Specifically, the signal acquisition module is preferably a FLIR thermal imaging camera and an ultraviolet-visible spectrophotometer, wherein the FLIR thermal imaging camera is used to detect temperature signals and the ultraviolet-visible spectrophotometer is used to detect absorbance signals.
[0026] In this invention, the preparation method of the PdPtRu nanoprobe is as follows: Hexadecyltrimethylammonium chloride (CTAC) was added to water and heated and stirred until dissolved. After cooling, NaOH solution was added to obtain a hexadecyltrimethylammonium chloride solution. H2PdCl4 solution, H2PtCl6 solution, and RuCl3·3H2O solution were added sequentially to the hexadecyltrimethylammonium chloride solution and stirred to disperse. After standing, ascorbic acid (AA) solution was added and stirred to mix. After the mixture was finished, it was centrifuged, the precipitate was collected, and the precipitate was post-treated to obtain PdPtRu nanoparticle (PP NPs) solution. The PdPtRu nanoparticle solution was mixed with potassium carbonate solution, and then anti-Salmonella typhimurium antibody was added and stirred. Subsequently, gelatin solution was added and incubated to obtain PdPtRu nanoprobes.
[0027] Specifically, the heating temperature is preferably 37°C, the centrifugation speed is preferably 10000 r, and the centrifugation time is preferably 10 min.
[0028] Specifically, the settling time is preferably 30 minutes; Specifically, when adding ascorbic acid (AA) solution and stirring, the stirring time is preferably 30 minutes; Specifically, when the PdPtRu nanoparticle solution is mixed with potassium carbonate solution and then the anti-Salmonella typhimurium antibody is added and stirred, the stirring time is preferably 413 Å. Specifically, the incubation time is preferably 1 hour.
[0029] It is understood that the PdPtRu nanoprobe in this invention uses hexadecyltrimethylammonium chloride (CTAC) as a structure directing agent to guide the aggregation and alignment of the metal precursor along its hydrophobic chain, thereby achieving in-situ reduction and forming a branched metal structure with surface protrusions and internal nanopores. This geometric feature generates a local electromagnetic field concentration on the surface of the nanoprobe, thereby amplifying the photothermal and catalytic responses and realizing multimodal output of colorimetric, photothermal, and catalytic signals in lateral flow immunochromatography (LFIA), thus improving the detection sensitivity, signal stability, and applicability of this invention.
[0030] In this invention, the concentrations of the H2PdCl4 solution, H2PtCl6 solution, and RuCl3·3H2O are 10 mmol / L; The volume ratio of the H2PtCl6 solution, H2PtCl6 solution, and RuCl3·3H2O is 1:1:1.
[0031] In this invention, the post-processing is as follows: The precipitate was washed with an ethanol / deionized water mixture and then added to water to prepare a 0.021 mg / mL PdPtRu nanoparticle solution.
[0032] Specifically, the volume ratio of ethanol to deionized water in the ethanol / deionized water mixture is preferably 1:1, and the washing frequency is preferably 3 times. In this invention, the mass concentration of the potassium carbonate solution is 1%, and the volume ratio of the PdPtRu nanoparticle solution to the potassium carbonate solution is 50:1.
[0033] In this invention, the preparation method of the lateral flow immunoassay strip is as follows: The sample pad was sealed with bovine serum albumin (BSA) solution and then dried. Anti-mouse IgG solution was used as the control line antibody, and capture antibody was used as the detection line antibody. After being diluted with phosphate-buffered saline, the solutions were spotted onto nitrocellulose membranes to form the control line (C line) and the detection line (T line). The dried sample pad, the spotted NC membrane, and the absorbent pad are overlapped and fixed onto the backing card, and then cut into 3 mm wide test strips to obtain lateral flow immunoassay strips.
[0034] Specifically, the preferred mass-to-volume ratio of the bovine serum albumin (BSA) solution is 2%, the preferred mass-to-volume concentration of the anti-mouse IgG solution is 1.0 mg / mL, and the preferred mass-to-volume concentration of the detection line antibody is 1 mg / mL.
[0035] Specifically, when using anti-mouse IgG solution as the control antibody and capture antibody as the detection antibody, and diluting them with phosphate-buffered saline before spotting them onto a nitrocellulose membrane, the preferred final concentration of the anti-mouse IgG solution and the detection antibody is 1 mg / mL. In this invention, the colorimetric solution comprises: sodium acetate-acetic acid (NaAc-HAc) buffer (pH 6.0), H2O2 solution (20 mM), and TMB solution (1 M).
[0036] Specifically, the concentration of the H2O2 solution is 1 mol / L, and the concentration of the TMB solution is 20 mmol / L.
[0037] The present invention also provides a method for detecting the concentration of pathogenic bacteria in a sample using the multimodal lateral flow immunoassay system based on PdPtRu nanoprobes, characterized in that the detection method includes: colorimetric detection, enzyme-catalyzed detection, and photothermal detection.
[0038] In this invention, the colorimetric detection method is as follows: the sample solution is mixed with the PdPtRu nanoprobe and added to the sample pad of the lateral flow immunoassay strip, incubated for 20 minutes, the absorbance signal of the T line color in the lateral flow immunoassay strip is detected using a signal acquisition module, and the absorbance signal is substituted into the colorimetric detection standard curve to obtain the concentration of pathogenic bacteria in the sample. The enzyme-catalyzed detection method is as follows: the sample solution is mixed with the PdPtRu nanoprobe and added to the sample pad of the lateral flow immunoassay strip. After incubation for 20 minutes, the lateral flow immunoassay strip is immersed in the colorimetric solution. Then, the absorbance signal of the T line color in the lateral flow immunoassay strip is detected using a signal acquisition module. The absorbance signal is substituted into the enzyme-catalyzed detection standard curve to obtain the concentration of pathogenic bacteria in the sample. The photothermal detection method is as follows: the sample solution is mixed with the PdPtRu nanoprobe and added to the sample pad of the lateral flow immunoassay strip. After incubation for 20 minutes, the T line of the lateral flow immunoassay strip is irradiated with a laser. After the incubation, the temperature signal of the T line is detected by the signal acquisition module. The temperature signal is then substituted into the photothermal detection standard curve to obtain the concentration of pathogenic bacteria in the sample.
[0039] Specifically, the colorimetric detection standard curve is as follows: Figure 9 As shown in Figure A; the photothermal detection standard curve is as follows: Figure 9 As shown in C; the enzyme catalysis detection standard curve is as follows: Figure 7 As shown in Figure A.
[0040] It is understood that the availability of multiple signal modes in this invention can flexibly adapt to different sample types and overcome the limitations of colorimetric detection in the presence of interference, thus greatly improving the applicability of this application.
[0041] In this invention, the pathogenic bacterium is Salmonella typhimurium.
[0042] Example 1 Preparation of PdPtRu nanoprobes: S1. Dissolve 90 mg CTAC in 90 mL of deionized water, heat to 37 °C and stir until completely dissolved. After cooling the solution to 25 °C, add 3.2 mL of 100 mmol / L NaOH to obtain the CTAC solution. S2. Add 4 mL of 10 mmol / L H2PdCl4, 4 mL of 10 mmol / L H2PtCl6, and 4 mL of 10 mmol / L RuCl3·3H2O to the CTAC solution in sequence, and stir to ensure uniform dispersion. After standing for 30 minutes, quickly add 8.0 mL of freshly prepared 0.3 mmol / L AA solution, stir for 30 minutes, centrifuge (10000 r) for 10 min to collect the precipitate, wash the precipitate 3 times with an ethanol / deionized water mixture (V:V=1:1), and then add it to deionized water to prepare a 0.021 mg / mL PdPtRu nanoparticle (PPR NPs) solution. S3. Mix 1 mL of 0.021 mg / mL PdPtRu nanoparticle solution with 20 μL of 1% potassium carbonate solution, then add 5 μL of 1 mg / mL anti-Salmonella typhimurium antibody. Stir the mixture gently at room temperature for 45 minutes. After stirring, add 20 μL of 30% gelatin solution and incubate for 1 hour to obtain PdPtRu nanoprobes.
[0043] Example 2 Preparation of lateral flow immunoassay strips: The sample pad was blocked with a 2% (w / v) bovine serum albumin (BSA) solution and then completely dried in an oven (37°C, 5 h). Subsequently, the control line antibody (anti-mouse IgG, 1.0 mg / mL) and the detection line antibody (capture antibody, 1 mg / mL) were diluted with phosphate-buffered saline (PBS) and then spotted onto a nitrocellulose membrane (NC membrane) using a spotting device to form the control line (C line) and the detection line (T line). Following the correct fluid direction, overlap and fix the sample pad, NC membrane, and absorbent pad onto the backing card. Cut the assembled membrane card into individual test strips with a width of 3 mm to obtain lateral flow immunoassay strips.
[0044] Example 3 Preparation of the colorimetric solution: Buffer solution preparation: Mix 0.2 M acetic acid solution with 2.2 M sodium acetate trihydrate, and adjust the pH of the solution to 6 with 1 M NaOH; Preparation of 20 mM TMB solution: Add 0.096 g TMB powder to 20 mL of anhydrous ethanol solution and sonicate until completely dissolved; Preparation of 1 M H2O2 solution: Dilute the 30% (w / w) H2O2 stock solution tenfold.
[0045] Comparative Example 1 Preparation of bimetallic palladium-platinum (PdPt) mesoporous nanospheres (PP NPs): S1. Dissolve 90 mg CTAC in 90 mL of deionized water, heat and stir until completely dissolved, cool the solution to 25 °C, and add 3.2 mL of 100 mmol / L NaOH to obtain CTAC solution; S2. Add 4 mL of 10 mmol / L H2PdCl4 and 4 mL of 10 mmol / L H2PtCl6 to the CTAC solution in sequence, and stir to ensure uniform dispersion. After standing for 30 minutes, quickly add 8.0 mL of freshly prepared 0.3 mmol / L AA solution, stir for 30 minutes, centrifuge to collect the precipitate, wash the precipitate with an ethanol / deionized water mixture, and add it to deionized water to prepare a 0.021 mg / mL PdPt nanoparticle (PP NPs) solution. Comparative Example 2 Preparation of PdPtRu nanoparticles with smooth surfaces: Add 4 mL of 10 mmol / L H₂PdCl₄, 4 mL of 10 mmol / L H₂PtCl₆, and 4 mL of 10 mmol / L RuCl₃·3H₂O sequentially to 90 mL of deionized water, stirring to ensure uniform dispersion. After standing for 30 minutes, quickly add 8.0 mL of freshly prepared 0.3 mmol / L AA solution, stir for 30 minutes, centrifuge to collect the precipitate, wash the precipitate with an ethanol / deionized water mixture, and then add it back to deionized water to prepare a 0.021 mg / mL PdPtRu nanoparticle (PPRNPs) solution. Test Example 1 The synthesis and characterization of the PPR NPs prepared in Example 1 and the PP NPs prepared in Comparative Example 1 were analyzed, and the results are as follows: Figure 1 As shown; Figure 1 In the image: A is a transmission electron microscope (TEM) image of PPR NPs; B is a high-angle annular dark-field scanning TEM image and elemental distribution map of PPR NPs; C is a line scan analysis of PPR NPs; D is a low-magnification TEM image of PPR NPs; E is a selected area electron diffraction pattern of PPR NPs; F is an X-ray diffraction pattern of PPR NPs; G, H, and I are elemental valence state analyses of PPR NPs, specifically: palladium (G), platinum (H), and ruthenium (I); J, K, and L are elemental valence state analyses of PPR NPs: palladium (J), platinum (K), and total (L).
[0046] Specifically, this invention successfully prepared trimetallic alloy nanoparticles (PPR NPs) with unique porosity by combining the reducing agent ascorbic acid with metal precursors H2PdCl4, H2PtCl4 and RuCl3·3H2O and a surfactant-directed method.
[0047] The morphology and nanostructure of the nanoparticles were extensively characterized using transmission electron microscopy (TEM). Figure 1A). The PPR NPs of the present invention achieve a balance between specific surface area and metal content (Pd / Pt / Ru = 58.9 / 34.3 / 6.8), which is confirmed by elemental distribution uniformity and quantitative inductively coupled plasma mass spectrometry (ICP-MS). Figure 1 B). Unlike common core-shell structures, the distribution widths of the three metals in PPR NPs are almost the same, indicating that the material adopts a trimetallic alloy structure. Figure 1 C). Surfactants mediate the self-assembly of amphiphilic molecules into vesicular micelles, whose surface electrostatic interactions drive the deposition of metal precursors, thereby modulating the porosity and morphological evolution of alloy nanoparticles. High-resolution transmission electron microscopy images show that face-centered cubic (FCC) lattice fringes exist in PPR nanoparticles, with a lattice spacing of 0.219 nm along the (111) crystal plane. PPR NPs nanoparticles have a metallic dendritic structure radiating outward from the central core, forming dendritic mesopores (>2 nm) with "lightning rod"-like tips. Figure 1 D). To further clarify the crystallinity and structure of PPR NPs, selected area electron diffraction (SAED) was employed. Figure 1 E) and wide-angle X-ray diffraction (XRD) patterns ( Figure 1 F). Both techniques showed distinct (111), (200), (220), and (311) diffraction peaks. The XRD peaks of the PPR NPs were located between face-centered cubic palladium (JCPDS 46-1043), platinum (04-0802), and ruthenium (06-0663), confirming the formation of the trimetallic alloy.
[0048] Furthermore, X-ray photoelectron spectroscopy (XPS) was used to compare the changes in electronic properties before and after quantitative Ru growth on PdPt micelles. Compared to PP NPs, the introduction of Ru improved the electronic properties of Pd 3d... 3 / 2 and Pd 3d 5 / 2 The binding energies of the orbitals are 334.97 eV and 340.47 eV, respectively. Figure 1 J) shifted slightly to 335.95 eV and 341.07 eV ( Figure 1 G), while Pt 4f 5 / 2 and Pt 4f 7 / 2 The binding energies of the orbitals range from 74.17 eV and 70.87 eV ( Figure 1 K) moved to 75.07 eV and 71.67 eV ( Figure 1 This shift in binding energy indicates a migration of electron density from PdPt to Ru, resulting in electron-deficient PdPt. The enhanced interaction between the electron-deficient catalyst and the substrate leads to improved catalytic activity. The peaks at 462.95 eV and 485.54 eV are attributed to Ru 3p, respectively.3 / 2 and Ru 3p 1 / 2 The spin orbital splitting indicates that Ru in PPR NPs is in the Ru position. n+ Oxidized state, there is no metallic Ru ( Figure 1 (I and 1L). Electron transfer results in high electron density and excellent catalytic activity at the Ru sites. The above results demonstrate the synthesis and properties of PPR NPs.
[0049] Test Example 2 The photothermal properties of the PPR NPs prepared in Example 1, single Pd nanoparticles, and the tipless PPR nanoparticles prepared in Comparative Example 2 were measured and evaluated under near-infrared (NIR) laser irradiation. Deionized water was used as a negative control in all experiments. The laser spot was adjusted to ensure uniform coverage of the sample surface. Real-time thermal images were recorded using a FLIR thermal imaging camera, and quantitative analysis was performed using FLIR software. The concentration-dependent photothermal effect was investigated by irradiating aqueous solutions of different concentrations (0–100 μg / mL) of PPR nanoparticles at a fixed power density of 2 W / cm² with an 808 nm laser for 10 minutes. To assess the effect of laser power, the PPR nanoparticle solutions were irradiated with an 808 nm laser at different power densities ranging from 0.9 to 1.9 W / cm². The photothermal conversion efficiency (η) was calculated by first recording the natural cooling curves of the PPR nanoparticle solutions after reaching their maximum temperature under laser irradiation. The photothermal conversion efficiency was calculated by combining the thermal transfer coefficient (hS), the absorbance of the sample at 808 nm, and the laser power density. This process was used to determine the photothermal conversion efficiency of PPR nanoparticles with different pore structures, and the measurement results are shown below. Figure 2 As shown; Figure 2 In the diagram: A represents the photothermal conversion efficiency measurement of PPR NPs; B represents the photothermal conversion efficiency measurement of Pd nanoparticles; C represents the photothermal conversion efficiency of PPR nanoparticles with smooth surfaces; D represents the photothermal measurement results of PPR NPs aqueous solutions at various concentrations; E represents the temperature statistics of photothermal measurements of PPR NPs aqueous solutions at different concentrations; F represents the temperature statistics of photothermal measurements of PPR NPs aqueous solutions at different powers; and G represents the photothermal cycling measurement of PPR NPs.
[0050] Specifically, after irradiation with an 808 nm laser, time-temperature curves were recorded, and PPR NPs were calculated. Figure 2 The photothermal conversion efficiency of A) is 55.84%, and the Pd nanoparticles ( Figure 2 B) was 44.55%, and PPR nanoparticles without sharp edges ( Figure 2C) is 41.34%. The photothermal conversion efficiency of PPR NPs is 11.29% higher than that of Pd nanoparticles and 14.5% higher than that of PPR nanoparticles without surface features.
[0051] These results demonstrate that rationally customized nanomaterials can significantly improve performance, with shape playing a more crucial role in photothermal properties. Furthermore, photothermal evaluation shows that PPR NPs possess dual-parameter tunability: concentration-dependent temperature regulation (…). Figure 2 E and 2D) and power response thermal regulation (0.9-1.9 W / cm²) Figure 2 F) established the importance of programmable thermal output for precision biosensing applications.
[0052] Furthermore, the rapid cooling of the solution after laser shutdown indicates that PPR NPs have good thermal conductivity, and repeated heating and cooling cycles show excellent reproducibility. Figure 2 G). These results indicate that PPR nanoparticles can serve as photothermal signal tags, and their signal variations can be controlled by concentration and laser power.
[0053] Test Example 3 Catalytic Activity Assay: The peroxidase-like (POD-like) activity of the nanoparticles was assessed based on the colorimetric reaction of 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue product upon catalytic oxidation with hydrogen peroxide (H₂O₂). In a typical reaction system, 3 μL of PPR nanoparticle (PPR NPs) solution (20 μg / mL), 5 μL of H₂O₂ solution (1 mol / L), and 5 μL of TMB solution (20 mmol / L) were added sequentially to 180 μL of sodium acetate-acetic acid (NaAc-HAc) buffer (pH 6.0). The reaction was carried out at room temperature, and the absorbance at 652 nm was continuously monitored using a UV-Vis spectrophotometer to assess catalytic activity. For comparison, the catalytic activity of PPR nanoparticles (PPR NPs) and PP nanoparticles (PP NPs) was assessed using the same methods and conditions. Substrate dependence analysis was performed to evaluate the kinetic properties of the catalysts. The reaction system contained 180 μL of NaAc-HAc buffer and 3 μL of catalyst (20 μg / mL). The concentrations of H2O2 and TMB were varied independently. The initial reaction rate (V0) was calculated by continuously monitoring the absorbance change over 90 seconds at different substrate concentrations. The relationship between V0 and substrate concentration ([S]) was fitted to the Michaelis-Menten equation. Key kinetic parameters, including the maximum reaction rate (Vmax) and the Michaelis constant (Km), were determined using Linnberg's double reciprocal plot analysis. Finally, the kinetic parameters of different catalysts (PPR nanoparticles and PP nanoparticles) were compared, and the results are shown below. Figure 3 As shown; Figure 3 In the table: A represents the colorimetric results of the precipitate and supernatant collected by centrifugation in step S2 of Example 1; B represents the absorbance detection results of the reaction system at different pH values; C represents the results of the cycling experiment; D represents the storage stability results; E represents the catalytic kinetic results using PPR NPs with H2O2 as the substrate; G represents the catalytic kinetic results using PP NPs with H2O2 as the substrate; F represents the catalytic kinetic results using PPR NPs with TMB as the substrate; H represents the catalytic kinetic results using PP NPs with TMB as the substrate. Specifically, by comparing the signals from the precipitate and the supernatant, it was confirmed that this activity was not caused by ion leakage. Figure 3 A). Subsequent experiments used NaAc-HAc buffer (pH 6) and TMB as chromogenic substrates, and quantified color development by intensity changes. Figure 3 B). PPR NPs retained 91.6% of their initial activity after 6 cycles (Figure 3C) and maintained 94.69% efficiency after two months of storage. Figure 3 (D), which confirms its excellent operational durability and shelf stability, enabling it to maintain label performance over time. The influence of active sites was subsequently evaluated using Mie kinetic analysis, comparing the catalytic effects of PPR and PdPt (PP) nanoparticles on TMB and H2O2. For the H2O2 substrate, the Kc of PPR nanoparticles... m The value increased by 3.22 times, V max The value increased by 5.98 times ( Figure 3 E and 3G). For TMB substrates, the K of PPR nanoparticles m The value increased slightly, while V max The value increased by 8.42 times ( Figure 3 F and 3H).
[0054] Test Example 4 The detection performance of the PdPtRu nanoprobe-based multimodal lateral flow immunoassay system (hereinafter referred to as PPRNPs-multiLFIA) and the conventional gold nanoparticle-immunochromatography (AuNPs-LFIA) of this invention were tested, and the standard curves were obtained: The detection performance of this invention was verified by detecting known concentrations of Salmonella typhimurium using the colorimetric detection method (hereinafter referred to as colorimetric mode), enzyme-catalyzed detection method (hereinafter referred to as catalytic mode), and photothermal detection method (hereinafter referred to as photothermal mode) of the PPR NPs-multiLFIA. Standard curves for different detection methods were obtained simultaneously. During the detection process, a FLIR thermal imaging camera was used to detect the temperature signal, and a UV-Vis spectrophotometer was used to detect the absorbance signal. The results are as follows: Figure 4-8 As shown; Figure 4In the image: A is a photographic image in colorimetric mode; B is a photographic image of the T-line in photothermal mode. Figure 5 In the diagram: A represents the signal response curve of PPR NPs-multiLFIA under a wide range of Salmonella typhimurium concentrations in colorimetric mode (colorimetric detection standard curve); B represents the signal linear range in colorimetric mode; C represents the linear curve in photothermal mode (photothermal detection standard curve); D represents the signal linear range in photothermal mode. Figure 6 In the image: A is a photographic image in catalytic mode; B is a visual image of the test strip using AuNPs-LFIA. Figure 7 In the diagram: A represents the signal curve under catalytic mode (enzyme catalysis detection standard curve); B represents the linear relationship under catalytic mode; C represents the signal response of AuNPs-LFIA; and D represents the signal linear range of AuNPs-LFIA. Figure 8 In the middle: A, B, and C are the specificity evaluation results of colorimetric (A), catalytic (B), and photothermal (C) modes.
[0055] Specifically, the experiment used concentrations ranging from 10... 7 Salmonella typhimurium was detected in 10 CFU / mL PBS. In colorimetric mode, the color intensity of the T line gradually decreased with decreasing bacterial concentration, and the limit of detection (LOD) observed visually was 5 × 10⁻⁶. 3 CFU / mL Figure 4 A). PPR nanoparticles-multiple LFIA exhibit a strong response over a wide concentration range ( Figure 9 A). In 10 3 Up to 5×10 4 Within the range of CFU / mL, bacterial concentration showed a linear correlation with signal intensity, with the linear equation being y = 1208.84x - 3395.46 (R²). 2 = 0.999) Figure 9 B). In photothermal mode, the signal decreases monotonically with increasing concentration ( Figure 4 B). The photothermal mode exhibits a strong response, and at 10 3 There is a clear inflection point at CFU / mL (LOD). Figure 9 C). From 5×10 3 Up to 10 6 CFU mL -1 The bacterial concentration showed a linear correlation with the signal intensity, with the linear equation being y = 20.47x - 60.05 (R²). 2 = 0.981)( Figure 11A). In catalytic mode, the color intensity of the T line decreases monotonically with decreasing bacterial concentration, and the detection limit observed by the naked eye is 5 × 10⁻⁶. 2 CFU mL -1 ( Figure 6 A). The catalytic mode exhibits a strong response across the entire range ( Figure 7 A). From 5×10 2 Up to 5×10 4 CFU mL -1 The bacterial concentration showed a linear correlation with the signal intensity, with the linear equation being y = 3653.96x - 7029.82 (R²). 2 = 0.992)( Figure 7 B). Compared with the colorimetric mode, the photothermal mode and catalytic mode improved the sensitivity by 5-fold and 10-fold, respectively. We compared PPR NPs-multiLFIA with gold nanoparticle-immunochromatography (...). Figure 6 B). The detection limit of gold nanoparticles-immunochromatography is 10. 5 CFU mL -1 It also exhibits a good signal response over a wide range of bacterial concentrations. Figure 7 C). In 5×10 5 Up to 5×10 7 CFU mL -1 Within the specified range, bacterial concentration and signal intensity show a linear correlation, with the linear equation being y = 18471.49x - 100090.21 (R0). 2 =0.998)( Figure 7 D). Compared with traditional gold nanoparticle-lateral flow immunochromatography, PPR NPs-multiLFIA showed 20-fold, 100-fold, and 200-fold improvements in sensitivity, respectively. Furthermore, the specificity of the detection was evaluated. *Escherichia coli* O157:H7 and *Staphylococcus aureus* (Gram-negative and Gram-positive bacteria) were selected as interfering species. The colorimetric mode (…) was only used in the presence of *Salmonella typhimurium*. Figure 8 A) Catalytic mode ( Figure 8 B) and photothermal mode ( Figure 8 A significant signal response was only observed under C), indicating that PPR NPs-multiLFIA has excellent specificity.
[0056] Test Example 5 The practical detection capability test of the PPR NPs-multiLFIA of this invention: Common leafy vegetables (lettuce) and fresh sliced fruit (apple) were selected as matrix samples. Sample solutions were prepared by washing and filtering these materials. Then, a high-concentration Salmonella typhimurium suspension was diluted in these sample solutions to prepare spiked samples of known concentrations. To evaluate the accuracy of the method, real pathogenic bacteria samples were cultured, and the recovery rate of LFIA detection (measured value / true value) was calculated using plate counting as the gold standard. During the LFIA detection process, the sample solution was first added to the sample pad. After a 20-minute incubation period, the conventional colorimetric signal was recorded. Subsequently, enhanced signals based on enzyme catalysis and photothermal effects were obtained and recorded and analyzed by immersing the test strip in the colorimetric solution or by laser irradiation, respectively. The results are as follows: Figure 9-13 As shown; Figure 9 In the middle: A, B, and C are photographic images of fruit samples under colorimetric mode (A), catalytic mode (B), and photothermal mode (C); Figure 10 In the middle: A, B, and C are data analysis graphs for fruit sample detection under colorimetric mode (A), catalytic mode (B), and photothermal mode (C); Figure 11 In the middle: A, B, and C are photographic images of lettuce samples under colorimetric mode (A), catalytic mode (B), and photothermal mode (C); Figure 12 In the middle: A, B, and C represent the data analysis of lettuce samples under colorimetric mode (A), catalytic mode (B), and photothermal mode (C); Figure 13 In the middle: A and B are comparisons of the recovery rates of two concentrations in lettuce and fruit samples using blind assays and standard methods. Specifically, washing solutions from freshly sliced fruit and fresh lettuce were used as sample matrices, and known concentrations of pathogens were added for detection. In the freshly sliced fruit sample, the T-line signal intensity monotonically decreased with increasing bacterial concentration. Figure 9 A, 9B, 9C). Colorimetry ( Figure 10 A) Catalysis ( Figure 10 B) and photothermal mode ( Figure 10 The detection limits for C) are 10 4 10 3 and 10 3 CFU mL -1 This indicates that it has good adaptability in detecting pathogens and can cover a wide concentration range. In lettuce samples, the signal intensity also decreased monotonically with decreasing bacterial concentration. Figure 11 A, 11B, 11C). Colorimetry ( Figure 12 A) Catalysis ( Figure 12 B) and photothermal mode ( Figure 12 The detection limits for C) are 10 4 10 3 and 10 3 CFU mL -1 The availability of multiple signal modes allows for flexible adaptation to different sample types and overcomes the limitations of colorimetric detection in the presence of interference. Furthermore, we cultured pathogen sample solutions with unknown concentrations and calculated recoveries by comparing standard plate counting (true values) with the detection values (measured values) of PPR NPs-multiLFIA to assess the feasibility of blind testing in samples. In lettuce samples, the recoveries of the three modes were 94.08%, 100.16%, and 98.45%, respectively, while in fruit samples, the recoveries were 97.45%, 93.4%, and 96.99% (at 10... 4 CFU mL -1 hour)( Figure 13 A). At higher concentrations (5 × 10⁻⁶), 4 CFU mL -1 Under these conditions, the recovery rates for lettuce samples were 96.4%, 98.34%, and 91.46%, respectively, while the recovery rates for fruit samples were 103.46%, 93.68%, and 93.6%, respectively. Figure 13 B). These results demonstrate that PPR NPs-multiLFIA possesses excellent anti-interference capabilities.
[0057] In summary, this invention develops a multimodal lateral flow immunoassay system based on PdPtRu nanoprobes. By designing a structurally tunable trimetallic palladium-platinum-ruthenium (PdPtRu) nanoprobe and using hexadecyltrimethylammonium chloride (CTAC) as a structure directing agent, the invention guides the aggregation and alignment of metal precursors along their hydrophobic chains, thereby achieving in-situ reduction and forming a branched metal structure with surface protrusions and internal nanopores. This geometric feature generates a localized electromagnetic field concentration on the nanoprobe surface, amplifying the photothermal and catalytic responses. This enables multimodal output of colorimetric, photothermal, and catalytic signals in lateral flow immunochromatography (LFIA), thereby improving the detection sensitivity, signal stability, and applicability of this invention.
[0058] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is determined by the appended claims.
[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.
Claims
1. A multimodal lateral flow immunoassay system based on PdPtRu nanoprobes, characterized in that, include: Lateral flow immunoassay strips, colorimetric solution, signal acquisition module, and PdPtRu nanoprobes; The lateral flow immunoassay strip includes a nitrocellulose membrane, a sample pad, and an absorbent pad. The acquisition module is used to acquire the absorbance and temperature signals of the lateral flow immunoassay strip after testing.
2. The multimodal lateral flow immunoassay system based on PdPtRu nanoprobes according to claim 1, characterized in that, The preparation method of the PdPtRu nanoprobe is as follows: Hexadecyltrimethylammonium chloride was added to water and heated and stirred until dissolved. After cooling, NaOH solution was added to obtain a hexadecyltrimethylammonium chloride solution. H2PdCl4 solution, H2PtCl6 solution and RuCl3·3H2O solution were added sequentially to the hexadecyltrimethylammonium chloride solution and stirred to disperse. After standing, ascorbic acid solution was added and stirred to mix. After the mixture was finished, centrifugation was performed, the precipitate was collected, and the precipitate was post-treated to obtain PdPtRu nanoparticle solution. The PdPtRu nanoparticle solution was mixed with potassium carbonate solution, and then anti-Salmonella typhimurium antibody was added and stirred. Subsequently, gelatin solution was added and incubated to obtain PdPtRu nanoprobes.
3. The multimodal lateral flow immunoassay system based on PdPtRu nanoprobes according to claim 1, characterized in that, The concentrations of the H2PdCl4 solution, H2PtCl6 solution, and RuCl3·3H2O were 10 mmol / L. The volume ratio of the H2PtCl6 solution, H2PtCl6 solution, and RuCl3·3H2O is 1:1:
1.
4. The multimodal lateral flow immunoassay system based on PdPtRu nanoprobes according to claim 1, characterized in that, The post-processing is as follows: The precipitate was washed with an ethanol / deionized water mixture and then added to water to prepare a 0.021 mg / mL PdPtRu nanoparticle solution.
5. The multimodal lateral flow immunoassay system based on PdPtRu nanoprobes according to claim 1, characterized in that, The potassium carbonate solution has a mass concentration of 1%, and the volume ratio of the PdPtRu nanoparticle solution to the potassium carbonate solution is 50:
1.
6. The multimodal lateral flow immunoassay system based on PdPtRu nanoprobes according to claim 1, characterized in that, The method for preparing the lateral flow immunoassay strip is as follows: The sample pad was sealed with bovine serum albumin (BSA) solution and then dried. Anti-mouse IgG solution was used as the control line antibody, and capture antibody was used as the detection line antibody. After being diluted with phosphate-buffered saline, the solutions were spotted onto nitrocellulose membranes to form the control line (C line) and the detection line (T line). The dried sample pad, the spotted NC membrane, and the absorbent pad are overlapped and fixed onto the backing card, and then cut into 3 mm wide test strips to obtain lateral flow immunoassay strips.
7. The multimodal lateral flow immunoassay system based on PdPtRu nanoprobes according to claim 1, characterized in that, The colorimetric solution includes: sodium acetate-acetic acid (NaAc-HAc) buffer (pH 6.0), H2O2 solution, and TMB solution.
8. A method for detecting the concentration of pathogenic bacteria in a sample using a multimodal lateral flow immunoassay system based on PdPtRu nanoprobes as described in any one of claims 1 to 7, characterized in that, The detection methods include: colorimetric detection, enzyme-catalyzed detection, and photothermal detection.
9. The detection method according to claim 8, characterized in that, The colorimetric detection method is as follows: the sample solution is mixed with the PdPtRu nanoprobe and added to the sample pad of the lateral flow immunoassay strip. After incubation for 20 minutes, the absorbance signal of the T line color in the lateral flow immunoassay strip is detected using a signal acquisition module. The absorbance signal is then substituted into the colorimetric detection standard curve to obtain the concentration of pathogenic bacteria in the sample. The enzyme-catalyzed detection method is as follows: the sample solution is mixed with the PdPtRu nanoprobe and added to the sample pad of the lateral flow immunoassay strip. After incubation for 20 minutes, the lateral flow immunoassay strip is immersed in the colorimetric solution. Then, the absorbance signal of the T line color in the lateral flow immunoassay strip is detected using a signal acquisition module. The absorbance signal is substituted into the enzyme-catalyzed detection standard curve to obtain the concentration of pathogenic bacteria in the sample. The photothermal detection method is as follows: the sample solution is mixed with the PdPtRu nanoprobe and added to the sample pad of the lateral flow immunoassay strip. After incubation for 20 minutes, the T line of the lateral flow immunoassay strip is irradiated with a laser. After the incubation, the temperature signal of the T line is detected by the signal acquisition module. The temperature signal is then substituted into the photothermal detection standard curve to obtain the concentration of pathogenic bacteria in the sample.
10. The detection method according to claim 9, characterized in that, The pathogenic bacterium is Salmonella typhimurium.