SERS (Surface Enhanced Raman Scattering) probe for monitoring redox reaction as well as preparation method and application of SERS probe

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) probe for monitoring a redox reaction as well as a preparation method and application of the SERS probe. A metal complex TEPE-Au is prepared firstly, and the chemical structural formula of the metal complex TEPE-Au is shown as a formula I; the TEPE-Au complex has an obvious Raman characteristic peak in a silent zone, shows high sensitivity and high resolution, and can be used as a silent zone reporter molecule. After being prepared into an SERS probe, the SERS probe is combined with an SERS detection technology, so that the SERS probe can be used for monitoring redox reaction, can also be used for detecting the concentration or content of alkaline phosphatase, and has a good application prospect in the fields of biological detection, chemical detection or medical diagnosis and the like. And the formula I of the # imgabs0 # is shown in the specification.

Description

Technical Field

[0001] The invention belongs to the field of chemical and biological detection, and particularly relates to a SERS probe for monitoring redox reactions and a preparation method and application thereof. Background Art

[0002] Redox reactions, as the most fundamental processes in life, play key roles in various physiological processes, including energy metabolism, cell signal transduction, regulation of biomolecular functions, and participation in physiological and pathological processes. Redox reactions play a vital role in both chemistry and environmental science. In chemistry, redox reactions represent a common type of chemical reaction involving the transfer of electrons between substances. In environmental science, redox reactions are widely used in the remediation of pollutants in water and soil, especially the degradation of organic pollutants. Among these reactions, compounds such as potassium persulfate (PPS) and ascorbic acid (AA) play a key role in advanced oxidation technologies. Recent studies have shown that both PPS and AA can be used for soil remediation and removal of organic pollutants in wastewater. Therefore, monitoring redox reactions represented by compounds such as PPS and AA helps to understand their role and efficiency in redox processes, thereby achieving the optimization of these processes for more effective removal of organic pollutants and environmental purification.

[0003] Since alkaline phosphatase (ALP) can catalyze the release of AA from its substrate, L-ascorbic acid-2-phosphate trisodium salt (AA2P), ALP can be indirectly detected by detecting the redox reaction in which AA participates. ALP is one of the important biological enzymes that regulates intracellular metabolism and plays a vital role in phosphate metabolism. ALP exists in tissues throughout the body and is a diagnostic indicator for biliary and bone diseases. Abnormal serum ALP levels are closely related to a variety of diseases, such as liver tuberculosis, obstructive jaundice, secondary liver cancer, osteomalacia, bone damage, osteocellular carcinoma, and malignant bone metastasis.

[0004] At present, methods for monitoring redox reactions include electrochemistry (authorization announcement number: CN106596687B), fluorescence (authorization announcement number: CN107446034B), etc. However, these methods have some disadvantages, such as complex operation, passivation caused by electrode contamination, photobleaching, poor photostability, insufficient sensitivity, etc. Surface-enhanced Raman spectroscopy (SERS) is a reliable, sensitive and non-invasive technology that has been widely used to monitor redox reactions in various studies. Compared with traditional SERS, frequency-shift SERS detects the shift of significant signals instead of resolving peaks in noise, thereby alleviating the requirements for spectral resolution. Studies have confirmed (publication number: WO2022147135A1) that the frequency of the surface-enhanced Raman peak is very sensitive to small changes in the structure of the material. This sensitivity makes it possible to investigate changes in the structure of the material under specific conditions by monitoring changes in the Raman shift. The interaction between the SERS probe and the target molecule induces a Raman shift of the SERS peak by changing the chemical and dielectric environment of the SERS probe. Frequency-shift SERS has been used to determine various analytes, such as melamine, aniline, miRNA, and cancer proteins. Due to its sensitivity to local environmental changes caused by molecular interactions, frequency-shift SERS can be used to monitor redox reactions. Currently, the research on frequency-shift SERS is mainly focused on the application of bioimaging and single analyte detection, while there are few reports on the use of frequency-shift SERS to monitor redox reactions.

[0005] The exploration of SERS based on biological imaging and detection is mainly focused on the Raman fingerprint region (<1800 cm -1 ), in this region, signal acquisition is easily interfered by the intrinsic Raman signal of biological molecules, resulting in increased background noise and reduced signal-to-noise ratio. In recent years, more and more research has focused on applying the silent region (1800–2800 cm -1 ) signal molecules to reduce these interferences. The acquisition and analysis of SERS data is crucial to determining the outcome of the detection process. In particular, the precise identification and specific extraction of tiny Raman frequency changes place higher demands on data processing. Given the latest advances in artificial intelligence, machine learning algorithms have been used to effectively address challenges such as background noise and signal overlap encountered during SERS data analysis. This significantly enhances the automation capabilities of SERS data analysis, thereby improving analysis efficiency, reducing subjective errors, and ultimately enhancing the accuracy and sensitivity of detection. Summary of the invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a metal complex.

[0007] Another object of the present invention is to provide a method for preparing the metal complex.

[0008] Another object of the present invention is to provide application of the metal complex.

[0009] The purpose of the present invention is achieved through the following technical solutions: A metal complex, named TEPE-Au complex, has a chemical structure as shown in Formula I: Formula Ⅰ.

[0010] The preparation method of the metal complex (TEPE-Au) comprises the following steps: 1,2-bis(4-ethynylphenyl)-1,2-diphenylethylene (bis-EDDP) is dissolved in a mixed solution of degassed tetrahydrofuran and methanol (THF / MeOH), and then (1,3-di-trimethylphenyl-1H-imidazole-2(3H)-ylidene)gold(I) chloride ((IMes)AuCl) and sodium hydroxide (NaOH) are added under a protective gas atmosphere, and the reaction is stirred. After the reaction is completed, the filtrate is filtered, the filtrate is collected, the solvent is evaporated by rotary evaporation, and then the filtrate is dissolved in acetone, centrifuged, the supernatant is taken, and added dropwise to a n-hexane solution. Finally, the obtained precipitate is dried to obtain a TEPE-Au complex, i.e., the metal complex.

[0011] The mass ratio of 1,2-bis(4-ethynylphenyl)-1,2-diphenylethylene (bis-EDDP), (1,3-di-trimethylphenyl-1H-imidazole-2(3H)-ylidene)gold(I) chloride ((IMes)AuCl), and sodium hydroxide (NaOH) is 15:(47-67):16; preferably 15:57:16.

[0012] The volume ratio of tetrahydrofuran to methanol in the mixed solution of tetrahydrofuran and methanol (THF / MeOH) is 2:1.

[0013] The protective gas is preferably nitrogen (N2).

[0014] The stirring reaction time is 8 to 14 h, preferably 12 h.

[0015] The centrifugation (washing) conditions are: centrifugation at 10,000 rpm for 2 to 5 min; preferably centrifugation at 10,000 rpm for 2 min.

[0016] The TEPE-Au complex is stored as follows: stored at 4° C. away from light.

[0017] The metal complex is used in preparing silent zone Raman reporter molecules (SERS reporter molecules) or surface enhanced Raman scattering probes (SERS probes).

[0018] The SERS probe comprises a silent zone SERS frequency shift probe.

[0019] A SERS probe for monitoring redox reactions comprises a SERS substrate and the above-mentioned metal complex (silent zone Raman reporter molecule).

[0020] The SERS substrate is gold microparticles (AuMPs).

[0021] The method for preparing the SERS probe for monitoring redox reactions comprises the following steps: The above metal complex (TEPE-Au) is dissolved in a solvent to obtain a TEPE-Au solution; then a gold microparticle (AuMPs) solution is added to the TEPE-Au solution, and after stirring for reaction (incubation), the TEPE-Au modified AuMPs (TEPE-Au-AuMPs) are collected after centrifugation to obtain a SERS probe for monitoring redox reactions.

[0022] The concentration of the TEPE-Au solution is 1-3 mg / mL, preferably 2 mg / mL.

[0023] The solvent is a solvent obtained by mixing dimethyl sulfoxide (DMSO) and an ethanol aqueous solution with a concentration of 95% by volume in a volume ratio of 1:9.

[0024] The content of AuMPs in the gold microparticle (AuMPs) solution is 50 to 200 particles / mL, preferably 100 particles / mL.

[0025] The volume ratio of the gold micron particle (AuMPs) solution to the TEPE-Au solution is 1:10.

[0026] The stirring reaction time is 20 to 40 minutes, preferably 30 minutes.

[0027] The gold microparticles (AuMPs) can be prepared by conventional methods in the art, such as the method described in the Chinese patent (patent number 202210875549.X, entitled "A method for detecting copper ions using a silent zone SERS probe and its application"); preferably, they are obtained by the following method: The aqueous solution of chloroauric acid is dissolved in a hydrochloric acid solution, and then polyvinylpyrrolidone (PVP) and N-(3-aminophenyl)-aniline (NAAN) are added, and the reaction is carried out at 2 to 8°C, and the reaction is centrifuged and washed to obtain gold microparticles (AuMPs).

[0028] The mass ratio of chloroauric acid (HAuCl4), polyvinylpyrrolidone (PVP) and N-(3-aminophenyl)-aniline (NAAN) is (10-20): (9-18): (18-54); preferably 20:18:18.

[0029] The concentration of the HAuCl4 solution is 5-25% by mass, preferably 10% by mass.

[0030] The concentration of the hydrochloric acid solution is preferably 1 mmol / L.

[0031] The volume ratio of the aqueous chloroauric acid solution to the hydrochloric acid solution is preferably 1:125.

[0032] The hydrochloric acid solution is an aqueous hydrochloric acid solution.

[0033] The molecular weight of the polyvinyl pyrrolidone (PVP) is 40,000 to 200,000, preferably 40,000.

[0034] The reaction time is 16 to 48 h, preferably 24 h.

[0035] The centrifugal washing conditions are: centrifugation at 1000 rpm for 2 to 5 min; preferably: centrifugation at 1000 rpm for 2 min; the centrifugal fluid used for centrifugation is N-methylpyrrolidone (NMP).

[0036] The gold microparticles (AuMPs) are stored in deionized water at 4°C.

[0037] The metal complex and / or the SERS probe for monitoring redox reactions are used in monitoring redox reactions or detecting alkaline phosphatase for purposes other than disease diagnosis and treatment.

[0038] A method for monitoring redox reactions using a silent zone SERS frequency shift probe comprises the following steps: S1. Qualitative testing (1) SERS detection: the sample to be tested is added dropwise onto a glass slide (glass slide) covered with the above-mentioned SERS probe for monitoring redox reactions, and the sample is allowed to react at room temperature. After the reaction is completed, the sample is naturally dried, and then SERS detection is performed to obtain a Raman spectrum. (2) Judgment: According to the Raman spectrum, the 2115 cm -1 , 2107 cm -1 and 2123 cm -1 If the peak intensity is in the silent zone 2123 cm -1 A new Raman peak was generated at 2115 cm-1 If the Raman peak at 2107 cm in the silent zone decreases, it means that the sample contains oxidizing substances. -1 A new Raman peak was generated at 2115 cm -1 If the Raman peak at 2115 cm in the silent zone decreases, it means that the sample contains reducing substances. -1 There is no obvious change, indicating that the sample to be tested does not contain redox substances or exceeds the detection limit; or S2. Quantitative detection (3) preparing at least 5 concentration gradients of ascorbic acid (AA) and potassium persulfate (PPS) aqueous solutions, respectively, and then dropping them onto a glass slide covered with the SERS probe for monitoring the redox reaction, respectively, and allowing to react at room temperature. After the reaction is completed, the solution is naturally dried, and then SERS detection is performed to obtain a Raman spectrum. Then, a 2115 cm -1 , 2107 cm -1 and 2123 cm -1 The peak intensity at , and calculate the peak intensity ratio I 2115 / I 2107 and I 2115 / I 2123 ; Finally, the concentration and peak intensity ratio of ascorbic acid (AA) aqueous solution I 2115 / I 2107 Draw a standard curve A, with the concentration of potassium persulfate (PPS) aqueous solution and the peak intensity ratio I 2115 / I 2123 Draw the standard curve and obtain the linear equation B; (4) The sample to be tested is added dropwise onto the glass slide of the SERS probe for monitoring redox reaction, and the reaction is allowed to stand at room temperature. After the reaction is completed, the sample is naturally dried, and then SERS detection is performed to obtain a Raman spectrum. The Raman spectrum is then used to obtain the 2115 cm -1 , 2107 cm -1 and 2123 cm -1 The peak intensity at , and calculate the peak intensity ratio I 2115 / I 2107 and I 2115 / I 2123 ; Finally, the concentration and / or content of the reducing substance or oxidizing substance is obtained by conversion according to the standard curve A or the linear equation B.

[0039] The dropping described in steps (1) to (4) is achieved by using a pipette to suck up the precipitate aqueous solution and drop it onto a glass slide, leaving it to stand at room temperature until the water evaporates completely, and then proceeding to the next step of SERS detection.

[0040] The oxidizing material in steps (2) and (4) is preferably potassium persulfate (PPS).

[0041] The reducing substance in steps (2) and (4) is preferably ascorbic acid (AA).

[0042] The concentration range of the ascorbic acid (AA) aqueous solution in step (3) is 1×10 -6 mol / L~1×10 -2 mol / L.

[0043] The concentration range of the potassium persulfate (PPS) aqueous solution in step (3) is 1×10 -6 mol / L~1×10 - 2 mol / L.

[0044] The ascorbic acid (AA) and potassium persulfate (PPS) aqueous solution described in step (3) is preferably prepared using DI pure water.

[0045] The reaction time in steps (1), (3) and (4) is 1 to 3 h, preferably 2 h.

[0046] The conditions for SERS detection described in steps (1), (3) and (4) are: using 633 nm laser excitation, recording the SERS spectrum changes on a single SERS probe, the power is 1 mW, and the accumulation time is 2 to 8 seconds.

[0047] A method for detecting alkaline phosphatase (ALP) using a silent zone SERS frequency shift probe comprises the following steps: (I) at least 5 alkaline phosphatase (ALP) solutions of different concentration gradients are mixed with L-ascorbic acid-2-phosphate trisodium salt (AA2P) solution, respectively, and reacted (oscillation reaction) at 36-38° C. (preferably 37° C.) to obtain a mixed solution after the reaction; then the mixed solution is added dropwise onto a glass slide covered with the SERS probe for monitoring the redox reaction, and allowed to react at room temperature, and allowed to dry naturally after the reaction is completed, and then SERS detection is performed to obtain a Raman spectrum; and a 2115 cm -1 , 2107 cm -1 The peak intensity at , and calculate the peak intensity ratio I 2115 / I 2107 ; Finally, the ALP concentration and ratio I 2115 / I 2107 Draw the standard curve and obtain the linear equation C; (II) adding a solution of trisodium L-ascorbic acid-2-phosphate (AA2P) to a sample solution to be tested, and reacting (oscillating reaction) at 36-38° C. (preferably 37° C.) to obtain a mixed solution after the reaction; then dropping the mixed solution onto a glass slide covered with the above-mentioned SERS probe for monitoring redox reactions, and allowing the mixture to react at room temperature. After the reaction is completed, the mixture is naturally dried, and then SERS detection is performed to obtain a Raman spectrum; a 2115 cm -1 , 2107 cm -1 The peak intensity at , and calculate the peak intensity ratio I 2115 / I 2107 Finally, according to the linear equation C obtained in step (I), the concentration and / or content of alkaline phosphatase (ALP) in the sample to be tested is calculated.

[0048] The alkaline phosphatase (ALP) solution described in step (I) is a solution prepared using a Tris-HCl buffer solution.

[0049] The Tris-HCl buffer solution is prepared by dissolving 60.6 mg Tris and 2 mg MgCl2 in 30 mL water, adjusting the pH to 8.0 with concentrated hydrochloric acid, and finally adding water to make the volume 50 mL.

[0050] The concentration of the alkaline phosphatase (ALP) solution described in step (I) ranges from 1 mU to 100 mU.

[0051] The concentration of the L-ascorbic acid-2-phosphate trisodium salt (AA2P) solution in steps (I) and (II) is 200-400 mmol / L, preferably 300 mmol / L.

[0052] The volume ratio of the L-ascorbic acid-2-phosphate trisodium salt (AA2P) solution and the alkaline phosphatase (ALP) solution described in step (I) is 1:9.

[0053] In steps (I) and (II), the reaction time at 36-38° C. is 1-2 hours, preferably 1 hour.

[0054] In steps (I) and (II), the reaction time at room temperature is 2 to 3 hours, preferably 2 hours.

[0055] The sample solution to be tested in step (II) is a solution prepared using a Tris-HCl buffer solution.

[0056] The volume ratio of the L-ascorbic acid-2-phosphate trisodium salt (AA2P) solution and the sample solution described in step (II) is 1:9.

[0057] The conditions for SERS detection in steps (I) and (II) are: using 633 nm laser excitation, recording SERS spectrum changes on a single SERS probe, power of 1 mW, and accumulation time of 2 to 8 seconds.

[0058] A method for redox reaction classification using machine learning combined with Raman spectroscopy, specifically comprising the following steps: (i) performing data enhancement on the Raman spectrum obtained in the above step (2), (4) or (II) to obtain more data; (ii) performing baseline correction, noise reduction, normalization and dimension reduction on the Raman spectrum data obtained in step (i), and collecting pre-processed data; (iii) The preprocessed data obtained in step (ii) is divided into data sets, the training set is used to train the machine learning model, and the test set is used to test the model to obtain various evaluations of the model.

[0059] The data enhancement described in step (i) is a data enhancement method of superimposing Gaussian noise, in which Gaussian noise with a mean of 0 and a variance of 10 is superimposed on the newly generated spectrum.

[0060] The baseline correction process described in step (ii) is performed using the airPLS algorithm.

[0061] The noise reduction process described in step (ii) is performed by using a Savitzky-Golay smoothing filter algorithm.

[0062] The dimensionality reduction process described in step (ii) is performed by using the principal component analysis (PCA) method.

[0063] The data set described in step (iii) is divided into: 60% training set and 40% test set.

[0064] The machine learning models described in step (iii) include: random forest (RF), decision tree (DT), gradient boosted decision tree (GBDT), K-nearest neighbor (KNN), support vector machine (SVM), logistic regression (LR), etc.

[0065] Compared with the prior art, the present invention has the following advantages and effects: 1. The present invention utilizes a layered gold particle self-assembly as a SERS substrate, which has a large number of Raman enhancement "hot spots", can significantly enhance the SERS signal, and can also realize single-particle SERS detection, greatly improving detection reproducibility.

[0066] 2. The present invention combines SERS detection technology with silent zone reporter molecules, and has the characteristics of high sensitivity and high specificity. It is expected to bring important progress in the field of redox reaction monitoring, and has great development space and broad application prospects in the fields of biology, chemical detection, medical diagnosis, etc.

[0067] 3. The present invention introduces Au (I) which is susceptible to environmental stimulation at the other end of the alkynyl group, and uses redox reaction to change its valence state, thereby changing the Raman characteristic peak of the alkynyl group located in the silent zone. The change of the characteristic peak of the alkynyl group is linearly related to the redox substance, thereby monitoring the oxidized or reduced substance.

[0068] 4. The present invention utilizes the displacement caused by the change in the chemical environment of the alkynyl group of the silent zone reporter molecule for redox monitoring. Compared with traditional SERS, frequency-shift SERS detects the shift of significant signals instead of resolving peaks in noise, thereby alleviating the requirements for spectral resolution.

[0069] 5. The present invention utilizes ALP to hydrolyze AA2P to generate AA, and then AA further changes Au (I) to Au (0), thereby changing the dielectric environment of the alkynyl group connected to Au, causing its Raman characteristic peak to shift. The change in the alkynyl characteristic peak is linearly related to ALP, thereby detecting ALP. The method of detecting ALP by frequency-shift SERS in the silent zone has not been reported, and the present invention can avoid the influence of the peak spectrum of common physiological molecules and chemical groups in the fingerprint zone by detecting in the Raman silent zone, greatly improving the accuracy and sensitivity of the detection.

[0070] 6. The present invention combines the advantages of frequency-shift SERS silent zone detection and machine learning data analysis to highly refine the information contained in the Raman spectrum, thereby further analyzing it so that it can classify redox reactions.

[0071] 7. The present invention provides a new SERS method for efficient monitoring and classification of redox reactions and ALP diagnosis. Through the careful design of the molecular structure, TEPE-Au has a highly sensitive Raman characteristic peak, showing high sensitivity and resolution. The application of machine learning algorithms realizes the automatic classification of reactions and provides a reliable solution for data processing. The design and preparation of TEPE-Au probes make it possible to accurately monitor redox reactions and provide an effective means for detecting small changes. The efficient use of machine learning algorithms improves the accuracy and reliability of data processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 3 are morphological images of gold micron particles AuMPs prepared by the present invention; wherein (A) is a representative SEM image of AuMPs; and (B) is a high-resolution SEM image of a single particle.

[0073] Figure 2 It is the Raman spectrum of the TEPE-Au complex prepared by the present invention.

[0074] Figure 3 It is the mass spectrum of the TEPE-Au complex prepared by the present invention.

[0075] Figure 4 The figure shows the effect of reducing agent ascorbic acid (AA) and oxidizing agent potassium persulfate (PPS) on the SERS spectrum of TEPE-Au complex; (A) is the reaction of TEPE-Au complex with AA; (B) is the reaction of TEPE-Au complex with PPS; (C) is the TEPE-Au complex.

[0076] Figure 5 The Raman spectra of potassium persulfate and ascorbic acid at different concentrations were detected using a SERS probe; (A) is potassium persulfate at different concentrations; (B) is ascorbic acid at different concentrations.

[0077] Figure 6 : These are the linear relationship diagrams between the SERS probe and the concentrations of potassium persulfate and ascorbic acid; wherein, (A) is the linear relationship diagram between the SERS probe and potassium persulfate; and (B) is the linear relationship diagram between the SERS probe and ascorbic acid.

[0078] Figure 7 The fluorescence (FL) spectra of TEPE-Au complex were measured under different redox stimuli.

[0079] Figure 8 The redox state of Au (I) is changed by redox stimulation, and the corresponding X-ray photoelectron spectroscopy (XPS) spectra are measured; (A) is the XPS spectrum of TEPE-Au after the reaction with AA; (B) is the XPS spectrum of TEPE-Au after the reaction with PPS.

[0080] Fig. 9 The following are the results of using SERS probe to detect standard ALP samples of different concentrations; (A) is the Raman spectrum; (B) is the linear curve.

[0081] Fig.10 It is the principal component analysis (PCA) result diagram of SERS spectral data; among them, (A) is the principal component analysis result of TEPE-Au before and after redox and its three categories (the calculated scores are presented in the form of a chart in the 3D projection: PC1vs PC2 vsPC3); (B) is the confusion matrix of the KNN classification results of the three categories.

[0082] Fig.11It is a schematic diagram of the preparation and application process of the SERS probe of the present invention. DETAILED DESCRIPTION

[0083] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually in accordance with conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0084] The conditions of the SERS test involved in the embodiment of the present invention are: using 633nm laser excitation, recording the SERS spectrum on a single AuMP, the power is 1mW, and the accumulation time is 4 seconds. For each sample, the SERS spectra of 3 AuMPs are obtained during the measurement for standard deviation calculation.

[0085] The N-(3-amidino)-aniline (NAAN) involved in the embodiments of the present invention can be synthesized according to the literature (Ma Y, Yung L. Synthesis of Self-Stabilized Poly (N-(3-Amidino)-Aniline) Particles and their CO2-Responsive Properties [J]. Particle & Particle Systems Characterization, 2015, 32 (7): 743-748.).

[0086] Example 1 (1) Synthesis of layered gold microparticles 25 μL of 32 mg / mL polyvinylpyrrolidone (PVP) (molecular weight of 40,000) was added to 959 μL of 1 mM HCl solution containing 8 μL of 10% chloroauric acid, the mixture was cooled to 4°C with ice water, and then 8 μL of 100 mg / mL NAAN solution (dissolved in 1 mM HCl solution) was added. After being thoroughly mixed by vortexing, the mixed solution was allowed to react at 4°C for 24 hours without further stirring. The reactants were centrifuged at 1000 rpm for 2 min, and the collected particles were washed with N-methylpyrrolidone (NMP) and deionized water (DI water) in turn to remove impurities. The final particles were dispersed in 100 μL of deionized water and stored in a 4°C refrigerator for further use. The morphology of the prepared layered gold microparticles AuMPs is as shown in Figure 1 shown.

[0087] (2) Synthesis of surface enhanced Raman scattering (SERS) reporter molecules for monitoring redox reactions 1,2-Bis(4-ethynylphenyl)-1,2-diphenylethylene (bis-EDDP) (15 mg, 0.04 mmol) was dissolved in 6 mL of degassed tetrahydrofuran / methanol (THF / MeOH) solution (v / v=2 / 1), and then (1,3-di-trimethylphenyl-1H-imidazole-2(3H)-ylidene)gold(I) chloride ((IMes)AuCl) (57 mg, 0.1 mmol) (CAS No.: 852445-81-9) and sodium hydroxide (NaOH) (16 mg, 0.4 mmol) were added under N2 atmosphere. The mixture was stirred overnight and filtered to remove the precipitate. The filtrate was collected and the solvent was removed by rotary evaporation to obtain a light yellow powder, which was then dissolved in acetone and centrifuged at 10000 rpm for 2 minutes. The obtained supernatant was added dropwise to the n-hexane solution, and the precipitate was collected and dried in a vacuum oven to obtain the TEPE-Au complex in the form of a light yellow solid. Store at 4°C away from light. The Raman spectra and mass spectra of the prepared silent zone SERS reporter molecules are shown in Figure 2 and Figure 3 Its chemical structure is shown in Formula I.

[0088] Formula Ⅰ.

[0089] Example 2 A surface enhanced Raman scattering (SERS) probe detection method for monitoring redox reactions based on silent region frequency shift reporter molecules, the specific steps are as follows: (1) Inject 10 uL of gold microparticles AuMPs prepared in Example 1 (about 100 particles / mL) into 100 uL of a mixed solvent (dimethyl sulfoxide (DMSO) and 95% ethanol aqueous solution mixed in a volume ratio of 1:9) containing 2 mg / mL TEPE-Au complex, stir for 30 minutes to allow TEPE-Au to attach to the AuMPs, collect the TEPE-Au modified AuMPs after centrifugation, and then take 10 μL and drop it on a glass slide. Finally, add 10 uL of oxidants or reductants (prepared with DI water) of different concentrations to the TEPE-Au modified AuMPs, react at room temperature for 2 hours, and then naturally dry after the reaction is completed, and measure their SERS signals. Set up three repetitions. The oxidant and reductant are potassium persulfate (PPS) and ascorbic acid (AA), respectively, and the final concentrations of both are: 1×10 -6 mol / L、1×10 -5 mol / L、1×10 -4 mol / L、1×10 -3mol / L、1×10 - 2 mol / L、1×10 -1 mol / L.

[0090] (2) Due to the Raman response of the alkyne group to its microenvironment, the change of the redox state of the central Au (I) in the TEPE-Au complex may lead to the shift of the Raman fingerprint of the alkyne group. To verify this hypothesis, the effects of the reducing agent ascorbic acid (AA) and the oxidizing agent potassium persulfate (PPS) on the SERS spectrum of the TEPE-Au complex were verified. The fingerprint spectrum is shown in Figure 4 As shown, the incubation of TEPE-Au complex with PPS did affect the fingerprint of benzene ring ( Figure 4 in (B)), while with TEPE-Au complex ( Figure 4 Compared with (C) in -1 In comparison, the SERS spectrum of the TEPE-Au complex after incubation with AA shifted from 2115 cm −1 To 2107 cm −1 , the observable -C≡C- bond redshift ( Figure 4 The above results indicate that the redox reaction of Au(I) in the TEPE-Au complex can induce changes in the SERS spectrum of the acetylenic bond. In addition, the quiet region frequency shift SERS probe linearly responds to the concentration of potassium persulfate and ascorbic acid from 1×10 -6 mol / L increases to 1×10 -1 mol / L (see Figure 6 ), the LOD of the detection result is 1×10 -6 mol / L (see Figure 5 middle curve 1 μM).

[0091] (3) In order to prove that the TEPE-Au reporter molecule did undergo a redox reaction, fluorescence characterization was also performed. The fluorescence (FL) spectrum of the TEPE-Au complex was measured under different redox stimuli. Figure 7As shown, the TEPE ligand has a maximum FL emission at 490 nm, while the FL emission of the TEPE-Au complex exhibits an obvious red shift at 505 nm. The addition of AA to the TEPE-Au complex solution can cause the emission spectrum to blue-shift to 496 nm, while PPS can induce the emission spectrum to red-shift to 513 nm. It is worth noting that the red-shifted FL of the TEPE-Au complex under oxidative stimulation is attributed to the conversion of Au (I) to Au (III), resulting in an enhanced electron-withdrawing ability of the acceptor. This enhanced electron-withdrawing ability may regulate the vibration frequency by affecting the π electron delocalization on -C≡C-, resulting in a blue-shifted SERS fingerprint of the -C≡C- group. AA can reduce Au (I) to Au (0), thereby inhibiting the coordination of Au to the alkynyl group, resulting in the SERS fingerprint of the -C≡C- group being similar to that of the TEPE ligand.

[0092] (4) In order to verify the change of the redox state of Au(I) by redox stimulation, the corresponding X-ray photoelectron spectroscopy (XPS) spectra were measured, such as Figure 8 After the reaction of TEPE-Au with a final concentration of 2 mg / mL and AA with a final concentration of 10 μM, the corresponding XPS spectrum of elemental Au can be deconvoluted into four peaks at 84.28, 87.98, 84.98 and 88.68 eV, corresponding to Au(0) 4f 7 / 2 、Au(0)4f 5 / 2 、Au(I) 4f 7 / 2 and Au(I)4f 5 / 2 ( Figure 8 (A) in Figure 1), revealing that AA partially reduced Au(I) to Au(0). In comparison, Au(III) 4f was observed after TEPE-Au was incubated with PPS. 7 / 2 、Au(III)4f 5 / 2 、Au(I)4f 7 / 2 、Au(I)4f 7 / 2 and Au(I)4f 5 / 2 The four peaks of Figure 8 These results clearly indicate that the change in the Au redox state is responsible for the shift in the fingerprint of the -C≡C- group.

[0093] Example 3 A surface enhanced Raman scattering (SERS) probe detection method for detecting alkaline phosphatase (ALP) based on a silent region frequency shift reporter molecule, the specific steps are as follows: 90 μL of L-ascorbic acid-2-phosphate trisodium salt (AA2P) (0.3 mol / L) and 10 μL of alkaline phosphatase (ALP) (concentrations: 1 mU / mL, 20 mU / mL, 40 mU / mL, 80 mU / mL, 100 mU / mL) were mixed in a 2 mL centrifuge tube containing 100uL of pH=8.0 Tris-HCl buffer. After reacting at 37°C for 1 hour, 10 μL of the resulting mixture was dropped onto a glass slide covered with TEPE-Au modified AuMPs (prepared in the same manner as step (1) of Example 2) to react at room temperature for 2 hours. After the reaction was completed, the mixture was naturally dried and the SERS signal of the TEPE-Au modified AuMPs was measured to obtain the Raman spectrum after the reaction. The 2115 cm -1 , 2107 cm -1 The peak intensity is then calculated as the peak intensity ratio I 2115 / I 2107 Finally, the ALP concentration and ratio I 2115 / I 2107 Draw the standard curve and obtain the linear equation ( Fig. 9 ).

[0094] Example 4 A surface enhanced Raman scattering (SERS) probe detection method for detecting alkaline phosphatase (ALP) in serum based on silent region frequency shift reporter molecules, the specific steps are as follows: Different concentrations of standard ALP samples were added to blank human serum samples (so that the final concentrations in the serum samples were 20 mU / mL, 40 mU / mL and 80 mU / mL, respectively, and the solvent was Tris-HCl buffer), and then 90 μL of ALP (0.3 mol / L) was added to 10 μL of the above human serum solutions containing different concentrations of ALP. After reacting at 37°C for 1 hour, 10 μL of the obtained mixture was dropped onto a glass slide covered with TEPE-Au modified AuMPs (preparation method is the same as step (1) of Example 2), and reacted at room temperature for 2 hours to obtain the sample to be tested. After drying at room temperature, SERS measurement was performed, and according to the above linear curve ( Fig. 9 The concentration of ALP was calculated by (B) and repeated three times. The human serum solution was a blood sample of a healthy volunteer provided by the First Affiliated Hospital of Jinan University (Guangzhou, China). The sample was allowed to stand at room temperature for more than half an hour, centrifuged at 3500-4000 rpm for 5-10 minutes, and the collected supernatant was diluted 1000 times with a Tris-HCl solution (the Tris-HCl solution was 60.6 mg Tris and 2 mg MgCl2 dissolved in 30 mL water, the pH was adjusted to 8.0 with concentrated hydrochloric acid, and finally water was added to make up to 50 mL).

[0095] The SERS measurement results showed that the ALP recovery rate was in the range of 99.85-104.75%, and the relative standard deviation (RSD) was 1.55-9.34% (n=3) (see Table 1). This shows that the SERS-based analytical technique provided in this example can detect ALP with high sensitivity and has broad application prospects in biological analysis and disease diagnosis.

[0096] Table 1 Detection of ALP content in serum samples based on SERS analysis technology Example 5 A redox classification method combining Raman spectroscopy and machine learning, the specific steps are as follows: The SERS spectral data in Example 2 were collected, and then the SERS spectral data preprocessing was divided into three steps: smoothing and denoising, baseline correction and normalization. First, we collected 145 redox reaction data and enhanced them to 435 redox reaction data using the data enhancement method of random additive Gaussian noise, and then used the Savitzky-Golay (SG) algorithm to denoise the collected spectral data. Subsequently, the adaptive iterative weighted penalized least squares (airPLS) algorithm was used to correct the baseline. Before establishing the classification model, each spectrum was preprocessed and normalization was applied. In addition, since the dimension of the spectrum is high and irrelevant information may exist, principal component analysis (PCA) is used to effectively reduce the dimension of the data and retain its key information. The purpose of applying PCA is to capture at least 95% of the cumulative contribution rate to ensure that a large part of the variance in the data is retained. After data preprocessing, a classification model was established using a classic machine learning algorithm, and all programs were implemented using the Scikit-learn library in the Python programming language. The PCA results and the model classification results are shown in the figure. Fig.10 And as shown in Table 2.

[0097] Table 2 Evaluation of classification results of various classic machine learning models The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A metal complex, characterized in that: The metal complex is a TEPE-Au complex, and its chemical structure is shown in Formula I: Formula Ⅰ.

2. The method for preparing the metal complex according to claim 1, characterized in that: The steps include: Dissolving 1,2-bis(4-ethynylphenyl)-1,2-diphenylethylene in a mixed solution of degassed tetrahydrofuran and methanol, then adding (1,3-di-trimethylphenyl-1H-imidazole-2(3H)-ylidene)gold(I) chloride and sodium hydroxide under a protective gas atmosphere, stirring for reaction, filtering after the reaction is completed, collecting the filtrate, rotary evaporating to remove the solvent, dissolving it in acetone, centrifuging, taking the supernatant, and dropping it into a n-hexane solution, and finally drying the obtained precipitate to obtain the metal complex; The mass ratio of 1,2-bis(4-ethynylphenyl)-1,2-diphenylethylene, (1,3-di-trimethylphenyl-1H-imidazole-2(3H)-ylidene)gold(I) chloride and sodium hydroxide is 15:47-67:16; The volume ratio of tetrahydrofuran to methanol in the mixed solution of tetrahydrofuran and methanol is 2:1; The stirring reaction time is 8 to 14 hours.

3. Use of the metal complex according to claim 1 in preparing silent zone Raman reporter molecules or surface enhanced Raman scattering probes.

4. A SERS probe for monitoring redox reactions, characterized in that: Comprising a SERS substrate and the metal complex according to claim 1; The SERS substrate is gold micron particles AuMPs.

5. The method for preparing a SERS probe for monitoring redox reactions according to claim 4, characterized in that: The steps include: The metal complex of claim 1 is dissolved in a solvent to obtain a TEPE-Au solution; then, the gold micron particle AuMPs solution is added to the TEPE-Au solution, stirred for reaction, and then the TEPE-Au modified AuMPs are collected after centrifugation to obtain a SERS probe for monitoring redox reactions; The concentration of the TEPE-Au solution is 1 to 3 mg / mL; The solvent is a mixture of dimethyl sulfoxide and an ethanol aqueous solution with a concentration of 95% by volume in a volume ratio of 1:9; The content of AuMPs in the gold micron particle AuMPs solution is 50 to 200 per mL; The volume ratio of the gold microparticle AuMPs solution to the TEPE-Au solution is 1:10; The stirring reaction time is 20 to 40 minutes.

6. Use of the metal complex according to claim 1 and / or the SERS probe for monitoring redox reactions according to claim 4 in monitoring redox reactions or detecting alkaline phosphatase for purposes other than disease diagnosis and treatment.

7. A method for monitoring redox reactions using a silent zone SERS frequency shift probe, characterized in that: The steps include: S1. Qualitative testing (1) SERS detection: drop the sample to be tested onto a glass slide covered with the SERS probe for monitoring redox reactions as claimed in claim 4, let it react at room temperature, dry it naturally after the reaction is completed, and then perform SERS detection to obtain a Raman spectrum; (2) Judgment: According to the Raman spectrum, the 2115 cm -1 , 2107 cm -1 and 2123 cm -1 If the peak intensity is in the silent zone 2123 cm -1 A new Raman peak was generated at 2115 cm -1 If the Raman peak at 2107 cm in the silent zone decreases, it means that the sample contains oxidizing substances. -1 A new Raman peak was generated at 2115 cm -1 If the Raman peak at 2115 cm in the silent zone decreases, it means that the sample contains reducing substances. -1 There is no obvious change, indicating that the sample to be tested does not contain redox substances or exceeds the detection limit; or S2. Quantitative detection (3) preparing at least 5 concentration gradients of ascorbic acid and potassium persulfate aqueous solutions, respectively, and then dropping them onto a glass slide covered with the SERS probe for monitoring redox reactions according to claim 4, allowing the reaction to proceed at room temperature, allowing the reaction to dry naturally after the reaction is completed, and then performing SERS detection to obtain a Raman spectrum; and then obtaining a 2115 cm -1 , 2107 cm -1 and 2123 cm -1 The peak intensity at , and calculate the peak intensity ratio I 2115 / I 2107 and I 2115 / I 2123 Finally, the concentration and peak intensity ratio of ascorbic acid aqueous solution I 2115 / I 2107 Draw a standard curve A, with the concentration of potassium persulfate aqueous solution and the peak intensity ratio I 2115 / I 2123 Draw the standard curve and obtain the linear equation B; (4) dropping the sample to be tested onto the glass slide of the SERS probe for monitoring redox reaction as claimed in claim 4, leaving it to react at room temperature, drying it naturally after the reaction is completed, and then performing SERS detection to obtain a Raman spectrum; and obtaining a 2115 cm -1 , 2107 cm -1 and 2123 cm -1 The peak intensity at , and calculate the peak intensity ratio I 2115 / I 2107 and I 2115 / I 2123 ; Finally, the concentration and / or content of the reducing substance or oxidizing substance is obtained by conversion according to the standard curve A or the linear equation B.

8. The method according to claim 7, characterized in that: The concentration range of the ascorbic acid aqueous solution in step (3) is 1×10 -6 mol / L~1×10 -2 mol / L; The concentration range of the potassium persulfate aqueous solution in step (3) is 1×10 -6 mol / L~1×10 -2 mol / L; The reaction time in steps (1), (3) and (4) is 1 to 3 hours; The conditions for SERS detection described in steps (1), (3) and (4) are: using 633 nm laser excitation, recording the SERS spectrum changes on a single SERS probe, the power is 1 mW, and the accumulation time is 2 to 8 seconds.

9. A method for detecting alkaline phosphatase using a silent zone SERS frequency shift probe, characterized in that: The steps include: (I) at least 5 alkaline phosphatase solutions with different concentration gradients are mixed with L-ascorbic acid-2-phosphate trisodium salt solution respectively, and reacted at 36-38° C. to obtain a mixed solution after the reaction; then the mixed solution is added dropwise onto a glass slide covered with the SERS probe for monitoring redox reaction according to claim 4, and allowed to react at room temperature, and dried naturally after the reaction is completed, and then SERS detection is performed to obtain a Raman spectrum; and a 2115 cm -1 , 2107 cm -1 The peak intensity at , and calculate the peak intensity ratio I 2115 / I 2107 ; Finally, the ALP concentration and ratio I 2115 / I 2107 Draw the standard curve and obtain the linear equation C; (II) adding a solution of trisodium L-ascorbic acid-2-phosphate to a sample solution to be tested, reacting at 36-38° C. to obtain a reaction mixture; then dropping the mixture onto a glass slide covered with the SERS probe for monitoring redox reactions according to claim 4, allowing the mixture to react at room temperature, allowing the mixture to dry naturally after the reaction is completed, and then performing SERS detection to obtain a Raman spectrum; obtaining a 2115 cm -1 , 2107 cm -1 The peak intensity at , and calculate the peak intensity ratio I 2115 / I 2107 Finally, the concentration and / or content of alkaline phosphatase in the sample to be tested is calculated according to the linear equation C obtained in step (I).

10. The method according to claim 9, characterized in that: The concentration range of the alkaline phosphatase solution in step (I) is 1 mU to 100 mU; The concentration of the L-ascorbic acid-2-phosphate trisodium salt solution in steps (I) and (II) is 200 to 400 mmol / L; The volume ratio of the L-ascorbic acid-2-phosphate trisodium salt solution to the alkaline phosphatase solution in step (I) is 1:9; In steps (I) and (II), the reaction time at 36-38° C. is 1-2 hours; In steps (I) and (II), the reaction is allowed to stand at room temperature for 2 to 3 hours; The conditions for SERS detection in steps (I) and (II) are: using 633 nm laser excitation, recording the SERS spectrum changes on a single SERS probe, the power is 1 mW, and the accumulation time is 2 to 8 seconds.

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