A sers probe for monitoring redox reaction and preparation method and application thereof

Abstract

The application discloses a SERS probe for monitoring redox reactions and a preparation method and application thereof. A metal complex TEPE-Au is prepared first in the application, and a chemical structural formula of the metal complex is shown as formula I. The TEPE-Au complex has obvious Raman characteristic peaks in a silent zone, and exhibits high sensitivity and high resolution, and can be used as a silent zone reporter molecule. After the TEPE-Au complex is made into a SERS probe and combined with a SERS detection technology, the SERS probe can be used for monitoring redox reactions, and can also be used for detecting the concentration or content of alkaline phosphatase, and has good application prospects in the fields of biological detection, chemical detection or medical diagnosis, etc.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemistry and biological detection, and particularly relates to a SERS probe for monitoring redox reactions and a preparation method and application thereof. BACKGROUND

[0002] Redox reactions, as the most basic processes in life, play a key role in various physiological processes, including energy metabolism, cell signal transduction, biological molecule function regulation, and participation in physiological and pathological processes. Redox reactions play a crucial role in both chemistry and environmental science. In chemistry, redox reactions represent a common type of chemical reaction involving electron transfer 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. In these reactions, compounds such as potassium persulfate (PPS) and ascorbic acid (AA) play a key role in advanced oxidation technology. 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 PPS and AA and other compounds helps to understand their role and efficiency in redox processes, thereby achieving optimization of these processes for more effective removal of organic pollutants and environmental purification.

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

[0004] Currently, the methods for monitoring redox reactions include electrochemistry (Patent No. CN106596687B), fluorescence (Patent No. CN107446034B), etc. However, these methods have some drawbacks, such as complex operation, passivation caused by electrode contamination, photobleaching, poor light stability, insufficient sensitivity, etc. Surface-enhanced Raman spectroscopy (SERS) is a reliable, sensitive and non-invasive technique that has been widely used to monitor redox reactions in various studies. Compared with traditional SERS, frequency-shift SERS detects the shift of the signal rather than distinguishing the peak value in the noise, thereby reducing the requirement for spectral resolution. Studies have confirmed (Publication No. WO2022147135A1) that the frequency of the surface-enhanced Raman peak is very sensitive to the slight changes in the structure of the substance. This sensitivity makes it possible to investigate the changes in the structure of the material under certain conditions by monitoring the changes in the Raman shift. The interaction between the SERS probe and the target molecule induces the Raman shift of the SERS peak by changing the chemical and dielectric environment of the SERS probe, and frequency-shift SERS has been used to determine various analytes, such as melamine, aniline, miRNA and cancer proteins. Due to the sensitivity of frequency-shift SERS to local environmental changes caused by molecular interactions, it can be used to monitor redox reactions. Currently, the research on frequency-shift SERS mainly focuses on the application of biological imaging and single-analyte detection, and 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 mainly focuses on the Raman fingerprint region (<1800 cm -1 ), in which the signal acquisition is easily disturbed by the inherent 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 signal molecules in the silent region (1800-2800 cm -1 ) to reduce these disturbances. The acquisition and analysis of SERS data are crucial for determining the results of the detection process. In particular, the accurate identification of small Raman frequency changes and specific extraction impose higher requirements on data processing. Given the latest developments in artificial intelligence, machine learning algorithms have been used to effectively address challenges such as background noise and signal overlap encountered during the SERS data analysis process. 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

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

[0007] Another object of the present application is to provide a preparation method of the metal complex.

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

[0009] The object of the present application is achieved by the following technical solutions:

[0010] A metal complex, named TEPE-Au complex, has a chemical structural formula as shown in Formula I:

[0011]

[0012] Formula I.

[0013] The preparation method of the metal complex (TEPE-Au) comprises the following steps:

[0014] bis-EDDP is dissolved in a mixed solution of degassed tetrahydrofuran and methanol (THF / MeOH), then (IMes)AuCl and NaOH are added under a protective gas atmosphere, the reaction is stirred, after the reaction is completed, the filtrate is collected, the solvent is removed by rotary evaporation, then it is dissolved in acetone, centrifuged, the supernatant is taken, added dropwise into a n-hexane solution, and finally the obtained precipitate is dried to obtain the TEPE-Au complex, i.e. the metal complex.

[0015] The mass ratio of bis-EDDP, (IMes)AuCl and NaOH is 15: (47-67): 16; preferably 15:57:16.

[0016] The volume ratio of tetrahydrofuran and methanol in the mixed solution of tetrahydrofuran and methanol (THF / MeOH) is 2:1.

[0017] The protective gas is preferably nitrogen (N2).

[0018] The stirring reaction time is 8-14 h; preferably 12 h.

[0019] The centrifugation (washing) condition is: 10000 rpm centrifugation for 2-5 min; preferably 10000 rpm centrifugation for 2 min.

[0020] The storage mode of the TEPE-Au complex is as follows: stored at 4℃ in the dark.

[0021] Use of the metal complex in preparation of silent zone Raman reporter molecules (SERS reporter molecules) or surface enhanced Raman scattering probes (SERS probes).

[0022] The SERS probe includes a silent zone SERS frequency shift probe.

[0023] A SERS probe for monitoring redox reactions, comprising a SERS substrate and the above-mentioned metal complex (silent zone Raman reporter molecule).

[0024] The SERS substrate is gold microparticles (AuMPs).

[0025] The preparation method of the SERS probe for monitoring redox reactions comprises the following steps:

[0026] The above-mentioned metal complex (TEPE-Au) is dissolved in a solvent to obtain a TEPE-Au solution; then gold microparticles (AuMPs) solution is added to the TEPE-Au solution, and after stirring reaction (incubation), TEPE-Au modified AuMPs (TEPE-Au-AuMPs) are collected by centrifugation to obtain a SERS probe for monitoring redox reactions.

[0027] The concentration of the TEPE-Au solution is 1-3 mg / mL; preferably 2 mg / mL.

[0028] The solvent is a solvent obtained by mixing dimethyl sulfoxide (DMSO) and 95% (v / v) ethanol aqueous solution at a volume ratio of 1:9.

[0029] The content of AuMPs in the gold microparticle (AuMPs) solution is 50-200 / mL; preferably 100 / mL.

[0030] The volume ratio of the gold microparticle (AuMPs) solution and the TEPE-Au solution is 1:10.

[0031] The stirring reaction time is 20-40 minutes; preferably 30 minutes.

[0032] The gold microparticles (AuMPs) can be prepared by conventional methods in the art, such as the method described in Chinese patent (Patent No. 202210875549.X, entitled "A method for detecting copper ions using a silent zone SERS probe and its application"); preferably obtained by the following method:

[0033] The chloroauric acid aqueous solution is dissolved in a hydrochloric acid solution, then polyvinylpyrrolidone (PVP) and N-(3-nitrophenyl)-aniline (NAAN) are added, and the reaction is carried out at 2-8℃, and then centrifugal washing is performed to obtain gold microparticles (AuMPs).

[0034] The mass ratio of the chloroauric acid (HAuCl4), polyvinylpyrrolidone (PVP) and N-(3-nitrophenyl)-aniline (NAAN) is (10-20):(9-18):(18-54); preferably 20:18:18.

[0035] The concentration of the HAuCl4 solution is 5-25% by mass; preferably 10% by mass.

[0036] The concentration of the hydrochloric acid solution is preferably 1 mmol / L.

[0037] The volume ratio of the chloroauric acid aqueous solution to the hydrochloric acid solution is preferably 1:125.

[0038] The hydrochloric acid solution is a hydrochloric acid aqueous solution.

[0039] The molecular weight of the polyvinylpyrrolidone (PVP) is 40000-200000; preferably 40000.

[0040] The reaction time is 16-48 h; preferably 24 h.

[0041] The centrifugal washing condition is 1000 rpm centrifugation for 2-5 min; preferably 1000 rpm centrifugation for 2 min; and the centrifugal liquid is N-methylpyrrolidone (NMP).

[0042] The gold microparticles (AuMPs) are stored by being dispersed in deionized water and stored at 4℃.

[0043] The metal complex and / or the SERS probe for monitoring redox reactions are used for monitoring redox reactions or detecting alkaline phosphatase for non-disease diagnosis and treatment purposes.

[0044] A method for monitoring redox reactions by using a silent zone SERS frequency shift probe, comprising the following steps:

[0045] S1, qualitative detection

[0046] (1) SERS detection: the sample to be detected is added dropwise to a glass slide (carrier glass) covered with the SERS probe for monitoring redox reactions, and the reaction is allowed to stand at room temperature, and after the reaction is completed, it is naturally dried, and then SERS detection is performed to obtain a Raman spectrum;

[0047] (2) Judgment: According to the Raman spectrum, the peak intensity at 2115 cm -1 , 2107 cm -1 and 2123 cm -1 is obtained. If a new Raman peak is generated at the silent region 2123 cm -1 , and the Raman peak at 2115 cm -1 is reduced, it indicates that the sample contains oxidizing substances; if a new Raman peak is generated at the silent region 2107 cm -1 , and the Raman peak at 2115 cm -1 is reduced, it indicates that the sample contains reducing substances; if there is no obvious change at the silent region 2115 cm -1 , it indicates that the sample does not contain redox substances or is beyond the detection limit.

[0048] or

[0049] S2, quantitative detection

[0050] (3) At least 5 concentration gradients of ascorbic acid (AA) and potassium persulfate (PPS) aqueous solution are prepared respectively, and then are added dropwise to the glass covered with the above-mentioned SERS probe for monitoring redox reaction, and are left to react at room temperature. After the reaction is completed, it is naturally dried, and then SERS detection is carried out to obtain the Raman spectrum. Then the peak intensity at 2115 cm -1 , 2107 cm -1 and 2123 cm -1 is obtained according to the Raman spectrum, and the peak intensity ratio I 2115 / I 2107 and I 2115 / I 2123 is calculated. Finally, the concentration of ascorbic acid (AA) aqueous solution and the peak intensity ratio I 2115 / I 2107 are used to draw a standard curve A, and the concentration of potassium persulfate (PPS) aqueous solution and the peak intensity ratio I 2115 / I 2123 are used to draw a standard curve to obtain a linear equation B.

[0051] (4) The sample to be tested is added dropwise to the above-mentioned SERS probe for monitoring redox reaction, and is left to react at room temperature. After the reaction is completed, it is naturally dried, and then SERS detection is carried out to obtain the Raman spectrum. Then the peak intensity at 2115 cm -1 , 2107 cm -1 and 2123 cm -1 is obtained according to the Raman spectrum, and the peak intensity ratio I 2115 / I 2107 and I 2115 / I 2123 ; and finally, the concentration and / or content of the reducing substance or oxidizing substance is obtained according to the standard curve A or linear equation B.

[0052] The dropwise adding in steps (1)-(4) is achieved by using a pipette to drop the precipitate aqueous solution onto a glass slide, and then standing at room temperature until the water is completely volatilized, and then performing the next step of SERS detection.

[0053] The oxidizing substance in steps (2) and (4) is preferably potassium persulfate (PPS).

[0054] The reducing substance in steps (2) and (4) is preferably ascorbic acid (AA).

[0055] The concentration of the ascorbic acid (AA) aqueous solution in step (3) is in the range of 1×10 -6 mol / L to 1×10 -2 mol / L.

[0056] The concentration of the potassium persulfate (PPS) aqueous solution in step (3) is in the range of 1×10 -6 mol / L to 1×10 - 2 mol / L.

[0057] The ascorbic acid (AA) and potassium persulfate (PPS) aqueous solutions in step (3) are preferably prepared using DI pure water.

[0058] The reaction time in steps (1), (3) and (4) is 1-3 h; preferably 2 h.

[0059] The SERS detection conditions in steps (1), (3) and (4) are: excitation with 633 nm laser, recording the SERS spectrum change on a single SERS probe, power is 1 mW, and accumulation time is 2-8 seconds.

[0060] A method for detecting alkaline phosphatase (ALP) using a silent zone SERS frequency shift probe, comprising the following steps:

[0061] (I) mixing at least 5 concentration gradients of alkaline phosphatase (ALP) solution with L-ascorbic acid-2-phosphate trisodium salt (AA2P) solution, respectively, and performing a reaction (oscillating reaction) at 36-38°C (preferably 37°C) to obtain a mixed solution after reaction; then dropping the mixed solution onto a glass slide covered with the above-mentioned SERS probe for monitoring the redox reaction, standing at room temperature for reaction, and then naturally air-drying after the reaction is completed, and then performing SERS detection to obtain a Raman spectrum; and obtaining 2115 cm -1 , 2107 cm-1 the peak intensity at 2115 cm-1 and the peak intensity at 2107 cm-1, and calculate the peak intensity ratio I 2115 / I 2107 ; finally, the ALP concentration and the ratio I 2115 / I 2107 are drawn, and a linear equation C is obtained.

[0062] (II) adding L-ascorbic acid-2-phosphoric acid trisodium salt (AA2P) solution into the sample solution to be measured, and reacting (oscillating reaction) at 36-38°C (preferably 37°C) to obtain a mixed solution after reaction; then, the mixed solution is added dropwise to the glass covered with the SERS probe for monitoring the redox reaction, and is left to react at room temperature, and after the reaction is completed, it is naturally dried, and then SERS detection is performed to obtain a Raman spectrum; according to the Raman spectrum, the peak intensity at 2115 cm-1 and the peak intensity at 2107 cm-1 are obtained, and the peak intensity ratio I -1 / I -1 is calculated; 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 measured are calculated. 2115 2107

[0063] The alkaline phosphatase (ALP) solution in step (I) is a solution prepared by using Tris-HCl buffer solution.

[0064] The preparation method of the Tris-HCl buffer solution is as follows: 60.6 mg of Tris and 2 mg of MgCl2 are dissolved in 30 mL of water, the pH is adjusted to 8.0 by using concentrated hydrochloric acid, and finally water is added to make up to 50 mL.

[0065] The concentration of the alkaline phosphatase (ALP) solution in step (I) ranges from 1 mU to 100 mU.

[0066] The concentration of the L-ascorbic acid-2-phosphoric acid trisodium salt (AA2P) solution in steps (I) and (II) is 200-400 mmol / L; preferably 300 mmol / L.

[0067] The volume ratio of the L-ascorbic acid-2-phosphoric acid trisodium salt (AA2P) solution to the alkaline phosphatase (ALP) solution in step (I) is 1:9.

[0068] In steps (I) and (II), the reaction time at 36-38°C is 1-2 hours; preferably 1 hour.

[0069] In steps (I) and (II), the reaction time at room temperature is 2-3 hours; preferably 2 hours.

[0070] ​​The sample solution to be detected in step (II) is a solution prepared by using a Tris-HCl buffer solution.

[0071] The volume ratio of the L-ascorbic acid-2-phosphoric acid trisodium salt (AA2P) solution to the sample solution to be detected in step (II) is 1:9.

[0072] The SERS detection conditions in steps (I) and (II) are as follows: 633 nm laser excitation, recording of SERS spectrum change on a single SERS probe, power of 1 mW, and cumulative time of 2-8 seconds.

[0073] A method for realizing redox reaction classification by combining machine learning with Raman spectrum, specifically comprising the following steps:

[0074] (i) performing data enhancement on the Raman spectrum obtained in steps (2), (4) or (II) above to obtain more data;

[0075] (ii) performing baseline correction, noise reduction, normalization and dimension reduction processing on the Raman spectrum data obtained in step (i) to collect the preprocessed data;

[0076] (iii) performing data set division on the preprocessed data obtained in step (ii), training a machine learning model with a training set, testing the model with a test set, and obtaining model evaluation.

[0077] The data enhancement in step (i) is a data enhancement method of superimposing Gaussian noise, and Gaussian noise with a mean of 0 and a variance of 10 is superimposed on the newly generated spectrum.

[0078] The baseline correction processing in step (ii) is a correction processing by using an airPLS algorithm.

[0079] The noise reduction processing in step (ii) is a noise reduction processing by using a Savitzky-Golay smoothing filter algorithm.

[0080] The dimension reduction processing in step (ii) is a dimension reduction processing by using a principal component analysis (PCA) method.

[0081] The data set division method in step (iii) is as follows: 60% training set and 40% test set.

[0082] The machine learning model in step (iii) includes random forest (RF), decision tree (DT), gradient boosting decision tree (GBDT), K-nearest neighbor (KNN), support vector machine (SVM), logistic regression (LR) and the like.

[0083] The present application has the following advantages and effects over the prior art:

[0084] 1、The application uses gold particle self-assembly body with a layered structure as a SERS substrate, which has a large number of Raman enhancement 'hot spots', can significantly enhance the SERS signal, and can realize single-particle SERS detection, greatly improving the detection reproducibility.

[0085] 2、The application combines SERS detection technology and silent zone reporter molecules, has high sensitivity and high specificity, and is expected to bring important progress to the field of redox reaction monitoring, and has great development space and broad application prospect in the fields of biological, chemical detection, medical diagnosis and the like.

[0086] 3、The application introduces Au (I) susceptible to environmental stimulation at the other end of the alkynyl group, and changes the valence state by using the redox reaction, so as to change the Raman characteristic peak of the alkynyl group in the silent zone, and the change of the alkynyl group characteristic peak is linearly related to the oxidation or reduction substance, and then the oxidation or reduction substance is monitored.

[0087] 4、The application uses the displacement of the silent zone reporter molecule alkynyl group caused by the change of the chemical environment to monitor the oxidation and reduction, compared with the traditional SERS, the frequency shift SERS detection shifts the signal, rather than distinguishing the peak value in the noise, thereby reducing the requirement for spectral resolution.

[0088] 5、The application uses ALP to hydrolyze AA2P to generate AA, and then AA further changes Au (I) to Au (0), changes the dielectric environment of the alkynyl group connected with Au, makes the Raman characteristic peak of the alkynyl group shift, and the change of the alkynyl group characteristic peak is linearly related to ALP, and then ALP is detected. The silent zone frequency shift SERS detection method for ALP has not been reported, and the application detects in the Raman silent zone, which can avoid the influence of the peak spectrum of common physiological molecules and chemical groups in the fingerprint zone, greatly improving the accuracy and sensitivity of detection.

[0089] 6、The application combines the advantages of frequency shift SERS silent zone detection and machine learning data analysis, highly refines the information contained in the Raman spectrum, and further analyzes it, so that it can classify the oxidation and reduction reactions.

[0090] 7、The application provides a novel SERS method for efficient monitoring and classification of redox reactions and ALP diagnosis. Through careful design of the molecular structure, TEPE-Au has highly sensitive Raman characteristic peaks, 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 the TEPE-Au probe 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 DRAWINGS

[0091] Figure 1 Figure 1 is a morphology diagram of gold microparticles AuMPs prepared by the application; wherein (A) is a representative SEM image of AuMPs; (B) is a high-resolution SEM image of a single particle.

[0092] Figure 2 Figure 2 is a Raman spectrum of the TEPE-Au complex prepared by the application.

[0093] Figure 3 Figure 3 is a mass spectrum of the TEPE-Au complex prepared by the application.

[0094] Figure 4 Figure 4 is a diagram showing the influence of the reducing agent ascorbic acid (AA) and the oxidizing agent potassium persulfate (PPS) on the SERS spectrum of the TEPE-Au complex; wherein (A) is the reaction of the TEPE-Au complex with AA; (B) is the reaction of the TEPE-Au complex with PPS; (C) is the TEPE-Au complex.

[0095] Figure 5 Figure 5 is a Raman light spectrum of the detection of different concentrations of potassium persulfate and ascorbic acid using the SERS probe; wherein (A) is different concentrations of potassium persulfate; (B) is different concentrations of ascorbic acid.

[0096] Figure 6 Figure 6 is a linear relationship diagram of the SERS probe with potassium persulfate and ascorbic acid; wherein (A) is a linear relationship diagram of the SERS probe with potassium persulfate; (B) is a linear relationship diagram of the SERS probe with ascorbic acid.

[0097] Figure 7 Figure 7 is a fluorescence (FL) spectrum of the TEPE-Au complex measured under different redox stimuli.

[0098] Figure 8The redox state of Au(I) is changed by a redox stimulus, and the corresponding X-ray photoelectron spectroscopy (XPS) spectrum is measured; wherein, (A) is the XPS spectrum after the reaction of TEPE-Au and AA; (B) is the XPS spectrum after the reaction of TEPE-Au and PPS.

[0099] Figure 9 The detection result graph of the SERS probe for detecting standard ALP samples with different concentrations is shown in Figure 2; wherein, (A) is a Raman light spectrum; (B) is a linear curve.

[0100] Figure 10 The principal component analysis (PCA) result graph of the SERS spectrum data is shown in Figure 3; wherein, (A) is the principal component analysis result of TEPE-Au before and after redox and the three categories thereof (the calculated scores are presented in the form of a graph in 3D projection: PC1 vs PC2 vs PC3); (B) is the confusion matrix of the KNN classification result of the three categories.

[0101] Figure 11 The preparation and application process of the SERS probe of the present application is shown in Figure 4. DETAILED DESCRIPTION

[0102] The present application will be further described in detail below in conjunction with examples, but the embodiments of the present application are not limited thereto. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field. The test methods in the following examples, unless otherwise specified, are usually carried out according to conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present application are commercially available.

[0103] The conditions of the SERS test involved in the embodiments of the present application are as follows: a 633 nm laser is used for excitation, and the SERS spectrum on a single AuMP is recorded, the power is 1 mW, and the cumulative time is 4 seconds. For each sample, 3 SERS spectra of AuMPs are obtained for standard deviation calculation.

[0104] The N-(3-amidino)-aniline (NAAN) involved in the embodiments of the present application 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.).

[0105] Example 1

[0106] (1) Synthesis of Layered Structure Gold Microparticles

[0107] Into 959 μL of 1 mM HCl solution containing 8 μL of 10% mass concentration of chloroauric acid, 25 μL of 32 mg / mL polyvinylpyrrolidone (PVP) (molecular weight of 40000) was added, and 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 mixing by vortexing, the mixed solution was left to react at 4°C for 24 hours without further stirring. The reaction was centrifuged at 1000 rpm for 2 min, and the collected particles were washed with N-methylpyrrolidone (NMP) and deionized water (DI water) in sequence 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 structure gold microparticles AuMPs is shown in FIG. 1. Figure 1

[0108] (2) Synthesis of Surface-Enhanced Raman Scattering (SERS) Reporter Molecule for Monitoring Redox Reaction

[0109] 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 chloro(1,3-di-mesityl-1H-imidazol-2(3H)-ylidene)gold(I) ((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 distillation to obtain a light yellow powder, which was dissolved in acetone and centrifuged at 10000 rpm for 2 min. The obtained supernatant was added dropwise to a n-hexane solution, and the precipitate was received and dried in a vacuum oven to obtain the TEPE-Au complex in the form of a light yellow solid. It was stored at 4°C in the dark. The Raman spectrum and mass spectrum of the prepared silent zone SERS reporter molecule are shown in FIGS. 2 and 3, respectively. Its chemical structural formula is shown in Formula I. Figure 2 Figure 3

[0110] Formula I.

[0111] Example 2

[0112] Example 2

[0113] ​​A surface-enhanced Raman scattering (SERS) probe detection method for monitoring redox reactions based on silent zone frequency shift reporting molecules, the specific steps are as follows:

[0114] (1) 10 uL of gold microparticles AuMPs prepared in Example 1 (about 100 / mL) were injected into 100 uL of mixed solvent (dimethyl sulfoxide (DMSO) and 95% ethanol aqueous solution with a volume ratio of 1:9) containing 2 mg / mL TEPE-Au complex, and after stirring for 30 minutes, TEPE-Au was attached to AuMPs. After centrifugation, TEPE-Au modified AuMPs were collected, and then 10 uL was dropped on a glass slide. Finally, 10 uL of different concentrations of oxidizing agents or reducing agents (DI water preparation) were added to the TEPE-Au modified AuMPs, and after reaction at room temperature for 2 hours, the SERS signals of the TEPE-Au modified AuMPs were measured. Set three times. The oxidizing agent and reducing agent are potassium persulfate (PPS) and ascorbic acid (AA), respectively, and the final concentration of both is: 1×10 -6 mol / L, 1×10 -5 mol / L, 1×10 -4 mol / L, 1×10 -3 mol / L, 1×10 - 2 mol / L, 1×10 -1 mol / L.

[0115] (2) Due to the Raman response of the alkynyl group to its microenvironment, changes in the central Au(I) redox state of the TEPE-Au complex can cause the shift of the Raman fingerprint spectrum of the alkynyl group. To verify this hypothesis, the effect of reducing agent ascorbic acid (AA) and oxidizing agent potassium persulfate (PPS) on the SERS spectrum of the TEPE-Au complex was verified. The fingerprint spectrum is shown in Figure 4 As shown in Figure 4 (B) in the fingerprint spectrum of the benzene ring, and compared with the TEPE-Au complex (C) in Figure 4 , the fingerprint spectrum of the -C≡C- bond shows a significant blue shift at 2123 cm -1 . As a comparison, the SERS spectrum of the TEPE-Au complex after incubation with AA, from 2115 cm −1 to 2107 cm −1 , a red shift of the -C≡C- bond was observed Figure 4The above results indicate that the redox reaction of Au(I) in TEPE-Au complex can induce the change of SERS spectrum of alkynyl bond. In addition, the SERS probe with the frequency shift in the silent region responds linearly to the concentration of potassium persulfate and ascorbic acid from 1×10 -6 mol / L increased 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).

[0116] (3) In order to prove that the TEPE-Au reporter molecule did undergo redox reaction, fluorescence characterization was also performed. The fluorescence (FL) spectra of the TEPE-Au complex were measured under different redox stimuli. Figure 7 As shown, the TEPE ligand has a maximum FL emission at 490 nm, while the FL emission of the TEPE-Au complex exhibits a significant red shift at 505 nm. The addition of AA to the TEPE-Au complex solution causes the emission spectrum to blue-shift to 496 nm, while PPS induces a 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 modulate the vibrational frequency by affecting the π electron delocalization on -C≡C-, thereby generating a blue-shifted SERS fingerprint of the -C≡C- group. AA can reduce Au (I) to Au (0), thereby inhibiting the coordination of Au with the alkynyl group, resulting in a SERS fingerprint of the -C≡C- group similar to that of the TEPE ligand.

[0117] (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, as shown in 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, which correspond 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 3), 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)4f5 / 2 Au (III) 4f 7 / 2 Au (III) 4f 7 / 2 Au (III) 4f 5 / 2 Au (III) 4f Figure 8 Au (III) 4f Au (III) 4f

[0118] Example 3 Au (III) 4f

[0119] A surface enhanced Raman scattering (SERS) probe detection method for detecting alkaline phosphatase (ALP) based on silent zone frequency shift reporter molecules, the specific steps are as follows: Au (III) 4f

[0120] 90 μL L-ascorbic acid-2-phosphoric acid trisodium salt (AA2P) (0.3 mol / L) and 10 μL alkaline phosphatase (ALP) (concentration: 1 mU / mL, 20 mU / mL, 40 mU / mL, 80 mU / mL, 100 mU / mL) were mixed in a 2 mL centrifuge tube containing 100 uL pH=8.0 Tris-HCl buffer. After 1 hour of reaction at 37℃, 10 μL of the resulting mixture was dropped onto a glass slide covered with TEPE-Au modified AuMPs (preparation method same as Example 2 step (1)) for 2 hours of reaction at room temperature. After the reaction was completed, it was naturally air-dried, and the SERS signal of the TEPE-Au modified AuMPs was finally measured to obtain the Raman spectrum after the reaction; according to the Raman spectrum, the peak intensity at 2115 cm -1 , 2107 cm -1 , the peak intensity ratio I 2115 / I 2107 was calculated, and finally the standard curve was drawn with ALP concentration and the ratio I 2115 / I 2107 , and the linear equation was obtained. Figure 9 .

[0121] Example 4 Au (III) 4f

[0122] A surface enhanced Raman scattering (SERS) probe detection method for detecting alkaline phosphatase (ALP) in serum based on silent zone frequency shift reporter molecules, the specific steps are as follows: Au (III) 4f

[0123] To the blank human serum sample, different concentrations of standard ALP sample were added respectively (so that the final concentrations in the serum sample were 20 mU / mL, 40 mU / mL and 80 mU / mL respectively, and the solvent was Tris-HCl buffer), then 90 μL of AA2P (0.3 mol / L) was added to 10 μL of the above human serum solution containing different concentrations of ALP, and after 1 hour of reaction at 37°C, 10 μL of the resulting mixture was dropped onto a glass slide covered with TEPE-Au modified AuMPs (preparation method same as step (1) of Example 2), and reacted for 2 hours at room temperature to obtain the sample to be tested. After drying at room temperature, SERS measurement was performed, and the concentration of ALP was calculated according to the linear curve (B) in the above Figure 9

[0124] The human serum solution was a blood sample of a healthy volunteer provided by the First Affiliated Hospital of Jinan University (Guangzhou, China). It was left to stand at room temperature for more than half an hour, and then centrifuged at 3500-4000 rpm for 5-10 minutes. The collected supernatant was diluted 1000 times with Tris-HCl solution (Tris-HCl solution: 60.6 mg Tris, 2 mg MgCl2 dissolved in 30 mL water, adjusted to pH 8.0 with concentrated hydrochloric acid, and finally diluted to 50 mL with water).

[0125] 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 indicates that the SERS-based analysis technology provided in this example can detect ALP with high sensitivity, and has broad application prospects in biological analysis and disease diagnosis.

[0126] Table 1 Detection of ALP content in serum samples by SERS-based analysis technology

[0127]

[0128] Example 5

[0129] A redox classification method combining Raman spectroscopy and machine learning, the specific steps are as follows:

[0130] ​The SERS spectral data collected in Example 2 were then pre-processed in three steps: smoothing and denoising, baseline correction, and normalization. First, we collected 145 redox reaction data and augmented them to 435 redox reaction data using a data augmentation method with random additive Gaussian noise, and then denoised the collected spectral data using the Savitzky-Golay (SG) algorithm. Subsequently, the adaptive iteratively reweighted penalized least squares (airPLS) algorithm was used to correct the baseline. Before building the classification model, each spectrum was pre-processed by applying normalization. In addition, since the dimensionality of the spectrum is high and there can be irrelevant information, principal component analysis (PCA) was used to effectively reduce the dimensionality of the data and retain its key information. The purpose of applying PCA was to capture at least 95% of the cumulative contribution rate, ensuring that a large portion of the variance in the data was retained. After data pre-processing, a classic machine learning algorithm was used to build a classification model, and all procedures were implemented using the Scikit-learn library in Python programming language. The PCA results and model classification results are shown in Table 2 and Figure 2. Figure 10 and Table 2.

[0131] Table 2 Classification results evaluation of various classic machine learning models

[0132]

[0133] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

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, wherein The steps include: 1,2-bis(4-ethynylphenyl)-1,2-diphenylethylene is dissolved in a mixed solution of degassed tetrahydrofuran and methanol, and then (1,3-di-trimethylphenyl-1H-imidazole-2(3H)-ylidene)gold(I) chloride and sodium hydroxide are added under a protective gas atmosphere, and the reaction is stirred. After the reaction is completed, the mixture is filtered, the filtrate is collected, the solvent is removed by rotary evaporation, and the filtrate is dissolved in acetone, centrifuged, and the supernatant is collected and added dropwise to a n-hexane solution. Finally, the obtained precipitate is dried to obtain the metal complex; The mass ratio of the 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 to 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, wherein: The steps include: The metal complex according to 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 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 microparticle AuMPs solution is 50 to 200 particles / 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: the sample to be tested is added dropwise onto a glass slide covered with the SERS probe for monitoring redox reactions according to claim 4, 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 quiet 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 is reduced, it means that the sample contains reducing substances; if the Raman peak at 2115 cm is reduced, it means that the sample contains reducing substances; -1 There is no obvious change, indicating that the sample does not contain redox substances or exceeds the detection limit; or S2. Quantitative detection (3) Ascorbic acid and potassium persulfate aqueous solutions with at least 5 concentration gradients were prepared respectively, and then added dropwise to a glass slide covered with the SERS probe for monitoring redox reaction according to claim 4, and allowed to react at room temperature. After the reaction was completed, the solution was naturally dried, and then SERS detection was performed to obtain a Raman spectrum. Then, the Raman spectrum was obtained at 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 of ascorbic acid aqueous solution and the peak intensity ratio 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) The sample to be tested is added dropwise onto the glass slide of the SERS probe for monitoring redox reaction according to claim 4, 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.

8. The method according to claim 7, wherein: 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) mixing alkaline phosphatase solutions of at least five concentration gradients with L-ascorbic acid-2-phosphate trisodium salt solutions, respectively, and 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 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 of the alkaline phosphatase solution in step (I) ranges from 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 and the alkaline phosphatase solution described 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 described 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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