Preparation method of Au (at) Au (at) Ag / Pt / M NPs and application of Au (at) Au (at) Ag / Pt / M NPs in klebsiella pneumoniae detection

Abstract

The invention discloses a preparation method of Au (at) Au (at) Ag / Pt / M NPs and application of the Au (at) Au (at) Ag / Pt / M NPs in klebsiella pneumoniae detection, and belongs to the technical field of analysis and detection. The invention particularly relates to development of a signal probe and combination and assembly of the signal probe and lateral flow immunochromatography (LFIA) for klebsiella pneumoniae detection. Au (at) Au (at) Ag / Pt / MNPs is a multi-element noble metal nanoparticle with plasma resonance property, peroxidase-like activity and light stability, shows strong detection potential in three modes of colorimetric (CM), catalytic colorimetric (CL) and surface enhanced Raman scattering (SERS), and is loaded into LFIA to construct a multi-mode LFIA (CM / CL / SERS-LFIA). The kit is expected to be used for direct multimode detection of klebsiella pneumoniae in clinical sputum samples, and a new early detection strategy is provided for respiratory tract infection diseases such as klebsiella pneumoniae.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and more specifically, to a preparation method of Au@Au@Ag / Pt / M NPs nanogap nanoprobes and their application in the detection of Klebsiella pneumoniae. Background Art

[0002] At present, in the face of some major sudden respiratory infectious diseases, quickly identifying the pathogen and taking corresponding measures in a timely manner is the top priority of prevention and treatment. Klebsiella pneumoniae (Kp) is one of the main pathogens causing bacterial lower respiratory tract infections, with characteristics such as complex conditions, easy drug resistance, and low early detection rate. In particular, the infection of drug-resistant Klebsiella pneumoniae has brought great challenges to clinical treatment. Therefore, developing a rapid, accurate, and low-cost detection method for Klebsiella pneumoniae is of great clinical significance for guiding clinical medication and reducing the mortality rate of patients.

[0003] At present, the early diagnosis of Klebsiella pneumoniae infection mainly relies on the detection of disease markers. For example, when a patient is infected with Klebsiella pneumoniae, the concentration of C-reactive protein in the blood is usually greater than 8.2 mg / L, which is an important indicator of the inflammatory response. Some specific biomarkers, such as Interleukin 6 (IL-6), Interleukin 8 (IL-8), and Tumor necrosis factor-alpha (TNF-α), etc., will also increase significantly in the early stage of Klebsiella pneumoniae infection. However, although these detections based on disease markers are effective, they are often carried out after the clinical symptoms appear, and the detection indicators lack specificity, making it difficult to achieve early warning and diagnosis in the initial stage of infection.

[0004] At the same time, traditional pathogen detection methods, such as microbial isolation and culture, smear microscopy, real-time quantitative nucleic acid amplification detection, ELISA, etc., although having good accuracy and specificity, have problems such as long time consumption, complex operation, high equipment cost, and limited sensitivity, resulting in many limitations in clinical application for guiding disease treatment plans. An ideal rapid analytical detection tool for Klebsiella pneumoniae infection should have: (1) fast detection speed, easy to operate, and suitable for non-professional testers; (2) reagents and instruments are economical and portable, enabling on-site sampling and instant reporting of results; (3) high sensitivity and good accuracy, capable of reliably detecting low-concentration pathogens in samples.

[0005] Based on the above problems and needs, lateral flow immunoassay (LFIA) is a rapid detection technology based on chromatographic separation and immune recognition. As an important point-of-care testing (POCT) tool, it has the advantages of being fast, simple, flexible and economical. However, the traditional colorimetric LFIA with gold nanoparticles (AuNP) as labels suffers from low sensitivity and quantitative ability in practical applications, which has always been a difficult technical process problem to solve.

[0006] With the development of nanomaterials, nanozymes, as a nanomaterial with a structure and function similar to natural enzymes, have attracted a lot of attention. Their outstanding advantages such as robust catalytic activity, adjustable properties, and good stability make them have broad application potential in the field of catalytic analysis. After the nanozyme is assembled with LFIA, it can mediate catalytic amplification of colorimetric signals and significantly improve the sensitivity of LFIA test strips. However, the background interference of complex biological systems is serious, and the colorimetric signal amplification mediated by nanozymes still cannot meet the sensitivity requirements of clinical accurate diagnosis, and the detection and quantification capabilities of low-abundance pathogens in the early stages of infection are still insufficient. At the same time, the colorimetric method has the disadvantages of being easily affected by ambient light and user subjective bias.

[0007] Surface enhanced Raman scattering (SERS) is a powerful vibrational spectroscopy technique with the advantages of extremely high sensitivity, fingerprint peak characteristics, simple operation, and rapid non-destructive detection. It can be used for ultra-sensitive detection of various biomarkers and pathogens, which can solve the above-mentioned problem of insufficient sensitivity based on colorimetric detection. However, the enhancement ability of traditional SERS substrates is limited. The conventional practice is to adsorb Raman marker molecules on the surface of metal SERS substrates. The marker molecules are easily affected by the complex sample environment and even fall off during subsequent biological modification and application. Therefore, the repeatability and stability of SERS signals are seriously affected. A shell-isolated nanoparticle enhanced Raman spectroscopy technology can effectively solve the above problems. The Raman reporter molecules are embedded between the core and the shell, effectively preventing the Raman reporter molecules from falling off from the substrate material and causing poor detection reproducibility. In addition, the SERS substrate with nanoscale internal gaps has uniformity and controllability in the establishment of nanogaps, thereby obtaining stable, highly enhanced and adjustable SERS signals.

[0008] Therefore, the present invention provides a method for preparing multi-noble metal nanoparticles Au@Au@Ag / Pt / M NPs having both plasmon resonance properties and peroxidase-like activity and its application in the detection of Klebsiella pneumoniae, providing a new idea for the sensitivity problem that urgently needs to be solved in Klebsiella pneumoniae infection. Summary of the invention

[0009] In view of this, the present invention provides a preparation method of Au@Au@Ag / Pt / MNPs and its application in the detection of Klebsiella pneumoniae.

[0010] The present invention first synthesizes Au@Au@Ag / Pt / MNPs through the seed-mediated method and the in-situ reduction method. Incorporating 4-MBA Raman reporter molecules into the Au@Au nanogap endows it with stronger Raman enhancement performance, and the AgPt shell endows it with nanozyme catalytic performance. Therefore, the developed Au@Au@Ag / Pt / MNPs combines dark color, catalytic color development, and surface-enhanced Raman performance. By modifying the specific antibody (Ab1) of Klebsiella pneumoniae on its surface to form a nanolabel, and then incubating it with the target Klebsiella pneumoniae. After a certain time, it is centrifuged and enriched, suspended in phosphate buffer (PBS), and the resuspended solution is dropped onto the test strip. Through capillary action, it flows laterally on the test strip. Then, the Klebsiella pneumoniae labeled with Au@Au@Ag / Pt / M NPs is captured by the specific antibody on the T line (Test line) of the test strip and thus shows color. Therefore, it is possible to simply judge whether Klebsiella pneumoniae exists through the color change. The excess Au@Au@Ag / Pt probe is captured by the secondary antibody on the C line (Control line) as a quality control evaluation of the test strip.

[0011] Due to the good nanozyme activity of the Au@Au@Ag / Pt / M nanolabel, the detection ability of the test strip is improved by performing an enzyme-catalyzed substrate color reaction on the T line. Specifically, when the target Klebsiella pneumoniae exists, the Klebsiella pneumoniae labeled with Au@Au@Ag / Pt / MNPs aggregates on the T line of the test strip. We immerse the T line of the test strip into a NaAC-HAC buffer solution with a pH of 4.0 and a 3-amino-9-ethylcarzole / hydrogen peroxide (3-amino-9-ethylcarzole / Hydrogen peroxide, AEC / H2O2) solution system. Based on the peroxidase activity of Au@Au@Ag / Pt / MNPs, H2O2 oxidizes AEC to form a stable brown-red precipitate product. This brown-red product is insoluble in water, realizing enzyme-catalyzed amplified colorimetric signal and further improving the color development sensitivity.

[0012] Similarly, due to the surface-enhanced Raman performance of the Au@Au@Ag / Pt / MNPs label, the quantitative detection of Klebsiella pneumoniae can be achieved by detecting the Raman signal of the T line. And because Raman detection is a very sensitive detection method, it can make up for the deficiency of the low sensitivity of visual colorimetry. Specifically, when the target Klebsiella pneumoniae exists, the Klebsiella pneumoniae labeled with Au@Au@Ag / Pt / M NPs aggregates on the T line of the test strip. We align the Raman laser with the T line. According to the 4-MBA Raman reporter molecule at 1588 cm-1 The characteristic peak signal at

[0013] To achieve the above object, the present invention adopts the following technical solutions:

[0014] The preparation method of Au@Au@Ag / Pt / MNPs is as follows:

[0015] (1) Mix 5 mL of 0.2 M CTAB solution with 10 mL of 5 mM HAuCl4 aqueous solution evenly, then quickly add 6 mL of 1 mM freshly prepared ice-cold NaBH4 solution, mix well, and place it in a water bath at 25 °C for 1 h to obtain a 3-5 nm Au nanocluster solution showing a brownish-yellow color;

[0016] (2) Mix 2 mL of 0.1 M CTAC solution, 1.5 mL of 1 M ascorbic acid solution, and 50 μL of the above-prepared Au nanocluster solution evenly, and quickly add 2 mL of 5 mM HAuCl4 aqueous solution under the action of ultrasound, and place it in a water bath at 25 °C for 50 min to obtain 10 nm AuNPs showing a red color;

[0017] (3) Mix 40 mL of 0.1 M CTAC solution, 2.6 mL of 0.1 M ascorbic acid solution, 3 mL of 10 nm AuNPs solution, and 40 mL of 5 mM HAuCl4 evenly, and after ultrasonic reaction for 8 min, the solution color is stable to purple-red; after standing in the dark for 2 days, centrifuge at 10000 rpm for 20 min and resuspend in 80 mL of 0.2 M CTAC solution to obtain a 30 nm AuNPs solution;

[0018] (3) Add 50 μL of 10 -3 M 4-MBA solution to 1 mL of the above-prepared 30 nm AuNPs solution and ultrasonically react for 10 min; centrifuge at 10000 rpm for 10 min, wash with 0.5 M CTAC solution to remove the excess 4-MBA solution, and resuspend in 1 mL of 0.5 M CTAC solution; add 180 μL of the above resuspension to a mixed solution of 4 mL of 0.5 M CTAC, 620 μL of 0.04 M ascorbic acid, and 120 μL of 5 mM HAuCl4, and ultrasonically react for 60 min; centrifuge at 8000 rpm for 8 min to remove the excess reaction solution and resuspend in 1 mL of H2O to synthesize interstitial Au@AuNPs;

[0019] (4) Take a 50 mL round-bottom flask, add 13.1 mL of ultrapure water, 0.4 mL of 0.5 M CTAC, and 1 mL of interstitial Au@AuNPs solution, place it in an ultrasonic bath to make it evenly dispersed, then add 50 μL of 0.02 M NaOH solution and 100 μL of 0.1 M ascorbic acid solution under magnetic stirring. After stirring for 2 min, add 1 mL of 10 mM AgNO3 solution at one time, and at the same time add 0.5 mL of 10 mM H2PtCl6 solution using a syringe at a rate of 40 seconds per drop. Then transfer it to 70 °C and heat for 2 h; let the solution obtained from the reaction cool naturally to room temperature, centrifuge, and resuspend it in 1 mL of H2O to obtain the Au@Au@Ag / PtNPs solution;

[0020] (5) Mix 500 μL of Au@Au@Ag / PtNPs solution with 50 μL of 10 -3 mol / L 4-MBA solution evenly, incubate with shaking for 30 min, then centrifuge at 8000 rpm for 10 min to remove the excess Raman reporter molecules, and then redisperse it in 500 μL of ultrapure water to obtain Au@Au@Ag / Pt / MNPs.

[0021] Furthermore, Au@Au@Ag / Pt / MNPs prepared by the described preparation method.

[0022] Furthermore, the application of the Au@Au@Ag / Pt / M NPs in the preparation of a Klebsiella pneumoniae detection reagent.

[0023] Furthermore, the application of the Au@Au@Ag / Pt / MNPs in the assembly of LFIA.

[0024] Furthermore, the application of the Au@Au@Ag / Pt / MNPs in the preparation of an LFIA kit for detecting Klebsiella pneumoniae.

[0025] Based on the necessity of early screening for Klebsiella pneumoniae infectious pneumonia, the present invention develops an Au@Au@Ag / Pt / MNPs sensing probe integrating nanozyme catalytic performance and plasmonic Raman enhancement function, combines it with LFIA, constructs a colorimetric, catalytic colorimetric, Raman detection multimode LFIA (CM / CL / SERS) and applies it to the detection of Klebsiella pneumoniae. Compared with traditional detection methods, the LFIA mediated by the Au@Au@Ag / Pt / MNPs is faster, more sensitive, and the detection results are presented multimodally. And the multiple detection results can verify each other to improve the detection accuracy and reliability, and different detection ranges and detection limits can meet different detection needs. Therefore, the LFIA mediated by the Au@Au@Ag / Pt / MNPs is an original detection method in this field and has potential clinical application prospects.

[0026] Development of an Au@Au@Ag / Pt / MNPs signal probe based on the dual characteristics of plasmonics and catalytic properties and construction of a multimode sensing platform (CM / CL / SERS) in combination with LFIA, which is expected to be applied to the early point-of-care detection of Klebsiella pneumoniae in clinical sputum.

[0027] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a preparation method of Au@Au@Ag / Pt NPs and its application in the detection of Klebsiella pneumoniae, having the following

[0028] Advantages:

[0029] (1) Currently, there is a lack of a method for directly detecting Klebsiella pneumoniae. The present invention can detect Klebsiella pneumoniae at low cost, rapidly, and sensitively;

[0030] (2) By using multifunctional nanoparticles Au@Au@Ag / Pt / MNPs with colorimetric, catalytic, and SERS functions, multiple signals can be output simultaneously to meet the requirements of different sensitivities and a wide detection range;

[0031] (3) The test strip integrates three signal output modes, and each mode has its unique advantages. Visual colorimetry is simple and rapid, catalytic colorimetry has an amplification ability, and SERS detection has high sensitivity and quantitative analysis ability, making the output signals diverse and complementary;

[0032] (4) Through the superposition and complementarity of the three signals, this multimode LFIA can maintain stable performance under different detection conditions, which is particularly important for point-of-care detection in complex and variable actual application scenarios. Description of the Drawings

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0034] Figure 1 Performance and morphology characterization of AuNPs of the present invention; (A) UV-visible absorption spectrum during the synthesis of 30 nm AuNPs; (B) TEM image of 10 nm AuNPs; (C) TEM image of 30 nm Au NPs.

[0035] Figure 2UV-visible absorption spectra (A) and Raman signal spectra (B) of the gap Au@Au nanomaterials and non-gap Au@Au nanomaterials of the present invention; TEM images of (C) gap Au@Au nanomaterials and (D) non-gap Au@Au nanomaterials.

[0036] Figure 3 TEM images of Au@Au@Ag / Pt / M NPs with different Ag / Pt ratios of the present invention; (A) 3:1; (B) 2:1; (C) 1:1; (D) 1:2; scale bar is 100 nm.

[0037] Figure 4 Au EDS (A), Ag EDS (B), Pt EDS (C), Au-Ag-Pt EDS (D), TEM (E), XRD (F), XPS full spectrum (G), Ag 3d XPS narrow spectrum (H), and Pt4f XPS narrow spectrum (I) of Au@Au@Ag / Pt / M NPs of the present invention.

[0038] Figure 5 UV-visible absorption spectra of Au@Au@Ag / Pt / M NPs of the present invention (Ag / Pt ratios are 3:1, 2:1, 1:1, 1:2, 1:3 respectively) (the insets are the corresponding optical photos).

[0039] Figure 6 UV-visible absorption spectra of five kinds of mixed solutions of the present invention (A), and the insets are optical photos (1: TMB, 2: TMB + H2O2, 3: Au@Au@Ag / Pt / M nanosubstrate + H2O2, 4: TMB + Au@Au@Ag / Pt / M nanosubstrate + TMB; 5: TMB + H2O2 + Au@Au@Ag / Pt / M nanosubstrate); (B) bar graph of the absorbance of the corresponding catalytic products at 650 nm.

[0040] Figure 7 Catalytic activity and Raman enhancement ability of Au@Au@Ag / Pt / M NPs of the present invention (Ag / Pt ratios are 3:1, 2:1, 1:1, 1:2, 1:3 respectively); (A-B) UV absorption spectra of the catalytic products and their absorption values at 650 nm; (C-D) Raman spectra and their Raman peak intensities at 1588 cm -1 -1.

[0041] Figure 8 Detection of Klebsiella pneumoniae by the multi-mode test strip of the present invention; wherein, (A) chromatographic result images of Klebsiella pneumoniae with a series of concentrations; (B) corresponding catalytic color development result images at the T line; (C) Raman spectra collected at the corresponding T line; (D) Raman signal-concentration standard curve. Detailed implementation mode

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Synthesis Preparation Method and Characterization of Au@Au@Ag / Pt / M Nanoparticles in Example 1

[0044] First, 5 mL of 0.2 M CTAB solution was mixed evenly with 10 mL of 5 mM HAuCl4 aqueous solution. Then, 6 mL of freshly prepared ice-cold NaBH4 solution of 1 mM was quickly added. After thorough mixing, it was placed in a water bath at 25 °C and reacted for 1 h to obtain a 3-5 nm Au nanocluster solution showing a brownish-yellow color. Next, 2 mL of 0.1 M CTAC (cetyltrimethylammonium chloride) solution, 1.5 mL of 1 M AA (ascorbic acid) solution, and 50 μL of the above-prepared Au nanocluster solution were mixed evenly. Under the action of ultrasound, 2 mL of 5 mM HAuCl4 aqueous solution was quickly added, and it was placed in a water bath at 25 °C and reacted for 50 min to obtain 10 nm AuNPs showing a red color. Finally, 40 mL of 0.1 M CTAC solution, 2.6 mL of 0.1 M AA solution, 3 mL of 10 nm AuNPs solution, and 40 mL of 5 mM HAuCl4 were mixed evenly. After ultrasonic reaction for 8 min, the solution color was stable to purplish-red. After standing in the dark for 2 days, it was centrifuged at 10000 rpm for 20 min and resuspended in 80 mL of 0.2 M CTAC solution to obtain a 30 nm AuNPs solution.

[0045] 50 μL of 10 -3 M 4-MBA (4-mercaptobenzoic acid) solution was added to 1 mL of the above-prepared 30 nm AuNPs solution, and ultrasonic reaction was carried out for 10 min. Subsequently, it was centrifuged at 10000 rpm for 10 min, and the excess 4-MBA solution was washed away with 0.5 M CTAC solution and resuspended in 1 mL of 0.5 M CTAC solution. Then, 180 μL of the above resuspension was added to a mixed solution of 4 mL of 0.5 M CTAC, 620 μL of 0.04 M AA, and 120 μL of 5 mM HAuCl4, and ultrasonic reaction was carried out for 60 min. Subsequently, it was centrifuged at 8000 rpm for 8 min to remove the excess reaction solution and resuspended in 1 mL of H2O to synthesize interstitial Au@AuNPs.

[0046] Take a 50 mL round-bottom flask, add 13.1 mL of ultrapure water, 0.4 mL of 0.5 M CTAC, and 1 mL of interstital Au@AuNPs solution, place it in an ultrasonic bath to make it disperse evenly, then add 50 μL of 0.02 M NaOH solution and 100 μL of 0.1 M AA solution under magnetic stirring. After stirring for 2 min, add 1 mL of 10 mM AgNO3 solution at one time, and at the same time add 0.5 mL of 10 mM H2PtCl6 solution (the ratio of Ag to Pt is 2:1) with a syringe at a rate of 40 drops / second. Subsequently, transfer it to 70 °C and heat for 2 h. Finally, naturally cool the solution obtained from the reaction to room temperature, centrifuge, and resuspend it in 1 mL of H2O to obtain 4-MBA-modified nanoparticles Au@Au@Ag / PtNPs (Au@Au@Ag / Pt / MNPs). In this experiment, a series of Au@Au@Ag / PtNPs with different Ag / Pt ratios were synthesized by controlling the amounts of AgNO3 and H2PtCl6 introduced during the synthesis process, aiming to endow the material with both good enzyme catalytic activity and Raman enhancement ability.

[0047] Mix 500 μL of Au@Au@Ag / PtNPs solution with 50 μL of 10 -3 mol / L 4-MBA solution evenly, incubate with shaking for 30 min, then centrifuge at 8000 rpm for 10 min to remove the excess Raman reporter molecules, and then redisperse it in 500 μL of ultrapure water to obtain Au@Au@Ag / Pt / MNPs further modified by 4-MBA.

[0048] Performing performance and morphology characterizations on the above-prepared AuNPs, interstital Au@AuNPs, and Au@Au@Ag / Pt / MNPs respectively, it can be seen that first, AuNPs with a size of about 10 nm were synthesized ( Figure 1 B), and then AuNPs with a size of about 30 nm were further synthesized ( Figure 1 C). According to the ultraviolet absorption spectrum ( Figure 1 A), as the AuNPs grow, when they grow to 10 nm, a characteristic absorption peak of Au appears at a wavelength of 520 nm. With further growth, the maximum ultraviolet absorption peak redshifts to 524 nm. Based on the 30 nm Au core, 4-MBA was further modified, and then the reduction of the Au shell was carried out. Through 4-MBA as the internal medium, an Au@Au nanogap was formed. From the TEM characterization results, it can be seen that its morphology is popcorn-like, and obvious gaps appear inside ( Figure 2 C). As a comparison, without modifying 4-MBA outside the 30 nm Au core and with the same remaining steps, a gapless Au nanomaterial was synthesized ( Figure 2 D), thus confirming the key role of 4-MBA as the internal medium in synthesizing nanogaps. And the ultraviolet results ( Figure 2(A) also further verified the successful synthesis of Au@Au nanogaps. Further, due to the gap enhancement effect, the SERS enhancement effect of Au@Au gap nanomaterials is significantly better than that of non-gap ones ( Figure 2 B).

[0049] Furthermore, Au@Au@Ag / Pt / MNPs were synthesized with different Ag-Pt ratios, and the TEM results showed that they all had a uniform morphology ( Figure 3 ). Further, EDS, XPS, and XRD characterizations were performed on all the elements contained in Au@Au@Ag / Pt / MNPs. As Figure 4 (A-E) shows, Au@Au@Ag / PtNPs mainly contain three elements: Au, Ag, and Pt. Moreover, it can be further found from the element distribution maps that since the lattice constants of Au and Ag (4.0786, 4.0862) are very close, Ag is first deposited on the surface of Au@Au to form a core-shell structure, and then Pt 4+ through the reaction 4Ag + Pt 4+ → 4Ag + + Pt forms Pt deposition on the surface, and the displaced Ag + will be reduced to Ag by AA in the reaction system, so that finally Ag and Pt are co-reduced to form an alloy and continuously deposit. Similarly, Figure 4 (F-I) all show the presence and chemical states of the three elements Au, Ag, and Pt. These results consistently indicate the successful synthesis of Au@Au@Ag / Pt / MNPs. The ultraviolet absorption spectrum shows that the AuNPs in the gap Au@AuNPs have a significant characteristic absorption peak at 524 nm. However, when further synthesizing Au@Au@Ag / Pt / MNPs, this characteristic absorption peak disappears because Pt produces a damping effect that makes the absorption peak of AuNPs disappear, which also indicates the effective deposition of the Pt layer, and there is no obvious difference in the ultraviolet absorption spectra formed by different Ag / Pt ratios ( Figure 5 ).

[0050] Example 2 Verification of the signal output mode ability of Au@Au@Ag / Pt / M nanoparticles

[0051] First, the verification of the surface-enhanced Raman ability of the nanomaterials: Take 5 μL of the Au@Au@Ag / Pt / MNPs solution modified with the Raman reporter molecule 4-MBA and drop it on a glass slide. Use a micro-Raman spectrometer with a 532 nm laser, a 50-fold long-focus objective lens, a collection time of 5 s, and integrate once to collect and detect its Raman spectrum.

[0052] Secondly, the classical enzyme reaction substrate 3,3',5,5'-tetramethylbenzidine (TMB) was used as the chromogenic substrate, and the catalytic performance of Au@Au@Ag / Pt / MNPs was investigated by ultraviolet absorption spectroscopy and enzyme reaction kinetics. 5 μL of Au@Au@Ag / Pt / MNPs solution was added to a mixed solution containing 1695 μL of NaAC-HAC buffer (pH = 4.5), 150 μL of 30% H2O2 and 150 μL of 10 mM TMB. After reacting at room temperature for 10 min, the ultraviolet absorption was measured.

[0053] It can be seen from the color development of each solution and the absorption spectrum diagram ( Figure 6 ), under the condition of using NaAC-HAC with pH 4.5 as the buffer, Au@Au@Ag / Pt / MNPs can catalyze the oxidation of TMB to oxTMB under the action of H2O2, making the solution change from colorless to blue, and a characteristic absorption peak can also be observed at 650 nm in the ultraviolet spectrum. When any one or two of Au@Au@Ag / Pt / MNPs, H2O2, and TMB are missing, the phenomenon of the solution turning blue cannot be observed, and the characteristic peak of oxTMB does not appear in the corresponding ultraviolet spectrum. These results indicate that Au@Au@Ag / Pt / MNPs has good peroxidase-like catalytic activity.

[0054] In addition, the catalytic performance of Au@Au@Ag / Pt / M can be adjusted by changing the ratio of Ag to Pt. Obviously, as the Ag / Pt ratio gradually decreases, more colorless TMB is oxidized to blue oxTMB by Au@Au@Ag / Pt / M, and the signal of the UV-Vis absorption peak at 652 nm is stronger ( Figure 7 A, B). The thicker the Pt shell, the more effectively the catalytic activity of the sensor is enhanced, because the whole catalytic oxidation process mainly depends on the Pt component. However, when the Ag / Pt ratio is 2:1, the catalytic efficiency reaches saturation.

[0055] The SERS performance of Au@Au@Ag / Pt / M was further investigated. As is well known, the 4-MBA Raman molecule is sandwiched in the internal gap between metal nanostructures, and the benzene ring generates a very strong SERS signal through the symmetric vibration of plasmon coupling. However, the overgrowth of the Pt shell may cover the plasmonic properties of the Au@Au and Ag layers, thus having a profound impact on the amplification of the SERS signal. As Figure 7As shown in C and D, the SERS signal intensity decreases with the decrease of the Ag / Pt shell ratio, indicating that the Pt shell has a negative impact on the SERS performance of the multilayer tags. Considering the SERS / catalytic performance of Au@Au@Ag / Pt / M comprehensively, the Ag / Pt ratio of 2:1 was used in subsequent experiments, making the prepared Au@Au@Ag / Pt / M exhibit the best SERS / catalytic signals.

[0056] Example 3 Assembly and verification of the detection performance of Klebsiella pneumoniae by Au@Au@Ag / Pt / M NPs probe and multimode LFIA

[0057] The anti-Klebsiella pneumoniae antibody Ab1 was directly linked to the surface of Au@Au@Ag / Pt / M NPs nanoparticles by adsorption: First, 20 μg of the antibody anti-Klebsiella pneumoniae polyclonal antibody 1 protein (Ab1, Yanofar Biotechnology Co., Ltd.) for detection was fully mixed with 5 μL of the above-prepared Au@Au@Ag / Pt / M NPs modified with 4-MBA, and gently oscillated and reacted at 25 °C and 200 rpm for 2 h. Subsequently, 50 μL of 10% BSA was added and reacted for 1 h to block the incompletely bound sites. After centrifugation, it was resuspended in 300 μL of the labeling composite solution (0.01 M PBS solution containing 2% sucrose, 0.5% Tween 20, 1% BSA) to obtain the label solution.

[0058] The prepared LFIA consists of four parts: a sample pad, an NC membrane, a water-absorbing pad, and a PVC bottom plate. When preparing the test strip, first, the NC membrane was pasted in the middle of the PVC bottom plate. After equilibrating at 37 °C for a period of time, 1.4 mg / mL of anti-Klebsiella pneumoniae polyclonal antibody 2 protein (Ab1, Abcam (Shanghai) Trading Co., Ltd.) and 1 mg / mL of goat anti-rabbit secondary antibody (Sigma-Aldrich (Shanghai) Co., Ltd.) were embedded on the NC membrane at a spraying speed of 1 μL / cm using a membrane scribing instrument to form the T line and the C line. After drying for 2 h, the sample pad was fixed at the head end of the PVC bottom plate, and the water-absorbing pad was fixed at the tail end of the PVC bottom plate, so that all parts overlapped with each other finally, enabling the sample solution to flow continuously along each part smoothly under capillary action after being dropped onto the sample pad. Finally, the assembled test strip was cut into 4 mm wide, loaded into the matching cartridge, and sealed with a desiccant for storage and standby.

[0059] Based on the Au@Au@Ag / Pt / M NPs nanoparticles with dual characteristics of plasmon and catalytic properties modified with Klebsiella pneumoniae antibody Ab1 prepared by the present invention as signal molecules, a sandwich recognition and capture system was established on the LFIA test strip.

[0060] The result of the assembled LFIA test strip is asFigure 8 As shown. By mixing 5 μL of the labeled solution, 20 μL of the chromatography buffer (borax pH standard solution containing 2% Tween-20 and 0.5% BSA), and 70 μL of the sample solution to be tested, when the target Klebsiella pneumoniae exists in the sample solution to be tested, due to the rapid recognition and binding reaction of antigen and antibody, the labeled / Klebsiella pneumoniae complex is first formed and further dropped onto the sample pad of the LFIA for reaction. The presence of a high-concentration Klebsiella pneumoniae solution can be directly judged by observing the color change on the T line with the naked eye, and its visual minimum detection limit is about 10 4 CFU / mL( Figure 8 A). However, the sensitivity by colorimetric observation is low, it is difficult to be used for the detection of low-concentration Klebsiella pneumoniae and quantitative detection cannot be achieved. Applying the catalytic colorimetric mode to judge the results, cutting the T line of the test strip and soaking it, the components of the soaking solution are set as 0.9 mL of NaAC-HAC, 200 μL of 50 mM AEC, and 200 μL of 30% H2O2. Under this condition, its visual minimum detection limit is about 10 3 CFU / mL, and its sensitivity is increased by one order of magnitude( Figure 8 B). To further improve the detection sensitivity, the Raman detection mode is adopted, and the detection limit is about 10 2 CFU / mL. At the same time, by fitting and analyzing the Raman signals collected from the T lines of each test strip( Figure 8 C), the quantitative detection of Klebsiella pneumoniae can be realized. As Figure 8 shown in D, the Raman signals show good linearity between 10 2 -10 7 CFU / mL, and R 2 = 0.989.

[0061] After testing, the multi-mode LFIA (basic colorimetry / catalytic colorimetry / surface-enhanced Raman scattering, CM / CL / SERS) has excellent signal output ability for the detection of Klebsiella pneumoniae, with good detection signal reproducibility and high specificity, and has good application prospects.

[0062] The present invention synthesizes hollow Au@Au@Ag / Pt / M NPs with plasma properties and catalytic activity, which are assembled with LFIA to form a multi-mode sensing platform, realizing highly specific, accurate, sensitive, rapid, and economical multi-modal detection of Klebsiella pneumoniae, and is expected to provide new ideas for the diagnosis of respiratory diseases such as Klebsiella pneumoniae infection.

[0063] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The preparation method of Au@Au@Ag / Pt / MNPs is characterized in that: The specific steps are as follows: (1) 5 mL of 0.2 M CTAB solution and 10 mL of 5 mM HAuCl4 aqueous solution were mixed evenly, and then 6 mL of 1 mM freshly prepared glacial NaBH4 solution was quickly added. After thorough mixing, the mixture was placed in a 25 °C water bath for 1 h to obtain a brown-yellow 3-5 nm Au nanocluster solution. (2) 2 mL of 0.1 M CTAC solution, 1.5 mL of 1 M ascorbic acid solution, and 50 μL of the Au nanocluster solution prepared above were mixed evenly, and 2 mL of 5 mM HAuCl4 aqueous solution was quickly added under the action of ultrasound, and placed in a 25 °C water bath for reaction for 50 min to obtain red 10 nm AuNPs; (3) 40 mL of 0.1 M CTAC solution, 2.6 mL of 0.1 M ascorbic acid solution, 3 mL of 10 nm AuNPs solution, and 40 mL of 5 mM HAuCl4 were mixed evenly and subjected to ultrasonic reaction for 8 min. The color of the solution was stable to purple red. After being placed away from light for 2 days, the solution was centrifuged at 10,000 rpm for 20 min and resuspended in 80 mL of 0.2 M CTAC solution to obtain a 30 nm AuNPs solution. (3) Add 50 μL of 10 -3 M4-MBA solution, and ultrasonically react for 10 minutes; centrifuge at 10000rpm for 10 minutes, wash with 0.5M CTAC solution to remove excess 4-MBA solution, and resuspend in 1mL 0.5M CTAC solution; add 180μL of the above resuspension to a mixed solution of 4mL 0.5M CTAC, 620μL 0.04M ascorbic acid, and 120μL 5mM HAuCl4, and ultrasonically react for 60 minutes; centrifuge at 8000rpm for 8 minutes, remove excess reaction solution, and resuspend in 1mL H2O to synthesize interstitial Au@AuNPs; (4) Take a 50mL round-bottom flask, add 13.1mL ultrapure water, 0.4mL 0.5M CTAC and 1mL interstitial Au@AuNPs solution, place it in an ultrasonic bath to disperse it evenly, then add 50μL 0.02M NaOH solution and 100μL 0.1M ascorbic acid solution under magnetic stirring, stir for 2min, add 1mL 10mM AgNO3 solution at one time, and add 0.5mL 10mM H2PtCl6 solution at a rate of 40 seconds / drop using a syringe, then turn to 70℃ and heat to react for 2h; the resulting solution is naturally cooled to room temperature and resuspended in 1mL H2O after centrifugation to obtain Au@Au@Ag / PtNPs solution; (5) 500 μL Au@Au@Ag / PtNPs solution was mixed with 50 μL 10 -3 mol / L 4-MBA solution was mixed evenly, incubated with shaking for 30 min, centrifuged at 8000 rpm for 10 min to remove excess Raman reporter molecules, and then redispersed in 500 μL ultrapure water to obtain Au@Au@Ag / Pt / MNPs.

2. Au@Au@Ag / Pt / MNPs prepared by the preparation method of claim 1.

3. Use of the Au@Au@Ag / Pt / MNPs described in claim 2 in the preparation of a Klebsiella pneumoniae detection reagent.

4. Use of the Au@Au@Ag / Pt / MNPs described in claim 2 in assembling LFIA.

5. Use of the Au@Au@Ag / Pt / MNPs described in claim 2 in the preparation of a LFIA kit for detecting Klebsiella pneumoniae.

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