Preparation method of a sweat pH sensing SERS chip based on core-shell internal standard nanoparticles

Abstract

This invention discloses a method for preparing Au@1,4-BDT@Au@4-MBA core-shell structured nanoparticles with sub-nanometer gaps. The method primarily uses a thiol molecule with a benzene ring as an internal standard, specifically 1,4-benzenedithiophenol (1,4-BDT), precisely encapsulated within the gap between a gold core and a gold shell. The gold core has a diameter of 35–40 nm, and the gold shell has a diameter of 45–60 nm, resulting in an overall particle size of 80–100 nm. The internal standard molecule located in the sub-nanometer gap layer generates a very strong electromagnetic enhancement signal, and because it is physically isolated by the outer gold shell layer, its signal is unaffected by external pH environments or solution matrices, providing an excellent reference. An external standard molecule (4-MBA) is then used for the outermost coating, serving as an acid-base sensing unit. A ratiometric calibration model is established based on the ratio of its characteristic peak to the internal standard peak, effectively reducing errors caused by uneven hotspots and matrix interference. This material can be applied to pH detection of human sweat based on SERS technology.

Description

Technical Field

[0001] This invention relates to a core-shell internal standard nanoparticle and its preparation method, particularly an Au@1,4-BDT@Au@4-MBA nanoparticle and its preparation method. Background Technology

[0002] Various methods exist for detecting sweat pH, including traditional colorimetric and electrochemical analysis. While colorimetric methods are simple, they suffer from limitations in quantitative accuracy and resistance to environmental interference. Electrochemical sensors, despite their high sensitivity, are highly susceptible to interference from complex biomolecules in sweat, leading to electrode surface contamination. In contrast, surface-enhanced Raman scattering (SERS), with its ultra-high sensitivity and unique molecular fingerprint recognition characteristics, shows great potential in the analysis of trace biological samples. To monitor pH, studies often introduce pH-sensitive probe molecules such as 4-mercaptobenzoic acid (4-MBA), 4-mercaptopyridine (4-MPY), or 4-aminothiophenol (4-ATP). pH is monitored by observing the changes in peak intensity and position caused by the varying degrees of protonation and deprotonation at different pH levels. However, these conventional single-probe sensors often exhibit "non-structural errors" during detection. Specifically, in complex sweat matrices, single-probe SERS sensors are prone to false positives or inaccurate signal fluctuations due to uneven distribution of plasmon "hot spots" and fluctuations in the detection environment (such as excitation light intensity and sampling location). This makes reliable quantitative analysis difficult using a single peak intensity or a single probe molecule, severely limiting the standardized application of surface-enhanced Raman scattering (SERS) technology in precise pH analysis. This invention addresses this by constructing a "core-gap-shell" structure, utilizing a physically isolated internal standard signal as a reference, fundamentally eliminating environmental noise interference. The internal standard molecule is precisely encapsulated within the gap between the gold core and the gold shell. This structure's advantage lies in generating a very strong electromagnetic enhancement signal from the internal standard molecule located in the sub-nanometer gap layer, unaffected by external factors. This multi-layered structure significantly increases the plasmon hot spot area. This solves the problem of "non-structural errors" caused by single probes, achieving accurate measurement of the desired Raman signal. This method is simple to synthesize, has good repeatability, and can be applied to human sweat pH detection based on SERS technology. Summary of the Invention

[0003] To address the aforementioned issues, this invention proposes Au@1,4-BDT@Au@4-MBA nanoparticles and their preparation method. The core advantage of this structure lies in the introduction of a pH probe internal standard calibration mechanism. The internal standard is encapsulated within the gap between the gold core and the gold shell. This nanoscale gap generates a very strong electromagnetic field, enabling the internal standard molecules located in the sub-nanometer gap layer to produce a strongly enhanced electromagnetic signal. Furthermore, the internal standard is encapsulated by an outer metal layer, ensuring its signal is unaffected by external pH environments or solution matrices, effectively canceling out external environmental noise interference. The external standard molecule (4-MBA) serves as an acid-base sensing unit. A ratiometric calibration model is established based on the ratio of its characteristic peak to the internal standard peak, effectively reducing errors caused by uneven hotspot distribution and matrix interference.

[0004] The present invention aims to solve the above-mentioned technical problems by providing Au@1,4-BDT@Au@4-MBA nanoparticles and their preparation method.

[0005] To solve the technical problem of this invention, the technical solution adopted is: Au@1,4-BDT@Au@4-MBA nanoparticles, particularly:

[0006] The Au@1,4-BDT@Au@4-MBA nanoparticles are based on an internal standard (1,4-BDT), specifically a thiol molecule with a benzene ring as the internal standard. 4-MBA, containing a carboxyl group, serves as the external standard and acts as an acid-base sensing unit for sweat pH detection.

[0007] The Au@1,4-BDT@Au is a gold sphere with a core-shell structure, wherein the gold core has a diameter of 35-40 nm, the gold shell has a diameter of 45-60 nm, and the overall Au@1,4-BDT@Au core-shell structure has a diameter of 80-100 nm.

[0008] The above-mentioned Au@1,4-BDT@Au@4-MBA method is for preparing core-shell structured gold spheres, and the specific steps are as follows:

[0009] Step 1: Prepare a solution of HAuCl4 and hexadecyltrimethylammonium chloride (CTAC) at a ratio of 1:100. Pour this solution into a prepared NaBH4 solution in ice water while stirring vigorously. After 3 minutes, dilute the gold seed solution 10 times in CTAC. Add 700–900 μL of the diluted seed solution and ascorbic acid (AA) to the CTAC solution. Pour in the HAuCl4 solution while stirring vigorously. Let the mixture stand for at least 10 minutes to obtain 8–10 nm gold spheres.

[0010] Step 2: Subsequently, CTAC, 8-10 nm gold spheres, and AA solution were mixed thoroughly at a volume ratio of 180–210:0.8–1.1:0.6–1. HAuCl4 was added dropwise over ten minutes, and the mixture was stirred for 1 hour. Finally, 200 μL of NaClO was added under rapid stirring, followed by HAuCl4 after 5 minutes. The mixture was stirred continuously for 45 minutes to obtain a solution of 35–40 nm gold spheres.

[0011] Step 3: Take an appropriate amount of 35-40 nm gold balls and centrifuge twice to reduce the CTAC concentration. Then add 1,4-BDT and stir for 0.5 h. Centrifuge three times to remove excess molecules and disperse in 0.1 mM CTAC solution for later use, to obtain Au@1,4-BDT.

[0012] Step 4: Add HAuCl4 to 10 mL of CTAC solution, add 500 μL of AA while stirring, and then add Au@1,4-BDT obtained in Step 3 and stir for 30 min. Centrifuge three times and dilute to CTAC, add 100 μL of 4-MBA and stir for 1 h to obtain Au@1,4-BDT@Au@4-MBA.

[0013] Step 5: Prepare Au@1,4-BDT@Au@4-MBA in Step 4 with 1% PVP ethanol solution at a volume ratio of 1–1.3:5–5.2. Incubate on a shaker for 30 min, then centrifuge and wash twice. Add dichloromethane and shake vigorously. After separation, add cyclohexane. Once a gold film appears, remove the cyclohexane and carefully transfer the gold film onto the silicon wafer using tweezers. Thus, Au@1,4-BDT@Au@4-MBA core-shell structured nanoparticles have been successfully prepared.

[0014] Compared to existing technologies, the advantages are as follows: First, by constructing a "shell-gap-core" structure and using a physically isolated internal standard signal as a reference, environmental noise interference is fundamentally eliminated. This ensures the signal is unaffected by external pH environments or solution matrices, providing an excellent reference. Furthermore, an external standard molecule (4-MBA) is used for the outermost coating, serving as an acid-base sensing unit. A ratiometric calibration model is established based on the ratio of its characteristic peak to the internal standard peak, effectively reducing errors caused by uneven hotspots and matrix interference. Second, the internal standard molecule is precisely encapsulated in the gap between the gold core and the gold shell. This structure has the advantage of generating a very strong electromagnetic field in the nano-gap, resulting in a very strong electromagnetic enhancement signal from the internal standard molecule located in the sub-nanometer gap layer, unaffected by external factors. This multi-layered structure significantly increases the plasmon hotspot region. Third, it solves the problem of "non-structural errors of single probes," achieving accurate measurement of the required Raman signal. This method is simple to synthesize, has good repeatability, and when applied to the pH detection of human sweat based on SERS technology, it has a wide pH detection range (pH: 1-10). Attached Figure Description

[0015] To more clearly illustrate the technical solutions in this invention, the accompanying drawings are briefly described below.

[0016] Figure 1 The results of characterization of the Au@1,4-BDT@Au nanoparticles synthesized in step 4 using scanning electron microscopy (SEM) are shown. Figure 1 In the image, the scanning electron microscope (SEM) icon has a bar of 100 nm.

[0017] Figure 2 The results of characterization of the Au@1,4-BDT@Au nanoparticles synthesized in step 4 using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) are shown. Figure 2 In the figure, the HAADF-STEM icon has a bar of 20 nm.

[0018] Figure 3 To conduct an anti-interference experiment on various interfering ions present in sweat at pH 4 using Au@1,4-BDT@Au@4-MBA.

[0019] Figure 4 The linear change in pH (1-10) of the obtained target product Au@1,4-BDT@Au@4-MBA was detected using Raman spectroscopy.

[0020] Figure 5 The paper demonstrates the detection of pH (4.5–7) Raman spectral signals in real human sweat. Detailed Implementation

[0021] In the embodiments of the present invention, 1,4-BDT is used as an internal standard molecule and 4-MBA is used as an external standard molecule.

[0022] The following embodiments will further illustrate the present invention with reference to the accompanying drawings.

[0023] Example 1: Synthesis of a solution of gold nanoparticles with a particle size of 40 nm, the specific steps are as follows:

[0024] (1) Add 50 μL of the prepared HAuCl4 solution to 5 mL of CTAC solution, and then pour in the prepared NaBH4 solution in ice water while stirring vigorously. After 3 minutes, dilute the gold seed solution 10 times in 100 mM CTAC, and add 700–900 μL of the diluted seed solution and 40 μL of ascorbic acid to 10 mL of CTAC solution. Pour in 50 μL of HAuCl4 solution while stirring vigorously. Let the mixture stand for at least 10 minutes to obtain 8–10 nm gold spheres; then mix 200 mL of CTAC, 700–900 μL of 8–10 nm gold spheres and 800–900 μL of AA solution evenly, and add HAuCl4 dropwise over 10 minutes, stirring for 1 h. Finally, add 200 μL of NaClO while stirring rapidly, and add HAuCl4 after 5 min, and continue stirring for 45 min to obtain a 35–40 nm gold sphere solution.

[0025] (2) In the above-obtained gold nanoparticle solution, an appropriate amount of 40 nm gold balls was centrifuged twice to reduce the CTAC concentration. Then, 1,4-BDT was added and stirred for 0.5 h. The mixture was centrifuged three times to remove excess molecules and dispersed in 0.1 mM CTAC solution for later use, thus obtaining Au@1,4-BDT.

[0026] (3) Add HAuCl4 to 0.1M CTAC solution, add ascorbic acid under stirring, and then add Au@1,4-BDT obtained in (2) and stir for 30 min. Centrifuge three times and adjust the volume to 0.1 M CTAC, add 100 μL of 4-MBA and stir for 1 h to obtain Au@1,4-BDT@Au@4-MBA core-shell structured nanoparticles. Characterize using scanning electron microscopy (SEM) to obtain... Figure 1 .

[0027] Example 2: Figure 2These images, characterized using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), clearly show that 1,4-BDT, as an internal standard, is precisely encapsulated in the gap between the gold core and the gold shell, with a uniform gap distribution and no metal bridging observed. The gold core has a diameter of 35–40 nm, and the gold shell has a diameter of 45–60 nm, with an overall particle size of 80–100 nm.

[0028] Example 3: Figure 3 This study employed Raman spectroscopy at pH 4 to detect interference ions present in sweat. Sweat is extremely complex, containing abundant electrolytes such as Na⁺, K⁺, and Ca²⁺, metabolic products such as lactic acid and urea, and various amino acids. These components readily interfere with the SERS signal through competitive adsorption or alteration of local refractive index. Multiple interfering ions were introduced for anti-interference experiments, all at a concentration of 0.01 M, far exceeding normal sweat levels. This near-immune anti-interference performance is primarily attributed to the "core-internal standard-shell" structure used in this study. The BDT molecules encapsulated within the gold shell serve as a stable signal reference, effectively offsetting systemic biases caused by solution refractive index fluctuations, ion competitive adsorption, or optical path deviations, thus demonstrating the chip's ability to detect complex matrices.

[0029] Example 4: Figure 4 The results of ratiometric fitting of pH (1-10) based on internal standard calibration are presented, showing obvious "piecewise linearity". The extremely high linear correlation coefficients in the two intervals confirm that the substrate has reliable quantitative analysis over a very wide range.

[0030] Example 5: Figure 5 The study used Raman spectroscopy to detect artificial sweat from real human sweat, demonstrating the evolution of Raman spectra in artificial sweat at different pH values ​​(4.5–7). It was observed that even in matrices containing lactic acid, urea, and high salt concentrations, the characteristic peaks of 4-MBA and BDT remained clearly distinguishable, exhibiting good signal stability. This demonstrates that the Au@1,4-BDT@Au@4-MBA design can accurately detect the physiological pH range (4.5–7) of human sweat.

Claims

1. A core-shell structured nanoparticle of Au@1,4-BDT@Au@4-MBA with sub-nanometer gaps, characterized in that: The sensor employs an Au@1,4-BDT@Au@4-MBA core-shell structure, in which 1,4-benzenedithiophenol (1,4-BDT) is precisely encapsulated as an internal standard molecule within the sub-nanometer gap between the gold core and the gold shell. 4-Mercaptobenzoic acid (4-MBA) was used as a pH-sensitive probe molecule to modify the outermost layer of the gold shell; A ratiometric pH monitoring model, unaffected by environmental interference, was constructed by utilizing the intensity ratio of the characteristic Raman peaks of internal standard molecules and probe molecules.

2. The preparation method according to claim 1, characterized in that step include: Step 1. Preparation of gold nanospheres by seed growth method: Gold seeds are obtained by injecting NaBH4 into a solution containing HAuCl4 and hexadecyltrimethylammonium chloride (CTAC) for reduction, and then gold nanosphere substrates with a diameter of 35~40 nm are obtained by secondary growth. Step 2. Self-assembly of internal standard molecules: 1,4-BDT molecules are modified onto the surface of the gold nanospheres, and excess molecules are removed by centrifugation and washing to form a saturated monolayer modified Au@1,4-BDT intermediate; Step 3. Sub-nanometer gap gold shell coating: Under the action of ascorbic acid (AA), the gold precursor is reduced in situ on the Au@1,4-BDT surface to form a continuous gold shell with a diameter of 45~60 nm, which physically isolates the internal standard molecules in the sub-nanometer gap. Step 4. External standard molecule modification: 4-MBA molecules are loaded onto the gold shell surface to obtain SERS active particles with a core-shell structure.

3. The method for preparing Au@1,4-BDT@Au@4-MBA as claimed in claim 2 is characterized in that... In step 1, a NaBH4 solution needs to be prepared in ice water.

4. The method for preparing Au@1,4-BDT@Au@4-MBA as claimed in claim 2 is characterized in that... During the secondary growth of the gold spheres in step 1, 1 mL of HAuCl is added dropwise at a uniform rate over ten minutes. 4。 5. The method for preparing Au@1,4-BDT@Au@4-MBA according to claim 2, characterized in that... In step 2, the 35-40 nm gold sphere solution is coated with an aromatic thiol internal standard molecule with a stable Raman scattering cross section.

6. The method for preparing Au@1,4-BDT@Au@4-MBA according to claim 2 is characterized in that... In step 3, the growth of the gold shell physically isolates the internal standard molecules within the sub-nanometer gaps.

7. The method for preparing Au@1,4-BDT@Au@4-MBA according to claim 2 is characterized in that... In step 4, thiol molecules with pH-responsive properties, such as 4-MBA, 4-MPY, or 4-ATP, are used for modification.

8. The method for preparing Au@1,4-BDT@Au@4-MBA according to claim 2, characterized in that: The stirring temperature is 30℃.

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