A liquid-phase SERS internal standard ratio quantitative detection method based on an integrating sphere independent double-cell detection system

Abstract

The application discloses a liquid-phase SERS internal standard ratio quantitative detection method based on an integral sphere independent double-pool detection system. The method utilizes two transparent containers which are in close contact and physically separated and are arranged in the integral sphere independent double-pool detection system, and the two containers accommodate a to-be-detected liquid sample and an internal standard liquid sample respectively; during detection, excitation laser simultaneously passes through the two containers, so that the to-be-detected sample and the internal standard sample synchronously generate SERS signals under the same light path condition; after the signals are acquired, the ratio of the internal standard characteristic peak intensity to the to-be-detected characteristic peak intensity is used for correction and quantitative analysis. The application has the advantages of high internal standard signal stability, high universality, suitability of the same internal standard for different to-be-detected substances, free regulation of the internal standard intensity, unlimited selection of the internal standard substance, simple operation, no need of surface modification, accurate ratio quantitative result and the like.

Description

Technical Field

[0001] This invention belongs to the field of Raman spectroscopy detection technology, specifically a quantitative detection method for the internal standard ratio of liquid phase SERS based on an integrating sphere independent dual-cell detection system. Background Technology

[0002] Surface-enhanced Raman scattering (SERS) technology can amplify the Raman signals of molecules adsorbed on rough metal surfaces such as gold and silver by several orders of magnitude, showing great potential in trace chemical analysis, biosensing, and environmental monitoring. However, SERS signal intensity is highly susceptible to complex factors such as fluctuations in excitation light power, the aggregation state of nanoparticles, minute changes in focusing position, and differences in sample matrix, making it difficult to directly use the absolute signal intensity for accurate quantitative analysis. Therefore, in SERS quantitative detection, an internal standard is usually introduced to correct the signal, and reliable quantification is achieved by using the ratio of the characteristic peak of the analyte to the characteristic peak of the internal standard.

[0003] Currently, the most commonly used internal standard strategy in liquid-phase SERS detection is to directly modify or co-adsorb internal standard molecules onto the surface of gold and silver nanoparticles, placing them in the same nanostructure environment as the analyte, and using the SERS signal of the internal standard molecule as a reference. (Cai, M.; Fan, J.; Wang, Q.; Zhang, Y.; Ma, Y.; Suo, J.; Zhang, M.; Zhang, S.; Chen, S. Triple Bond-Mediated Ratiometric Surface-Enhanced RamanScattering Nanoprobe for in Vitro and Ex Vivo Quantitative Monitoring of Mercury. Chemical Engineering Journal 2025, 515, 163427. https: / / doi.org / 10.1016 / j.cej.2025.163427.) While this method can partially compensate for the differences in reinforcement effects between particles in principle, it has a series of inherent drawbacks:

[0004] First, the internal standard signal has poor stability. The binding state of the internal standard molecule and the nanoparticle is highly dependent on the local physicochemical environment on the particle surface. The degree of particle aggregation, solution ionic strength, pH value, and coexisting substances can all cause the desorption, configuration change, or chemical transformation of the internal standard molecule, resulting in unpredictable fluctuations in the internal standard signal, which seriously limits the accuracy of ratio quantification.

[0005] Second, it lacks versatility. When detecting different analytes, the internal standard molecule may compete for adsorption or co-adsorb with the analyte, causing the peak intensity of the internal standard to vary with the type and concentration of the analyte. Therefore, the same internal standard system is difficult to apply to multiple analytes. If the analyte is changed, it is often necessary to re-screen suitable internal standard molecules and optimize co-modification conditions, increasing the difficulty of method development.

[0006] Third, the intensity of the internal standard is difficult to control. The intensity of the internal standard signal is determined by the coverage of internal standard molecules on the surface of the nanoparticles and the intrinsic scattering cross section, making it difficult to adjust flexibly according to the strength of the analyte signal. Once the surface modification is completed, the internal standard signal is basically fixed and cannot be freely controlled, making it difficult to meet the detection needs of different concentration ranges.

[0007] Fourth, the selection of internal standards is limited. Traditional strategies require that the internal standard molecule be able to stably anchor on the metal nanosurface and not interact uncontrollably with the analyte. This greatly limits the types of internal standards that can be used and is not conducive to the universality and standardization of the method.

[0008] Besides surface-modified internal standards, attempts have been made to utilize solvent characteristic peaks or external standards with separate optical paths. However, the former is often ineffective under SERS conditions because the solvent peak signal is inherently weak and easily interfered with; the latter, due to time-separated or component-separated measurements of the analyte and internal standard, cannot eliminate instantaneous fluctuations and optical path differences during the detection process, resulting in limited correction effects. Currently, no method has been achieved in liquid-phase SERS detection that allows the internal standard and analyte to be physically independent yet excited and acquired simultaneously along the same optical path.

[0009] In summary, existing liquid chromatography SERS internal standard techniques generally suffer from problems such as unstable internal standard signals, poor versatility, difficulty in adjusting the intensity as needed, and limited selection of internal standards. There is an urgent need to develop a new method for quantifying the internal standard ratio with an absolutely stable internal standard signal, no cross-interference with the analyte, flexible intensity adjustment, and applicability to different analytes, so as to meet the needs of accurate, universal, and convenient liquid chromatography SERS quantitative analysis. Summary of the Invention

[0010] The purpose of this invention is to provide a quantitative detection method for the internal standard ratio of liquid phase SERS based on an integrating sphere independent dual-cell detection system, in order to solve the problems of existing surface-modified internal standard methods, such as the internal standard signal being easily affected by the local physicochemical environment of nanoparticles and fluctuating, poor internal standard stability, insufficient versatility, difficulty in flexibly controlling the internal standard intensity, and severely limited selection of internal standard substances.

[0011] The technical solution for achieving the objective of this invention is as follows:

[0012] A method for quantitative detection of the internal standard ratio in liquid phase SERS based on an integrating sphere independent dual-cell detection system includes the following steps:

[0013] S1, Preparation and purification of bacterial cellulose nanomaterial BCN;

[0014] S2, Preparation of the specifically functionalized SERS substrate material BCN-AgNFs@DEB: A dilute sulfuric acid solution of BCN and a trioctylamine protective agent were added sequentially to a silver ammonia solution, followed by rapid ultrasonic treatment to fully emulsify the system into a homogeneous emulsion. The emulsion was placed in a constant temperature environment at 25 °C, and a hydrazine dispersion was rapidly injected as a reducing agent, followed by constant temperature stirring. After the reaction, the mixture was centrifuged and washed. An ethanol solution of 1,4-diethynylbenzene was added to the washed and purified centrifuged product, and the mixture was ultrasonically treated for 10 min for functionalization modification. The supernatant was removed by centrifugation again, and the solid product was washed. Finally, the modified solid product was uniformly dispersed in deionized water to obtain a BCN-AgNFs@DEB aqueous dispersion, which was then stored in a sealed container at low temperature for later use.

[0015] S3, construct Hg 2+ Quantitative detection standard curve: Prepare multiple sets of mercuric nitrate standard aqueous solutions with different concentration gradients as standard test samples; take 4 mL of each concentration of mercuric nitrate standard aqueous solution and place them in sealed glass bottles. Transfer 50 μL of BCN-AgNFs@DEB aqueous dispersion to each bottle, sonicate to uniformly disperse the substrate material, and let it stand at room temperature for a period of time to ensure that the substrate and Hg are in harmony. 2+ To ensure a fully specific reaction, a separate glass bottle containing 4 mL of organic solvent was used as an internal standard reference solution. The glass bottle containing the test standard solution and the glass bottle containing the organic solvent internal standard reference solution were placed side-by-side in an integrating sphere independent dual-cell detection system, and Raman spectra were acquired. Twenty spectra were repeatedly acquired for each sample group, and the average spectrum was used as the final detection spectrum. (The last sentence appears to be incomplete and possibly refers to a different method or method.) 2+ Logarithm of concentration (Hg) 2+ Using the concentration (μM) as the x-axis and the characteristic peak intensity ratio I / I as the y-axis, a linear relationship was fitted to establish Hg 2+ Quantitative detection standard curve;

[0016] S4, Hg in actual water sample 2+ Quantitative detection: Take 4 mL containing Hg 2+ The water sample to be tested was placed in a sealed glass bottle, and 50 μL of BCN-AgNFs@DEB aqueous dispersion was added. After ultrasonic mixing, the mixture was allowed to stand at room temperature for a period of time to ensure that the substrate material and Hg in the sample were homogeneous. 2+ Complete reaction; Raman spectra of the water sample and the organic solvent internal standard reference solution were simultaneously acquired using the same process and parameters as in step S3, and the intensity ratio I / I of the characteristic peaks of the water sample and the organic solvent internal standard reference solution was calculated; the obtained intensity ratio was substituted into the pre-constructed Hg... 2+ The standard curve for quantitative detection was used to calculate the Hg content in the water sample.2+ The actual concentration.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) The internal standard signal has extremely strong stability.

[0019] (2) High versatility, the same internal standard is applicable to different analytes.

[0020] (3) The strength of the internal standard can be freely adjusted.

[0021] (4) The choice of internal standard is not restricted.

[0022] (5) It is easy to operate and requires no surface decoration.

[0023] (6) The ratio quantification results are accurate. Attached Figure Description

[0024] Figure 1 In the figure, (a, b) represent SEM images of BCN-AgNFs at different magnifications; (c) represents the particle size distribution of BCN-AgNFs.

[0025] Figure 2 (a, b) SEM images of BCN-AgNFs@DEB at different magnifications; (c) Raman signals of BCN-AgNFs and BCN-AgNFs@DEB; (d) UV-Vis absorption spectra of BCN-AgNFs and BCN-AgNFs@DEB.

[0026] Figure 3 This is a schematic diagram of the detection method described in this invention.

[0027] Figure 4 The SERS signals for the same analyte (BCN-AgNFs@DEB) but different internal standards (benzene, dichloromethane, acetonitrile, N,N-dimethylformamide).

[0028] Figure 5 SERS signals for different analytes (ethanol, acetone, isopropanol, sodium sulfate, sodium carbonate) and the same internal standard (acetonitrile).

[0029] Figure 6 SERS signals for the same analyte (ethanol) with different concentrations of internal standard (0%, 20%, 50%, 70%, and 100% acetonitrile aqueous solution).

[0030] Figure 7 For different concentrations (10 -7 M, 5×10 -7 M, 10 -6 M, 2×10 -6 M, 5×10-6 M, 6×10 -6 M, 7×10 -6 M, 8×10 -6 M, 10 -5 M, 2×10 -5 M, 5×10 -5 M, 10 -4 M)Hg 2+ The SERS signal.

[0031] Figure 8 For I 2146 / I 2256 Value and Hg 2+ The relationship between concentrations (quantitative curve). Detailed Implementation

[0032] The technical solution of this application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely to help understand this disclosure and should not be regarded as specific limitations on this application.

[0033] It should be noted that, unless otherwise specified in the examples, the conditions should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0034] The reference to "embodiment" in this application means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments.

[0035] The technical term "and / or" in this application is only a description of the relationship between related objects, indicating that there can be three kinds of relationships. For example, A and / or B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0036] In addition, the character " / " in this application generally indicates that the objects before and after it are in an "or" relationship.

[0037] The integrating sphere independent dual-pool detection system described in this invention is built based on patent CN 108563006 A, as follows: Figure 3 As shown, this method utilizes two closely fitted and physically separated transparent containers within an integrating sphere independent dual-cell detection system to hold the liquid sample to be tested and the internal standard liquid sample, respectively. During detection, the excitation laser passes through both containers simultaneously, enabling the sample to be tested and the internal standard sample to generate SERS signals synchronously under the same optical path conditions. After acquiring the signals, the ratio of the characteristic peak intensity of the internal standard to the characteristic peak intensity of the sample to be tested is used for correction and quantitative analysis.

[0038] Preparation of BCN-AgNFs@DEB:

[0039] 1.5 g of bacterial cellulose (BC) was homogenized using a high-shear homogenizer and dispersed in 100 mL of deionized water. The homogenized BC solution was continuously stirred in an ice-water bath, and an equal volume of 65 wt% H₂SO₄ was slowly added. The mixture was then heated to 45 °C and stirred for 2 h. After cooling to room temperature, the solution was diluted with deionized water at a volume ratio of 1:4 and filtered through a 600-mesh polyester filter to remove un-acidified fibers. The filtrate was titrated with 1 mol / L KOH solution to pH 8, and dialyzed with deionized water to remove salts. The dialysate was freeze-dried to obtain purified BCN.

[0040] 0.021 g of silver nitrate was dissolved in 10 mL of deionized water. Ammonia was added dropwise while stirring until the precipitate was completely dissolved. Then, ammonia was slowly added to adjust the pH to 11, yielding a silver ammonia solution. 1 mL of a 10 wt% dilute sulfuric acid solution of BCN (0.01 g of BCN) and 2 mL of trioctylamine were added to this silver ammonia solution, and the mixture was sonicated for 30 s to form an emulsion. Additionally, 40 μL of hydrazine was dissolved in 10 mL of deionized water and stirred thoroughly to ensure uniform dispersion, preparing a hydrazine solution as a reducing agent. The emulsion was placed on a magnetic stirrer at 25°C and 500 rpm. The prepared hydrazine solution was rapidly added to the emulsion, and after reacting for 60 s, the solution was transferred to a 50 mL centrifuge tube. After centrifugation, the supernatant was discarded, and the mixture was washed with alternating ethanol and water for 3 cycles to obtain BCN-AgNFs. SEM and particle size distribution are shown below. Figure 1 As shown. Add 5 mL of 10 mL of [unspecified ingredient] to the centrifuged product. -3 A solution of 1,4-diethynylbenzene in ethanol was sonicated for 10 min. After centrifugation to remove the supernatant, the solution was washed twice with deionized water and dispersed in 5 mL of deionized water to obtain BCN-AgNFs@DEB, which was stored in a refrigerator for later use. SEM images are shown below. Figure 2 As shown in a and b in the figure, the appearance of the modified DEB Raman characteristic signal and the redshift of the ultraviolet absorption peak both prove the successful modification of DEB, as shown in the figure. Figure 2 As shown in c and d.

[0041] Verification of the advantages of the internal standard method:

[0042] In a preferred embodiment of the present invention, 5×10 -6M is an aqueous solution of mercuric nitrate. Add 4 mL of the above mercuric nitrate aqueous solution to a 5 mL glass bottle. Pipette 50 μL of BCN-AgNFs@DEB aqueous dispersion into the glass bottle, mix, and sonicate to ensure uniform dispersion of BCN-AgNFs@DEB. Let stand for 2 hours to allow the reaction to complete. Add 4 mL of (benzene, dichloromethane, acetonitrile, N,N-dimethylformamide) to another glass bottle. Place both bottles side-by-side in an integrating sphere independent dual-cell detection system. Irradiate with a 532 nm, 100% laser power for 1 s, accumulating 10 times to obtain the Raman spectra of the analyte and internal standard. Figure 4 As shown, this demonstrates that we can use any liquid substance that generates a signal under 532 nm laser excitation as an internal standard for the analyte, and the internal standard substance can be flexibly selected according to the different analytes.

[0043] In a preferred embodiment of the present invention, 4 mL of the water sample to be tested (which may be ethanol, acetone, isopropanol, sodium sulfate, or sodium carbonate) is added to a 5 mL glass bottle, and 4 mL of pure acetonitrile is added to another glass bottle. The two glass bottles are placed side by side in an integrating sphere independent dual-cell detection system, and irradiated with a 532 nm laser at 100% power for 1 s, accumulated 10 times, to obtain the Raman spectra of the analyte and the internal standard (acetonitrile). Figure 5 As shown, this method demonstrates that it can be used for the same internal standard calibration of different substances.

[0044] In a preferred embodiment of the present invention, 4 mL of ethanol is added to a 5 mL glass bottle, and 4 mL of acetonitrile solutions of different concentrations (0 wt%, 20 wt%, 50 wt%, 70 wt%, 100 wt%) are added to another glass bottle. The two glass bottles are placed side by side in an integrating sphere independent dual-cell detection system and irradiated with a 532 nm laser at 100% power for 1 s, accumulated 10 times, to obtain the Raman spectra of ethanol and internal standards (acetonitrile) at different concentrations. Figure 6 As shown, this method demonstrates that the concentration of the internal standard can be adjusted to meet different requirements for the strength of the internal standard.

[0045] Plotting the SERS quantitative curve:

[0046] Prepare aqueous solutions of mercuric nitrate at different concentrations: 10 -7 M, 5×10 -7 M, 10 -6 M, 2×10 -6 M, 5×10 -6 M, 6×10 -6 M, 7×10 -6 M, 8×10 -6 M, 10 -5M, 2×10 -5 M, 5×10 -5 M, 10 -4 M. Add 4 mL of the above-mentioned mercuric nitrate aqueous solution to a 5 mL glass bottle. Pipette 50 μL of BCN-AgNFs@DEB aqueous dispersion into the glass bottle, mix, and sonicate to ensure uniform dispersion of BCN-AgNFs@DEB. Let stand for 2 hours to allow the reaction to complete. Add 4 mL of acetonitrile to another glass bottle. Place both bottles side-by-side in an integrating sphere independent dual-cell detection system. Irradiate with a 532 nm laser at 100% power for 1 s, accumulating 10 repetitions to obtain the Raman spectra of the analyte and internal standard. Figure 7 As shown. Twenty spectra were collected for each sample, and the average spectrum was taken. The result is expressed as Log[Hg] 2+ [ ] (μM) as the x-axis, with I 2146 / I 2256 The value is the ordinate, and a quantitative curve is plotted, such as... Figure 8 As shown.

[0047] Real water sample test:

[0048] Add 4 mL of Hg to a 5 mL glass bottle. 2+ For real liquid samples, 50 μL of BCN-AgNFs@DEB aqueous dispersion was pipetted into a glass bottle and added dropwise. The mixture was then sonicated to ensure uniform dispersion of BCN-AgNFs@DEB. The mixture was allowed to stand for 2 hours to allow the reaction to complete. In another glass bottle, 4 mL of acetonitrile was added. The two bottles were placed side-by-side in an integrating sphere independent dual-cell detection system and irradiated with a 532 nm laser at 100% power for 1 second, repeated 10 times to obtain the Raman spectra of the analyte and internal standard. Twenty spectra were collected for each sample, and the average spectrum was taken. I... 2146 / I 2256 Substituting the values ​​into the quantitative curve, Hg can be calculated. 2+ Concentration. We used the standard addition method to detect the presence of Hg. 2+ Real water samples (tap water, lake water, tea, and fish) were collected. The corresponding concentrations of Hg were detected in all of these samples. 2+ The recovery rates reached 92.87%–104.43%, as shown in Table 1. This confirms the high feasibility and accuracy of this method in real sample testing.

[0049] Table 1. Hg in real samples 2+ Concentration detection

[0050]

[0051] This invention develops a quantitative detection method for internal standard ratios in liquid-phase SERS based on an integrating sphere independent dual-cell detection system. By completely isolating the internal standard and the analyte in space, it thoroughly avoids the problem of internal standard signal fluctuations caused by changes in the local physicochemical environment of nanoparticles in traditional surface-modified internal standard methods, providing an extremely stable internal standard reference for ratio quantification. The internal standard can be arbitrarily replaced as needed, without surface modification compatibility limitations; the same internal standard system can be applied to different analytes, and the internal standard intensity is not affected by the type of analyte; the internal standard signal intensity can be freely adjusted by adjusting the internal standard concentration to adapt to the ratio calculation requirements of different analyte signals; and this invention has the advantages of high stability, high versatility, simple operation, and flexible adjustment of internal standard intensity and internal standard substance. It provides a reliable and novel approach for the quantitative detection of SERS internal standard ratios.

[0052] The applicant declares that the above description is only a specific implementation of this application, but the protection scope of this application is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application fall within the protection and disclosure scope of this application.

Claims

1. A method for quantitative detection of the internal standard ratio of liquid-phase SERS based on an integrating sphere independent dual-cell detection system, characterized in that, Includes the following steps: S1, Preparation and purification of bacterial cellulose nanomaterial BCN; S2, Preparation of the specifically functionalized SERS substrate material BCN-AgNFs@DEB: A dilute sulfuric acid solution of BCN and a trioctylamine protective agent were added sequentially to a silver ammonia solution, followed by rapid ultrasonic treatment to fully emulsify the system into a homogeneous emulsion. The emulsion was placed in a constant temperature environment at 25 °C, and a hydrazine dispersion was rapidly injected as a reducing agent, followed by constant temperature stirring. After the reaction, the mixture was centrifuged and washed. An ethanol solution of 1,4-diethynylbenzene was added to the washed and purified centrifuged product, and the mixture was ultrasonically treated for 10 min for functionalization modification. The supernatant was removed by centrifugation again, and the solid product was washed. Finally, the modified solid product was uniformly dispersed in deionized water to obtain a BCN-AgNFs@DEB aqueous dispersion, which was then stored in a sealed container at low temperature for later use. S3, construct Hg 2+ Quantitative detection standard curve: Prepare multiple sets of mercuric nitrate standard aqueous solutions with different concentration gradients as standard test samples; take 4 mL of each concentration of mercuric nitrate standard aqueous solution and place them in sealed glass bottles. Transfer 50 μL of BCN-AgNFs@DEB aqueous dispersion to each bottle, sonicate to uniformly disperse the substrate material, and let it stand at room temperature for a period of time to ensure that the substrate and Hg are in harmony. 2+ To ensure a fully specific reaction, a separate glass bottle containing 4 mL of organic solvent was used as an internal standard reference solution. The glass bottle containing the test standard solution and the glass bottle containing the organic solvent internal standard reference solution were placed side-by-side in an integrating sphere independent dual-cell detection system, and Raman spectra were acquired. Twenty spectra were repeatedly acquired for each sample group, and the average spectrum was used as the final detection spectrum. (The last sentence appears to be incomplete and possibly refers to a different method or method.) 2+ Logarithm of concentration (Hg) 2+ Using the concentration (μM) as the x-axis and the characteristic peak intensity ratio I / I as the y-axis, a linear relationship was fitted to establish Hg 2+ Quantitative detection standard curve; S4, Hg in actual water sample 2+ Quantitative detection: Take 4 mL containing Hg 2+ The water sample to be tested was placed in a sealed glass bottle, and 50 μL of BCN-AgNFs@DEB aqueous dispersion was added. After ultrasonic mixing, the mixture was allowed to stand at room temperature for a period of time to ensure that the substrate material and Hg in the sample were homogeneous. 2+ Complete reaction; Raman spectra of the water sample and the organic solvent internal standard reference solution were simultaneously acquired using the same process and parameters as in step S3, and the characteristic peak intensity ratio I / I of the water sample and the organic solvent internal standard reference solution was calculated; the obtained intensity ratio was substituted into the pre-constructed Hg... 2+ The standard curve for quantitative detection was used to calculate the Hg content in the water sample. 2+ The actual concentration.

2. The detection method as described in claim 1, characterized in that, In S1, the purified bacterial cellulose nanomaterial BCN is obtained by acid hydrolysis of bacterial cellulose BC with concentrated sulfuric acid at low temperature, neutralization, dialysis, and freeze drying.

3. The detection method as described in claim 2, characterized in that, In S1, the purified bacterial cellulose nanomaterial BCN was obtained by low-temperature acid hydrolysis of bacterial cellulose BC with concentrated sulfuric acid, neutralization, dialysis, and freeze-drying. The specific process is as follows: the bacterial cellulose BC was subjected to high-shear homogenization to obtain a uniformly dispersed BC solution; the homogenized BC solution was placed in an ice-water bath and stirred continuously, and an equal volume of 65wt% concentrated sulfuric acid was slowly added, followed by heating to 45 ℃ and stirring for 2 h; after the reaction was completed, it was naturally cooled to room temperature, and deionized water was added at a volume ratio of 1:4 to dilute the reaction solution. The system was filtered to remove coarse fiber impurities that were not completely acidified; the filtrate was collected, and the filtrate was slowly titrated with 1 mol / L KOH solution until the pH value was 8 to neutralize the residual acidic substances; the neutralized solution was placed in a dialysis bag and continuously dialyzed with deionized water to remove salt impurities in the system. After dialysis, the obtained dialysate was freeze-dried to obtain the purified bacterial cellulose nanomaterial BCN.

4. The detection method as described in claim 1, characterized in that, In S2, the silver ammonia solution is prepared by the following steps: silver nitrate solid is completely dissolved in deionized water, and ammonia is added dropwise under stirring at room temperature until the initially formed silver oxide precipitate in the solution is completely dissolved; ammonia is added to adjust the pH of the mixed solution to 11 to obtain the silver ammonia solution.

5. The detection method as described in claim 1, characterized in that, The mass ratio of silver, BCN, and 1,4-diethynylbenzene in the silver ammonia precursor solution is 50:105:

30.

6. The detection method as described in claim 1, characterized in that, In S3, Raman spectra were acquired using parameters of 532 nm laser, 100% laser power, 1 second irradiation time, and 10 cumulative scans.

7. The detection method as described in claim 1, characterized in that, In S3, the multiple sets of mercuric nitrate standard aqueous solutions with different concentration gradients are set with 12 consecutive concentration gradients, the concentrations being 10 in sequence. -4 M, 5×10 -5 M, 2×10 -5 M, 10 -5 M, 8×10 -6 M, 7×10 -6 M, 6×10 -6 M, 5×10 -6 M, 2×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M.

8. The detection method as described in claim 1, characterized in that, The organic solvent is any one of benzene, dichloromethane, acetonitrile, and N,N-dimethylformamide.

9. The detection method as described in claim 1, characterized in that, The organic solvent is an acetonitrile solution with a concentration of at least 20 wt%.

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