A hollow alloy nanoparticle detection probe, its preparation method and application

Abstract

This invention belongs to the field of microbial detection technology, specifically relating to a hollow alloy nanoparticle detection probe, its preparation method, and its applications. This invention is the first to synthesize silver-palladium-platinum hollow alloy nanoparticles. Their uniform alloying structure and hollow structure endow them with excellent catalytic and photothermal properties. Combined with rapid detection methods, they can be widely used in medical diagnostics, bio-colorimetric sensing, and other fields. The prepared silver-palladium-platinum hollow alloy nanoparticles are used to prepare hollow alloy nanoparticle detection probes. The preparation operation is simple, low-cost, and the synthesis conditions are mild. It can be directly synthesized at room temperature and is easy to promote. It can achieve simple and rapid multi-target colorimetric and photothermal detection of iron bacteria in different scenarios, and has significant application value in medical diagnostics, biosensing analysis, and other fields, especially for the detection of iron bacteria in oil pipelines.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a hollow alloy nanoparticle detection probe, its preparation method, and its application. Background Technology

[0002] Iron bacteria are a type of bacteria that can convert ferrous iron (Fe²⁺) into ferrous iron (Fe₂ 2+ ) oxidized to trivalent iron (Fe) 3+ Microorganisms (such as Gallionella, Leptothrix, and Sphaerotilus) obtain energy from iron bacteria in pipelines. These microorganisms form biofilms within the pipeline, secreting metabolic products such as ferric hydroxide, leading to pipeline corrosion (microbially induced corrosion, MIC), blockages, or water quality deterioration. Iron bacteria naturally present in injected water or formation water, or introduced during pipeline maintenance or construction, can also form iron bacteria within oil pipelines. Stagnant water, low-flow-rate sections, or corrosion products (such as FeCO3 and FeS) in oil pipelines provide growth conditions for iron bacteria. The coexistence of iron bacteria with other corrosive microorganisms (such as sulfate-reducing bacteria SRB) can also form complex biofilms, further damaging the pipeline.

[0003] Traditional methods for detecting iron bacteria typically involve culture using specific media (such as Kligler iron agar). However, iron bacteria have long culture cycles (several weeks), low sensitivity, and many species are unculturable. Alternatively, inference can be made indirectly through iron deposits or corrosion products, but this method lacks specificity. Electrochemical and sensor technologies are being used for detection, indirectly reflecting microbial corrosion activity by monitoring changes in potential and current within the pipe. Some biosensors can specifically identify iron bacterial metabolites (such as Fe). 3+ (Concentration), but the current preparation methods are complex and costly, and the sensitivity is low.

[0004] Alloy nanoparticles are frequently used in the field of medical detection technology. Metal alloy nanoparticles are composed of two or more metals (such as main group metals, noble metals, transition metals, etc.) with particle sizes in the nanometer (nm) range. Their key characteristic is the combination of the physicochemical properties of different metals (such as catalytic activity, photothermal properties, and electrical conductivity) to form composite functional nanomaterials with multiple advantages. Alloy nanoparticles exhibit unique properties that are unmatched by other traditional materials. Compared to natural enzymes, alloy nanoparticle materials not only exhibit excellent catalytic activity, photothermal properties, and electrical conductivity, but also have advantages such as simple preparation, low cost, corrosion resistance, stability, and high applicability. Compared to binary alloy nanoparticles, ternary alloy nanoparticles can achieve superior properties that are difficult to obtain with single metals or binary alloy nanoparticles by adjusting the ratio and microstructure of the three metals, through a richer composition-structure-performance control space. However, there are relatively few reports on ternary hollow alloy nanoparticles that combine high catalytic activity and excellent photothermal properties.

[0005] Alloy nanoparticles can be used to prepare detection probes. Iron bacteria may exist in pipelines at low biomass levels (e.g., attached to biofilms), and existing detection probes have insufficient lower limits of detection (LoD), making it difficult to capture early contamination. Iron bacteria in biofilms may be in a metabolically dormant state, leading to reduced probe binding efficiency.

[0006] Existing probes have significant shortcomings in specificity, sensitivity, and field applicability, especially in complex environments such as oil pipelines. Developing next-generation probes is key to solving the challenge of iron bacteria detection, enabling early warning of corrosion risks and optimization of pipeline maintenance strategies. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a hollow alloy nanoparticle detection probe, its preparation method, and its application. Using silver nanoparticles as a sacrificial template, ascorbic acid to provide an acidic environment, and sodium borohydride as a reducing agent, silver nanoparticles are etched stepwise using sodium tetrachloropalladate and sodium tetrachloroplatinate to obtain silver-palladium-platinum hollow alloy nanoparticles. The synthesized hollow alloy particle solution is then incubated with iron bacteria-labeled antibodies and bovine serum albumin to finally obtain the hollow alloy nanoparticle detection probe, which can be applied to the detection of oil pipelines in Tiexi Street. The technical solution adopted is as follows:

[0008] A method for preparing a detection probe made of hollow alloy nanoparticles includes the following steps:

[0009] (1) Dissolve silver nitrate in water, add ascorbic acid solution, and stir to obtain silver nanoparticle solution; collect silver nanoparticle product by centrifugation, wash and use it as a sacrificial template for the synthesis of ternary hollow and gold nanoparticles.

[0010] (2) Dissolve silver nanoparticles in water, add polyvinylpyrrolidone, ascorbic acid, sodium borohydride and sodium tetrachloropalladium in sequence to the silver nanoparticle solution, stir, centrifuge, and collect the solid product; wash the product to obtain silver palladium nanoparticles.

[0011] (3) Dissolve silver palladium nanoparticles in water, add polyvinylpyrrolidone, ascorbic acid, sodium borohydride and sodium tetrachloroplatinate to the silver palladium nanoparticle solution in sequence, stir, centrifuge, and collect the solid product; wash the product to obtain silver palladium platinum hollow alloy nanoparticles.

[0012] (4) Dissolve the silver-palladium-platinum hollow alloy nanoparticles in water, add the silver-palladium-platinum hollow alloy nanoparticle solution to an EP tube, add iron-labeled bacterial antibody to the tube, vortex to mix, and incubate.

[0013] (5) Add bovine serum albumin to the EP tube in step (4), vortex to mix, and incubate; after incubation, centrifuge and discard the supernatant;

[0014] (6) Add a reconstitution solution to the precipitate and resuspend to obtain the detection probe.

[0015] Preferably, in step (1), the mixture is stirred at 100°C for 0.5 to 2 hours.

[0016] Preferably, in step (2), the mixture is stirred at room temperature for 0.5 to 2 hours; the product is washed with water 2 to 3 times.

[0017] Preferably, in step (3), the mixture is stirred at room temperature for 0.5 to 2 hours; the product is washed with water 2 to 3 times; and the molar ratio of silver, palladium and platinum in the silver-palladium-platinum hollow alloy nanoparticles is 1:7.4:2.6.

[0018] Preferably, in step (4), the mass ratio of iron bacteria to the volume ratio of the silver-palladium-platinum hollow alloy nanoparticle solution in the EP tube is 20 μg / ml; the incubation time is 5–15 min.

[0019] Preferably, in step (5), the volume ratio of the added bovine serum albumin to the volume of the silver-palladium-platinum hollow alloy nanoparticle solution in step (4) is 1:10; the incubation time is 20-30 min.

[0020] Preferably, in step (5), after incubation, the sample is centrifuged at 10000g and 4℃ for 5-10 minutes.

[0021] Preferably, in step (6), the reconstitution solution is 50mM bovine serum albumin, and the volume ratio of the added reconstitution solution to the volume of the silver-palladium-platinum hollow alloy nanoparticle solution in step (4) is 3:10.

[0022] Another problem to be solved by the present invention is to provide a detection probe prepared by a method for preparing a hollow alloy nanoparticle detection probe.

[0023] The third problem to be solved by this invention is to provide a detection probe for the detection of iron bacteria in oil pipelines, especially for the detection of *Isophytes* genus.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1) This invention synthesizes silver-palladium-platinum hollow alloy nanoparticles for the first time. Their uniform alloying structure and hollow structure endow them with excellent catalytic and photothermal properties. They can be combined with rapid detection methods to realize their wide application in medical diagnosis, biocolorimetric sensing and other fields.

[0026] 2) The prepared silver-palladium-platinum hollow alloy nanoparticles were used to fabricate hollow alloy nanoparticle detection probes. The preparation process is simple, low-cost, and the synthesis conditions are mild. It can be directly synthesized at room temperature and is easy to promote. It can achieve simple and rapid (detection time 11–15 min) multi-target colorimetric and photothermal detection of iron bacteria in various scenarios, and has significant application value in medical diagnostics, biosensing analysis, and other fields, especially for the detection of iron bacteria in oil pipelines. Attached Figure Description

[0027] Figure 1 The image shows a transmission electron microscope (TEM) image of the silver nanoparticles prepared in Example 1.

[0028] Figure 2 The image shows a transmission electron microscope (TEM) image of the silver-palladium nanoalloy particles prepared in Example 1.

[0029] Figure 3 Transmission electron microscopy (TEM) image of the silver-palladium-platinum hollow alloy nanoparticles prepared in Example 1.

[0030] Figure 4 The energy element mapping diagram is shown for the silver-palladium-platinum hollow alloy nanoparticles prepared in Example 1.

[0031] Figure 5 The UV-Vis absorption spectra of the silver nanoparticles, silver-palladium nanoalloy nanoparticles, and silver-palladium-platinum hollow alloy nanoparticles prepared in Example 1 are shown.

[0032] Figure 6 The image shows the peroxidase-like activity of the silver-palladium-platinum hollow alloy nanoparticles prepared in Example 1.

[0033] Figure 7 The image shows the oxidase activity of the silver-palladium-platinum hollow alloy nanoparticles prepared in Example 1.

[0034] Figure 8The silver-palladium-platinum hollow alloy nanoparticles prepared in Example 1 were subjected to an intensity of 808 nm / cm at a power of 2 W. 2 Curve showing the change in solution temperature over time under laser irradiation.

[0035] Figure 9 The working curves of the silver-palladium-platinum hollow alloy nanoparticles prepared in Example 1 are used to detect H2O2 standard solutions of different concentrations.

[0036] Figure 10 The working curves of the silver-palladium-platinum hollow alloy nanoparticles prepared in Example 1 were used to detect TMB standard solutions of different concentrations.

[0037] Figure 11 The working curves of the hollow alloy nanoparticle detection probe prepared in Example 1 were obtained for detecting iron bacteria standard solutions of different concentrations. Detailed Implementation

[0038] The accompanying drawings are for illustrative purposes only; the following detailed description is intended to provide a thorough and clear description of the technical solutions described in this invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0039] A method for preparing a detection probe made of hollow alloy nanoparticles includes the following steps:

[0040] (1) Preparation of silver nanoparticle solution: 4 mL of 50 mM ascorbic acid was added to 150 mL of water, boiled, and then 0.3 mL of 50 mM silver nitrate solution was added. After reacting for 1 hour, silver nanoparticle solution was obtained. The product was washed with water 3 times and used as a sacrificial template for the synthesis of ternary hollow and gold nanoparticles.

[0041] (2) Take 1 mL of silver nanoparticle solution and add it to 20 mL of water. Then add 5 mL of 0.3 M polyvinylpyrrolidone, 0.7 mL of 50 mM ascorbic acid, 0.6 mL of 50 mM sodium borohydride, and 7 mL of 2 mM sodium tetrachloropalladium in sequence. Stir at room temperature for 1.5 hours and wash the product with water 3 times to obtain silver palladium nanoparticles.

[0042] (3) Take 10 mL of silver palladium nanoparticle solution and add it to 5 mL of water. Then add 5 mL of 0.3 M polyvinylpyrrolidone, 0.5 mL of 50 mM ascorbic acid, 0.5 mL of 50 mM sodium borohydride, and 5 mL of 2 mM sodium tetrachloroplatinate in sequence. Stir at room temperature for 1.5 hours and wash with water 3 times to obtain silver palladium platinum hollow alloy nanoparticles.

[0043] (4) Take 1 mL of hollow alloy particle solution and add it to an EP tube. Add 20 μg of iron bacteria labeled antibody to the tube, vortex to mix, and incubate for 15 min. Preferably, select iron bacteria of the genus *Synthetium*.

[0044] (5) Add 100 μL of 10% BSA (bovine serum albumin) to the EP tube, vortex to mix, and incubate for 30 min; after incubation, centrifuge at 10000g and 4℃ for 10 min, and discard the supernatant.

[0045] (6) Add 100 μL of reconstitution solution to the precipitate and resuspend to obtain the detection probe.

[0046] Catalytic and photothermal properties of silver-palladium-platinum hollow alloy nanoparticles:

[0047] (1) Peroxidase-like activity: Using silver-palladium-platinum hollow alloy nanoparticles as nanozymes, when added to a mixed solution of H2O2 and TMB, the solution changed from colorless to blue. Combined with the UV-Vis absorption spectrum, it was confirmed that it has peroxidase-like activity. The specific steps were as follows: 100 μL of alloy nanoparticle solution (4 nM), 50 μL of H2O2 solution (3 mM), and 50 μL of TMB solution (1 mM) were added to 400 μL of Hac-NaAc solution (10 mM, pH 3.6), mixed evenly at room temperature, and allowed to stand for 5 minutes. At the same time, H2O2 solution, TMB solution, H2O2 solution + TMB solution, and H2O2 solution + TMB + alloy nanoparticle solution were used as control groups. The absorbance value at a wavelength of 652 nm (oxidized TMB (oxTMB)) was recorded using a UV-Vis absorption spectrometer. The results show that the solution turns blue only when alloy nanoparticles, TMB, and H2O2 are present simultaneously, and a characteristic absorption peak appears at 650 nm. Meanwhile, the solution in the control group does not turn blue. These results indicate that the silver-palladium-platinum hollow alloy nanoparticles have peroxidase-like activity.

[0048] (2) Peroxidase Activity: Using silver-palladium-platinum hollow alloy nanoparticles as nanozymes, when added to TMB solution, the solution changed from colorless to blue. Combined with UV-Vis absorption spectroscopy, this demonstrated that it possessed peroxidase-like activity. The specific steps were as follows: 100 μL of alloy nanoparticle solution (4 nM) and 50 μL of TMB solution (1 mM) were added to 400 μL of Hac-NaAc solution (10 mM, pH 3.6), mixed thoroughly at room temperature, and allowed to stand for 5 minutes. Simultaneously, the TMB solution and alloy nanoparticle solution were used as control groups. The absorbance at 652 nm (oxidized TMB (oxTMB)) was recorded using a UV-Vis absorption spectrometer. The results showed that the solution turned blue only when both alloy nanoparticles and TMB were present, and a characteristic absorption peak appeared at 652 nm. Meanwhile, the control group solutions did not turn blue. These results indicate that the silver-palladium-platinum hollow alloy nanoparticles possess peroxidase activity.

[0049] (3) Photothermal properties: 1 mL of silver-palladium-platinum hollow alloy nanoparticle solution was heated to 808 nm / cm at a power of 2 W. 2 The solution was irradiated with a laser for 10 minutes, then the laser was turned off. The temperature change of the solution was recorded using an infrared thermal imager over 20 minutes. The aqueous solution was used as a control group. The results showed that only the temperature of the silver-palladium-platinum hollow alloy nanoparticle solution increased in the first 10 minutes and decreased rapidly in the last 10 minutes. These results prove that the silver-palladium-platinum hollow alloy nanoparticles have photothermal properties.

[0050] The silver nanoparticles, silver-palladium nanoalloy nanoparticles and silver-palladium-platinum hollow alloy nanoparticles prepared in steps (1)-(3) were characterized by transmission electron microscopy (TEM). Figure 1 This is a transmission electron microscope (TEM) image of silver nanoparticles. Figure 2 This is a transmission electron microscope image of silver-palladium nanoalloy particles. Figure 3 This is a transmission electron microscope (TEM) image of silver-palladium-platinum hollow alloy nanoparticles. (Source: [Insert image here]) Figure 1 , 2 As shown in section 3, silver nanoparticles were successfully synthesized as a two-step process using silver nanoparticles as a sacrificial template to form hollow silver-palladium-platinum alloy nanoparticles. Figure 4 The UV-Vis absorption spectra of silver nanoparticles, silver-palladium nanoalloy nanoparticles, and silver-palladium-platinum hollow alloy nanoparticles are shown. With the addition of sodium tetrachloropalladate and sodium tetrachloroplatinate, the characteristic absorption peak (400 nm) of silver nanoparticles disappears, indicating the formation of silver-palladium-platinum hollow alloy nanoparticles. Figure 5 The energy element mapping diagram of the silver-palladium-platinum hollow alloy nanoparticles further demonstrates that the nanoparticles contain all three elements—silver, palladium, and platinum—and that they are uniformly distributed. These results indicate the successful synthesis of the silver-palladium-platinum hollow alloy nanoparticles.

[0051] Test Example 1: Using the silver-palladium-platinum hollow alloy nanoparticles prepared in Example 1 as nanozymes and H2O2 as the analyte, its colorimetric detection application was verified. The specific experiment is as follows:

[0052] 100 μL of silver-palladium-platinum hollow alloy nanoparticle solution, 50 μL of 3nMTMB, and 50 μL of H2O2 solutions of different concentrations (final concentration 0.1-3 mM) were added to 500 μL of Hac-NaAc solution (10 mM, pH 3.6). The mixture was homogeneous at room temperature, and the absorbance was recorded at 652 nm using a UV-Vis absorption spectrometer after 5 minutes. A working curve was plotted by comparing the absorbance of H2O2 solutions of different concentrations (e.g., ...). Figure 9 (As shown) The linear equation of the curve is Y = 0.54X + 1.38 (R² = 0.998), the linear range is 0.1-3 mM, and the detection limit is 0.1 nM. These results indicate that the silver-palladium-platinum hollow alloy nanoparticles provided by this invention can serve as a peroxidase-like enzyme and have promising applications in the field of colorimetric sensing.

[0053] Test Example 2: Using the silver-palladium-platinum hollow alloy nanoparticles prepared in Example 1 as nanozymes and TMB as the analyte, their colorimetric detection application was verified. The specific experiment is as follows:

[0054] 100 μL of a silver-palladium-platinum hollow alloy nanoparticle solution and 50 μL of TMB solutions of different concentrations (final concentration 0.12-0.98 mM) were added to 500 μL of Hac-NaAc solution (10 mM, pH 3.6). The mixture was homogeneous at room temperature, and the absorbance was recorded at 652 nm using a UV-Vis absorption spectrometer after 5 minutes. A working curve (e.g., ...) was plotted by comparing the absorbance of different TMB concentrations. Figure 10 (As shown) The linear equation of this curve is Y = 1.05X + 1.22(R) 2 =0.933), the linear range is 0.12-0.98 mM, and the detection limit is 0.12 mM. These results indicate that the silver-palladium-platinum hollow alloy nanoparticles provided by this invention can serve as an oxidase-like enzyme and have promising applications in the field of colorimetric sensing.

[0055] Test Example 3: Using the silver-palladium-platinum hollow alloy nanoparticle probe prepared in Example 1 as the detection probe, and iron bacteria solution as the analyte, its colorimetric detection application was verified. The specific experiment is as follows:

[0056] 100 μL of silver-palladium-platinum hollow alloy nanoparticle solution was mixed with 200 μL of iron bacteria solutions of different concentrations (4.9 × 10⁻⁶). 3 9.8×10 3 1.96×10 4 3.92×104 7..84×10 4 1.57×10 5 CFU·mL -1 The above solutions were added to 400 μL of PBS solution (10 mM, pH 7.5), incubated at room temperature for 15 minutes, and then centrifuged at 3000 g / min for 5 minutes. The supernatant was collected, and its absorbance at 400 nm was recorded using a UV-Vis absorption spectrometer. A working curve was plotted by comparing the ratios of absorbance of different supernatants to the absorbance of the original detection probe (e.g., ...). Figure 11 (As shown) The linear equation of this curve is Y = 0.00247X + 0.0133(R) 2 =0.998), with a linear range of 4.9 × 10. 3 -1.57×10 5 CFU·mL -1 The detection limit is 4.9 × 10⁻⁶. 3 CFU·mL -1 The above results indicate that the silver-palladium-platinum hollow alloy nanoparticles provided by this invention can be used as detection probes and have good application prospects in the field of colorimetric sensing.

[0057] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for preparing a hollow alloy nanoparticle detection probe, characterized in that, The method comprises the following steps: (1) taking silver nitrate dissolved in water, adding ascorbic acid solution, stirring, and obtaining silver nanoparticle solution; centrifuging to collect silver nanoparticle product, and washing to obtain a sacrificial template for synthesizing ternary hollow alloy nanoparticles; (2) dissolving silver nanoparticles in water, adding polyvinylpyrrolidone, ascorbic acid, sodium borohydride and sodium tetrachloropalladate in the silver nanoparticle solution in sequence, stirring, centrifuging, and collecting solid product; washing the product to obtain silver-palladium nanoparticles; (3) dissolving silver-palladium nanoparticles in water, adding polyvinylpyrrolidone, ascorbic acid, sodium borohydride and sodium tetrachloroplatinate in the silver-palladium nanoparticle solution in sequence, stirring, centrifuging, and collecting solid product; washing the product to obtain silver-palladium-platinum hollow alloy nanoparticles; (4) dissolving silver-palladium-platinum hollow alloy nanoparticles in water, taking silver-palladium-platinum hollow alloy nanoparticle solution and adding into an EP tube, adding iron bacterium marker antibody into the tube, vortexing, and incubating; (5) adding bovine serum albumin into the EP tube of step (4), vortexing, and incubating; after incubation, centrifuging, and discarding supernatant; (6) adding reconstitution solution into the precipitate, resuspending to obtain the detection probe.

2. The method for preparing a hollow alloy nanoparticle detection probe according to claim 1, characterized in that, In step (1), stirring is performed at 100℃ for 0.5-2h.

3. The method for preparing a hollow alloy nanoparticle detection probe according to claim 1, characterized in that, In step (2), stirring is performed at room temperature for 0.5-2h; the product is washed with water for 2-3 times.

4. The method for preparing a hollow alloy nanoparticle detection probe according to claim 1, characterized in that, In step (3), stirring is performed at room temperature for 0.5-2h; the product is washed with water for 2-3 times; the molar ratio of silver, palladium and platinum in the silver-palladium-platinum hollow alloy nanoparticles is 1:7.4:2.

6.

5. The method for preparing a hollow alloy nanoparticle detection probe according to claim 1, characterized in that, In step (4), the mass ratio of iron bacteria to silver-palladium-platinum hollow alloy nanoparticle solution in the EP tube is 20μg / ml; the incubation time is 5-15min.

6. The method for preparing a hollow alloy nanoparticle detection probe according to claim 1, characterized in that, In step (5), the volume ratio of bovine serum albumin to silver-palladium-platinum hollow alloy nanoparticle solution in step (4) is 1:10; the incubation time is 20-30min.

7. The method for preparing a hollow alloy nanoparticle detection probe according to claim 1, characterized in that, In step (5), after incubation, centrifuging at 10000g and 4℃ for 5-10min.

8. The method for preparing a hollow alloy nanoparticle detection probe according to claim 1, characterized in that, In step (6), the reconstitution solution is 50mM bovine serum albumin; the volume ratio of reconstitution solution to silver-palladium-platinum hollow alloy nanoparticle solution in step (4) is 3:

10.

9. The detection probe prepared by the preparation method of the hollow alloy nanoparticle detection probe according to any one of claims 1-8.

10. The detection probe prepared by the preparation method of the hollow alloy nanoparticle detection probe according to any one of claims 1-8 for detecting iron bacteria in a petroleum pipeline.

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