Gold-silver alloy nano-probe based on dual-signal collaborative detection, preparation method and application of gold-silver alloy nano-probe

Abstract

The invention discloses a gold-silver alloy nanoprobe based on double-signal cooperative detection, a preparation method and application thereof, and belongs to the technical field of sensing. Then mixing the gold nanorod, a Raman report molecule p-methylthiobenzonitrile and a chiral stimulating factor L-cysteine, and jointly incubating at room temperature to obtain a mixed solution; sequentially adding silver nitrate, chloroauric acid tetrahydrate and ascorbic acid into the mixed solution, and magnetically stirring; and placing the mixed solution under a constant temperature condition to react for a certain time, so that the gold-silver alloy shell fully grows, and finally obtaining the spiral structure alloy nanoprobe with Raman signals and CD signals. The probe realizes the dual detection functions of Raman signals and circular dichroism signals, and can detect the lactic acid concentration with high sensitivity and high selectivity by combining the specific catalytic action of the lactate oxidase, and meanwhile, the Raman signals and CD signals of the silent zone can further expand the application range of the probe.

Description

Technical Field

[0001] The present invention belongs to the field of sensor technology, and in particular relates to a gold-silver alloy nanoprobe based on dual-signal collaborative detection, a preparation method and an application thereof. Background Art

[0002] As an important biomarker, lactate has a wide range of application values ​​in medical diagnosis, exercise physiology, and food industry. For example, in the medical field, abnormally elevated lactate concentrations are closely related to a variety of diseases (such as sepsis, heart failure, and cancer); in exercise physiology, lactate levels are an important indicator for assessing exercise intensity and fatigue; in the food industry, lactate content directly affects food quality and safety. Therefore, the development of a rapid, sensitive, and reliable lactate detection method is of great practical significance.

[0003] At present, the detection methods of lactate mainly cover multiple fields such as electrochemical method, chromatography and optical sensing method. As traditional means of lactate detection, electrochemical method and chromatography have obvious limitations in practical applications due to their inherent invasive characteristics, and cannot detect the dynamic changes of lactate in some biological samples. Optical sensing has attracted widespread attention due to its significant advantages such as non-destructiveness, high sensitivity and rapid response. However, existing optical sensing technology still faces many challenges in achieving efficient and accurate detection. Taking the fluorescence detection method as an example, its inherent photobleaching phenomenon not only limits the continuity and stability of the detection, but also seriously affects the real-time monitoring of the dynamic changes of lactate. At the same time, biological tissues themselves have spontaneous fluorescence signals, which will overlap with the imaging signals of fluorescent probes, causing great interference to the analysis. In addition, detection methods based on single signal output are often easily interfered by environmental factors, making it difficult to ensure the reliability and repeatability of the detection results. Summary of the invention

[0004] In view of the above-mentioned defects existing in the prior art, the present invention provides a gold-silver alloy nanoprobe based on dual-signal synergistic detection, a preparation method and an application thereof. The probe uses gold nanorods as seeds and prepares core-shell structured nanoparticles having a gold core and a gold-silver alloy shell through a controllable synthesis process; the Raman reporter molecule p-methylthiobenzonitrile (4-MBN) and the chiral stimulator L-cysteine ​​(L-Cys) are co-encapsulated inside the core-shell structure. The design of the core-shell structure produces a significant electromagnetic field enhancement effect at the nanoscale, namely the so-called "hot spot" effect, so that the nanoparticles can simultaneously generate enhanced Raman scattering signals and significant CD signals. This dual signal enhancement mechanism enables the probe to have dual detection functions. Combined with lactate oxidase, it can realize qualitative and quantitative detection of lactic acid; the Raman signal and CD signal generated by the nanoparticle are both in the biological silent zone. First, in terms of Raman spectroscopy, the signal appears at 1800-2800 cm -1 The biological silent zone effectively avoids the characteristic Raman peaks of biological molecules (such as proteins, lipids and nucleic acids), significantly reducing biological background interference; secondly, in circular dichroism detection, the signal is located in the visible light region (usually 400-700 nm), avoiding the absorption band of common chromophores in biological samples; this dual silent zone characteristic gives the nanoprobe a unique advantage in the detection of complex biological samples, not only greatly improving the signal-to-noise ratio of the detection, but also ensuring the accuracy and reliability of the detection results. The preparation process of this method is simple and controllable, the detection response is fast, and the dual-signal synergistic detection is accurate and reliable. It can achieve high-sensitivity detection of lactic acid under the premise of eliminating background interference.

[0005] The present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a method for preparing a gold-silver alloy nanoprobe based on dual-signal collaborative detection, which specifically comprises the following steps: Step 1: Preparation of gold nanorods; Step 2: Mix the gold nanorods, the Raman reporter molecule p-methylthiobenzonitrile (4-MBN) and the chiral stimulator L-cysteine ​​(L-Cys) according to a certain amount of substances, and incubate them together at room temperature for at least 3 hours to obtain a mixed solution; Step 3: adding silver nitrate, chloroauric acid tetrahydrate and ascorbic acid to the mixed solution of step 2 in sequence, and performing magnetic stirring; Step 4: placing the mixed solution obtained in step 3 under constant temperature conditions to react for a certain period of time to allow the gold-silver alloy shell to fully grow, and finally obtaining a spiral structure alloy nanoprobe with Raman signal and CD signal.

[0006] Furthermore, in step 2, the molar ratio of the gold nanorods, the Raman reporter molecule p-methylthiobenzonitrile and the chiral stimulator L-cysteine ​​is 100-200:1:1.

[0007] Furthermore, in step three, the molar ratio of gold nanorods, silver nitrate, chloroauric acid and ascorbic acid is 100-200:1:1:200.

[0008] Furthermore, in step three, the magnetic stirring condition is stirring at 1500 rpm for 2 min.

[0009] Furthermore, in step 4, the constant temperature condition is 65-80°C, and the certain time is 1 hour.

[0010] In a second aspect, the present invention provides a gold-silver alloy nanoprobe based on dual-signal collaborative detection, which is prepared by the method described in the first aspect.

[0011] In the third aspect, the present invention provides an application of a gold-silver alloy nanoprobe based on dual-signal collaborative detection in lactate detection. Specifically, the prepared nanoprobe is fully mixed with lactate oxidase in a volume ratio of 10-20:1-5 to obtain a lactate detection system for lactate detection.

[0012] The principle of quantitative detection of lactic acid in the present invention is as follows: The present invention realizes the synergistic detection of dual-function optical signals by encapsulating the Raman reporter molecule 4-MBN and the chiral stimulator L-Cys in a nanoprobe; 4-MBN gives the probe a characteristic Raman signal, while L-Cys induces a significant circular dichroism (CD) signal. The designed gold-silver alloy shell not only provides a stable nanogap structure, but also forms a strong and uniform plasma hotspot, which plays a key role in achieving quantitative and repeatable optical signal enhancement. During the detection process, when lactic acid is present in the sample to be tested, lactic acid reacts with lactate oxidase to generate hydrogen peroxide (H2O2). Since silver has a high reactivity to H2O2, the silver component of the nanoprobe will be selectively etched, resulting in a change in the nanogap structure, thereby reducing the intensity of the plasma hotspot. This process is directly reflected in the synchronous attenuation of the Raman signal and the CD signal. By real-time monitoring of the changes in the intensity of the dual signals, the accurate determination of the lactic acid concentration can be achieved.

[0013] Compared with the prior art, the advantages of the present invention are as follows: The gold-silver alloy nanoprobe based on dual-signal collaborative detection, preparation method and application thereof of the present invention realizes the dual detection function of Raman signal and circular dichroism signal by co-encapsulating Raman reporter molecule and chiral stimulating factor in the nanoprobe, and can detect lactic acid concentration with high sensitivity and high selectivity in combination with the specific catalytic action of lactate oxidase, while the Raman signal and CD signal in the silent zone can further expand the application scope of the probe. The present invention not only has a simple and controllable preparation process, but also significantly improves the accuracy and reliability of detection through dual-signal collaborative detection, and has significant creativity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0015] Figure 1 A schematic diagram showing the structure and optical properties of the nanoprobe prepared in the present invention; Among them, (a) transmission electron microscopy (TEM) image, showing the morphology and structural characteristics of the nanoprobe; (b) CD spectrum; (c) Raman spectrum; Figure 2 This is a characterization diagram of the response performance of the nanoprobe of the present invention to hydrogen peroxide; Figure 3 The characterization result of the qualitative analysis of lactic acid by the lactic acid detection system of the present invention; Among them, (a) is a comparison diagram of Raman spectra before and after the reaction between the detection system and the lactic acid standard; (b) is a comparison diagram of CD spectra before and after the reaction between the detection system and the lactic acid standard; Figure 4 Schematic diagram of Raman spectrum response characteristics of the lactic acid detection system of the present invention; Among them, (a) Raman spectra changes after different concentrations of lactic acid solution (0-100 μM) interacted with the detection system; (b) 2226 cm -1 The linear relationship curve between Raman signal intensity and lactic acid concentration; Figure 5 It is a schematic diagram of the circular dichroism spectral response characteristics of the lactic acid detection system of the present invention; Among them, (a) the changes in the circular dichroism spectra after the detection system was exposed to different concentrations of lactic acid solutions (0-90 μM); (b) the linear relationship curve between the circular dichroism signal intensity and lactic acid concentration at 514 nm; Figure 6A schematic diagram of the anti-interference performance of the lactic acid detection system of the present invention in complex samples; Among them, (a) the Raman spectrum change of lactic acid sample containing hydrogen peroxide interference after the detection system interacts with it; (b) 2226 cm -1 Quantitative comparison of Raman signal intensities at . DETAILED DESCRIPTION

[0016] In order to clearly and completely describe the technical solution and its specific working process of the present invention, the specific implementation methods of the present invention are as follows in conjunction with the accompanying drawings of the specification: Embodiment 1: This embodiment provides a method for preparing a gold-silver alloy nanoprobe based on dual-signal collaborative detection, which specifically includes the following steps: Take 10 mL of 0.1 mM gold nanorod solution, add 1 mL of mixed solution containing L-Cys and 4-MBN (L-Cys and 4-MBN concentrations are both 40 μM), stir and react at 30°C for at least 3 hours to ensure that the molecules are fully modified; then add 70 μL of 10 mM silver nitrate solution and 191 μL of 6.8 mM chloroauric acid solution in sequence, and continue stirring for 15-30 minutes to fully disperse the precursor; then stir at 1500 rpm in a magnetic stirrer for 2 minutes, add 200 μL of 0.1 M ascorbic acid solution, and continue stirring for 2 minutes; transfer the mixed solution to a 70°C constant temperature water bath for reaction for 30 minutes to complete the growth of the gold-silver alloy shell; after the reaction is completed, centrifuge and wash three times at 6000 rpm to remove unreacted reagents to obtain a pure nanoprobe solution; the morphology and optical properties of the nanoprobe are shown in Figure 1 shown.

[0017] Figure 1 (a) shows the transmission electron microscope (TEM) image of the nanoprobe, which clearly reveals its unique spiral surface morphology. This characteristic structure is the structural basis for the nanoprobe to generate circular dichroism (CD) signals. Figure 1 (b) shows the CD spectrum of the nanoprobe. Two obvious characteristic peaks can be observed in the visible light region, located at 514 nm and 580 nm, respectively, which further confirms the optical properties of the nanoprobe and its applicability in biological detection.

[0018] Figure 1 (c) shows the Raman spectrum analysis results of the nanoprobe. -1 ) was observed at 2226 cm -1 The characteristic peak corresponds to the stretching vibration mode of cyano (C≡N). The existence of this characteristic peak indicates that the nanoprobe has significant application potential in the detection of complex biological samples and can effectively avoid the interference of biological background signals.

[0019] Embodiment 2: Taking Raman spectroscopy as an example, the feasibility of using nanoprobes to detect lactic acid was verified.

[0020] The steps for verifying the ability of the nanoprobe to respond to H2O2 are as follows: 200 μL of the prepared nanoprobe solution was mixed with 0-100 μM H2O2 standard solution in a volume ratio of 1:2, and reacted at room temperature for 30 min. The results were then detected using an ATR3110 portable Raman spectrometer. The detection parameters were set as follows: excitation wavelength 785 nm, laser power 180 mW, and integration time 10 s. Figure 2 As shown, at 2226 cm -1 At the characteristic peak, the Raman signal intensity gradually decreases with the increase of H2O2 concentration, indicating that the nanoprobe has an excellent concentration-dependent response to H2O2. Lactic acid can react with lactate oxidase to generate H2O2. Based on this principle, it is feasible to detect lactic acid by combining the nanoprobe with lactate oxidase and detecting the amount of H2O2 generated.

[0021] Embodiment 3: Qualitative analysis of lactic acid; first, construct a lactic acid detection system, take 200 μL of the prepared nanoprobe solution, add 700 μL of PBS buffer solution (pH 7.4), then add 100 μL of 20 U / mL lactate oxidase solution, and mix thoroughly to obtain a lactic acid detection system; prepare a lactic acid solution with a concentration of 3 mM, mix the PBS buffer solution (blank control) and lactic acid solution with the detection system at a volume ratio of 1:18, and after fully reacting at 37 °C for 30 min, perform Raman spectroscopy and CD spectroscopy analysis on the two mixed solutions. Figure 3 As shown in the figure, compared with the blank control group, the Raman signal and CD signal of the sample containing lactic acid decreased significantly. This phenomenon confirms that the detection system can not only realize the qualitative identification of lactic acid, but also show excellent dual-signal synergistic detection ability.

[0022] The Raman spectrum was detected by ATR3110 portable Raman spectrometer from AOPUS, and the detection parameters were set as follows: excitation wavelength 785 nm, laser power 180 mW, and integration time 10 s. The CD spectrum was scanned and detected in the 400-700 nm band using Chirascan CD spectrometer from Applied Photophysics, UK.

[0023] Embodiment 4: This embodiment provides an application of a gold-silver alloy nanoprobe based on dual-signal synergistic detection in lactic acid detection. First, a lactic acid detection system is constructed. Specifically, 200 μL of the prepared nanoprobe solution is added to 700 μL of PBS buffer solution (pH 7.4), and then 100 μL of a 20 U / mL lactate oxidase solution is added. After sufficient mixing, a lactic acid detection system is obtained; the system can be stored at 4 °C and needs to be restored to room temperature before use. By optimizing the volume ratio of lactic acid sample to detection system, high-sensitivity detection of lower concentration lactic acid samples can be further achieved.

[0024] Raman spectroscopy detection performance characterization; A lactic acid standard solution with a concentration gradient of 0-100 μM was prepared (the solvent was PBS buffer solution, pH 7.4). The lactic acid solution and the lactic acid detection system were mixed in a volume ratio of 1:9 and reacted at 37 °C for 30 minutes. The detection was performed using an ATR3110 portable Raman spectrometer from AOPUS. The detection parameters were set as follows: excitation wavelength 785 nm, laser power 180 mW, integration time 10 s. The experimental results showed that at 2226 cm -1 At the characteristic peak, the Raman signal intensity has a good linear negative correlation with the lactic acid concentration, such as Figure 4 As shown, the quantitative detection capability of the detection system for lactate concentration was confirmed.

[0025] Characterization of circular dichroism spectroscopy detection performance; Prepare a lactic acid standard solution with a concentration gradient of 0-90 μM (the solvent is PBS buffer solution, pH 7.4). Mix the lactic acid solution and the lactic acid detection system in a volume ratio of 1:9 and react at 37°C for 30 minutes. Use the Chirascan CD spectrometer of Applied Photophysics in the UK to scan and detect in the 400-700 nm band. The experimental results show that at the characteristic wavelength of 514 nm, the circular dichroism signal intensity is significantly linearly negatively correlated with the lactic acid concentration, such as Figure 5 As shown, the dual-signal detection reliability of the detection system is further verified.

[0026] Validation of actual sample testing methods; Considering that actual samples such as serum may contain reactive oxygen species such as H2O2, which will have a non-specific etching effect on the silver component of the nanoprobe, this embodiment uses a two-step detection method to ensure the specificity of the detection. The specific operation is as follows: First, prepare a mixed simulation solution containing 0.1 mM hydrogen peroxide and 3 mM lactic acid, mix the simulation solution with the nanoprobe in a volume ratio of 1:9, and react for at least 30 minutes to ensure signal stability; then add 100 μL of lactate oxidase solution and continue the reaction for 30 minutes. The ATR3110 portable Raman spectrometer was used for detection, and the experimental results showed that Figure 6 As shown, after the addition of lactate oxidase, the optical signal of the probe decreased significantly, which confirmed that the system can effectively distinguish the signal changes caused by lactate-specific reactions, thereby achieving accurate detection of complex real samples.

[0027] The preferred embodiments of the present invention are described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0028] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0029] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for preparing a gold-silver alloy nanoprobe based on dual-signal collaborative detection, characterized in that: The specific steps include: Step 1: Preparation of gold nanorods; Step 2: Mix the gold nanorods, the Raman reporter molecule p-methylthiobenzonitrile and the chiral stimulator L-cysteine ​​according to a certain amount of substances, and incubate them together at room temperature for at least 3 hours to obtain a mixed solution; Step 3: adding silver nitrate, chloroauric acid tetrahydrate and ascorbic acid to the mixed solution of step 2 in sequence, and performing magnetic stirring; Step 4: placing the mixed solution obtained in step 3 under constant temperature conditions to react for a certain period of time to allow the gold-silver alloy shell to fully grow, and finally obtaining a spiral structure alloy nanoprobe with Raman signal and CD signal.

2. The method for preparing a gold-silver alloy nanoprobe based on dual-signal collaborative detection according to claim 1, characterized in that: In step 2, the molar ratio of the gold nanorods, the Raman reporter molecule p-methylthiobenzonitrile and the chiral stimulator L-cysteine ​​is 100-200:1:

1.

3. The method for preparing a gold-silver alloy nanoprobe based on dual-signal collaborative detection according to claim 1, characterized in that: In step 3, the molar ratio of gold nanorods, silver nitrate, chloroauric acid and ascorbic acid is 100-200:1:1:

200.

4. The method for preparing a gold-silver alloy nanoprobe based on dual-signal collaborative detection according to claim 1, characterized in that: In step 3, the magnetic stirring condition is stirring at 1500 rpm for 2 min.

5. The method for preparing a gold-silver alloy nanoprobe based on dual-signal collaborative detection according to claim 1, characterized in that: In step 4, the constant temperature condition is 65-80°C and the certain time is 1 hour.

6. A gold-silver alloy nanoprobe based on dual-signal collaborative detection, characterized in that: It is prepared by the method according to any one of claims 1 to 5.

7. The use of a gold-silver alloy nanoprobe based on dual-signal coordinated detection in lactic acid detection as claimed in claim 6, characterized in that: Specifically, the prepared nanoprobe is fully mixed with lactate oxidase in a volume ratio of 10-20:1-5 to obtain a lactate detection system for lactate detection.

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