SERS / fluorescent bimodal composite probe, preparation method and application thereof, and chiral glucose detection method

Abstract

The invention belongs to the technical field of biological medicine detection, and particularly relates to an SERS / fluorescent bimodal composite probe, a preparation method and application thereof and a chiral glucose detection method, the composite probe is a gold nanorod modified by a tetraphenylethylene nuclear derivative, and the tetraphenylethylene nuclear derivative is a TPE nuclear molecular probe with AIE attributes; the tetraphenyl ethylene core derivative is prepared by reacting tetrabromo tetraphenyl ethylene with bis (pinacolato) diboron, oxidizing, and reacting with p-mercaptophenylboronic acid. Compared with the prior art, the method has the advantages that the limitations of long time consumption, insufficient sensitivity or specificity deficiency of the traditional chiral molecule identification method in the prior art are solved; and SERS (Surface Enhanced Raman Scattering) and fluorescence analysis are lack of methods and materials suitable for chiral molecule identification. According to the scheme, the composite nanoprobe can form a sandwich structure with molecules of an object to be detected, and rapid detection of chiral glucose is realized according to SERS intensity, fluorescence intensity and color change of the composite nanoprobe.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection technology, and specifically relates to a SERS / fluorescence dual-modal composite probe, a preparation method and application thereof, and a method for detecting chiral glucose. Background Art

[0002] Chirality, a fundamental characteristic of living systems, is also a key form of biological information storage. Although the enantiomers of chiral substances have identical chemical compositions, their biological activities and pharmacological effects can differ significantly, or even be opposite, due to the homochiral nature of biomolecules. Therefore, enantiomeric identification is of great significance in biochemistry, synthetic chemistry, and analytical chemistry, particularly in pharmacy, biology, environmental science, catalytic synthesis, and disease diagnostics.

[0003] Currently, most of the methods reported at home and abroad for determining chiral glucose molecules use technologies such as circular dichroism (CD), nuclear magnetic resonance (NMR), electrochemical analysis, and high-performance liquid chromatography (HPLC). However, these methods have the limitations of being time-consuming, insufficiently sensitive, or lacking specificity, making it difficult to meet the needs of accurate identification of chiral molecules in complex systems.

[0004] Surface-enhanced Raman spectroscopy (SERS) has become an important tool for chemical identification due to its high sensitivity and molecular fingerprint specificity. SERS enhances Raman signals, providing rich molecular vibrational information without the need for complex pre-processing, offering the advantage of non-destructive testing. Fluorescence analysis generally offers advantages such as high sensitivity and good selectivity, enabling the analysis of trace and micro-amounts of substances in complex samples. It is currently widely used in fields such as biomedicine and organic chemistry.

[0005] SERS and fluorescence analysis have been widely applied and studied in the prior art. For example, CN113740311B discloses a metal-dielectric composite probe SERS substrate and its preparation method, and CN119985443A discloses a gold-silver alloy nanoprobe based on dual-signal synergistic detection, its preparation method, and its application. However, their application in the qualitative and quantitative detection of chiral molecules has not been seen. Furthermore, CN115260205B discloses a bispyrenyl-doped extended porphyrin, its bispalladium metal complex, its preparation method, and its application, specifically disclosing compounds that can be used for chiral molecule recognition and detection. However, according to the detailed description, these compounds are used in HPLC to achieve enantiomer resolution, which still has the limitations of being time-consuming, insufficient sensitivity, or lacking specificity.

[0006] Therefore, in response to the increasing demand for rapid detection, developing a simple and stable method for the accurate distinction of chiral substances is both a challenge and a key goal. Summary of the Invention

[0007] The present invention aims to address at least one of the above-mentioned problems by providing a SERS / fluorescence dual-modal composite probe, its preparation method, and application, as well as a method for detecting chiral glucose. This approach addresses the limitations of conventional chiral molecule identification methods in the prior art, including CD, NMR, electrochemical analysis, and HPLC, which are time-consuming, insensitive, or lacking specificity, making it difficult to accurately identify chiral molecules in complex systems. Furthermore, SERS and fluorescence analysis lack methods and materials suitable for chiral molecule identification. The proposed composite nanoprobe, GNRs@TPEND, exhibits AIE properties and can form a sandwich structure with analyte molecules. Based on its SERS intensity, fluorescence intensity, and color changes, it enables rapid detection of chiral glucose in the biomedical field.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The first aspect of the present invention discloses a SERS / fluorescence dual-modal composite probe, wherein the composite probe is a gold nanorod modified with a tetraphenylethylene core derivative, wherein the tetraphenylethylene core derivative is a TPE core molecular probe with AIE properties;

[0010] The tetraphenylethylene core derivative is prepared by reacting tetrabromotetraphenylethylene with biboronic acid pinacol ester, followed by oxidation, and then reacting with p-mercaptophenylboronic acid (three-step organic synthesis).

[0011] Preferably, the average length of the gold nanorods is between 60 and 100 nm.

[0012] The second aspect of the present invention discloses a method for preparing the SERS / fluorescence dual-modal composite probe as described above, comprising the following steps:

[0013] S1: Preparation of gold nanorods: Synthesis of gold nanorods by seed growth method;

[0014] S2: Synthesis of tetraphenylethylene core derivative TPEND:

[0015] S21: adding tetrabromotetraphenylethylene, bipyralidoborane, and the first palladium catalyst to the first mixed solution to react, and obtaining TPE-Bpin2-2Br through post-treatment;

[0016] S22: adding TPE-Bpin2-2Br and sodium periodate to the second mixed solution to react, and obtaining TPE-B(OH2)2-2Br through post-treatment;

[0017] S23: adding TPE-B(OH2)2-2Br, p-mercaptophenylboronic acid, and the second palladium catalyst to the third mixed solution to react, and obtaining TPEND through post-treatment;

[0018] S3: Modification of gold nanorods with tetraphenylethylene core derivatives:

[0019] The gold nanorods were dispersed in pure water and TPEND was added, stirred and mixed, dialyzed and centrifuged to obtain the gold nanorods modified with the tetraphenylethylene core derivative.

[0020] Preferably, step S1 specifically includes the following steps:

[0021] S11: Prepare a gold nanoparticle seed solution: add 0.01±0.002 mol / L tetrachloroauric acid aqueous solution to 0.1±0.05 mol / L hexadecylammonium bromide aqueous solution, and after uniform dispersion, quickly add 0.01±0.002 mol / L sodium borohydride solution in an ice-water bath (0±3°C), stir evenly, and allow to stand to obtain a gold nanoparticle seed solution;

[0022] The volume ratio of tetrachloroauric acid aqueous solution, hexadecylammonium bromide aqueous solution and sodium borohydride solution is 0.25±0.05:9.75±0.25:0.6±0.02;

[0023] S12: preparing a gold nanorod solution: adding a 0.01±0.002 mol / L tetrachloroauric acid aqueous solution to a 0.1±0.002 mol / L hexadecyltrimethylammonium bromide aqueous solution, mixing well, adding 0.01±0.002 mol / L AgNO3, 1 M HCl, and 0.1±0.002 mol / L ascorbic acid, and then adding the gold nanoseed solution, stirring well, and allowing to stand to obtain a gold nanorod solution;

[0024] The volume ratio of tetrachloroauric acid aqueous solution, hexadecyltrimethylammonium bromide aqueous solution, AgNO3, HCl, ascorbic acid and gold nanoseed solution is 0.5±0.05mL:10±0.05mL:0.1±0.05mL:0.2±0.05mL:80±1μL:10~15μL;

[0025] S13: Purifying the gold nanorod solution: centrifuging the gold nanorod solution, collecting the sol and washing it with pure water, cooling it to crystallize hexadecyltrimethylammonium bromide, and obtaining gold nanorods.

[0026] Preferably, step S2 specifically includes the following steps:

[0027] S21: Potassium acetate is added to anhydrous dioxane and mixed and bubbled to obtain a first mixed solution; under a protective atmosphere, tetrabromotetraphenylethylene, diboronic acid pinacol ester and [1,1-bis(diphenylphosphino)ferrocene] palladium dichloride (CAS: 72287-26-4) are added to the first mixed solution, degassed, and refluxed at 100-120° C. for 68-78 hours; after the reaction, anhydrous dioxane is removed by vacuum distillation, ice water is added and stirred, and then filtered, washed with water and vacuum dried in sequence; re-dissolved in chloroform and purified by chromatography, the solvent is removed by vacuum distillation, resuspended in methanol, filtered and washed to obtain TPE-Bpin2-2Br;

[0028] The usage ratio of potassium acetate, anhydrous dioxane, tetrabromotetraphenylethylene, pinacol diboron and [1,1-bis(diphenylphosphino)ferrocene] palladium dichloride is 7.6±0.1g:150±5mL:5g:5.88g:200mg;

[0029] S22: Sodium periodate is mixed with a mixture of THF / water / ethyl acetate to obtain a second mixed solution. TPE-Bpin2-2Br is added to the second mixed solution under a protective atmosphere. After degassing, the mixture is refluxed at 80-90° C. for 24-30 hours until the solution turns clear yellow. After cooling in an ice bath, 1M HCl solution is added, stirred, and allowed to stand overnight. The organic solvent is evaporated under vacuum, and water is added, stirred, and the precipitate is collected, washed, and dried under vacuum to obtain TPE-B(OH2)2-2Br.

[0030] The dosage ratio of sodium periodate, mixed solution and TPE-Bpin2-2Br is 6.8 g:30±5 mL:2 g;

[0031] S23: TPE-B(OH2)2-2Br is mixed with p-mercaptophenylboronic acid, and tetrakis(triphenylphosphine)palladium is added under a protective atmosphere, followed by the addition of a third mixed solution consisting of toluene and a 2 mol / L potassium carbonate / potassium fluoride mixed solvent. The mixture is stirred and reacted at 85-100° C. for 48-60 h. Water is added to terminate the reaction, and dichloromethane is added for extraction. The lower organic phase is separated, rotary evaporated, and purified by chromatography to obtain TPEND.

[0032] The usage ratio of TPE-B(OH2)2-2Br, p-mercaptophenylboric acid, tetrakis(triphenylphosphine)palladium, toluene and potassium carbonate / potassium fluoride mixed solvent is 1 g: 0.31 g: 25 mg: 50-70 mL: 2-5 mL.

[0033] Preferably, step S3 specifically includes the following steps:

[0034] After gold nanorods were dispersed in pure water, 12.8±0.5 mg / mL TPEND was added, the mixture was stirred and mixed, and dialyzed for 20-30 h using a dialysis membrane with a molecular weight cutoff of 8-14 kDa and centrifuged to obtain gold nanorods modified with tetraphenylethylene core derivatives.

[0035] The third aspect of the present invention discloses an application of the SERS / fluorescence dual-modal composite probe described above in chiral glucose detection.

[0036] A fourth aspect of the present invention discloses a method for detecting chiral glucose, comprising the following steps:

[0037] The SERS / fluorescence dual-modal composite probe described above was added to a K3[Fe(CN)6] buffer solution, the pH was adjusted, and then the glucose solution to be tested was added, and chiral recognition of glucose was performed by SERS and fluorescence detection.

[0038] Preferably, the concentration of the K3[Fe(CN)6] buffer solution is 0.1±0.02 mM; the concentration of the SERS / fluorescence dual-modal composite probe is 1±0.2 mg / mL; the volume ratio of the SERS / fluorescence dual-modal composite probe to the glucose solution to be tested is 1:1;

[0039] and / or,

[0040] The SERS was carried out under the conditions of 785 nm laser, 40 mW power and 1-60 s integration time, using 1569 ± 2 cm -1 As the characteristic peak for determining the chiral glucose signal; the fluorescence detection is carried out at an excitation wavelength of 365nm, and 460±2nm is used as the characteristic peak for determining the chiral glucose signal.

[0041] Preferably, the detection method quantitatively determines the concentration of chiral glucose in the glucose solution to be tested by a machine learning method, wherein the machine learning method includes support vector regression.

[0042] The working principle of the present invention is:

[0043] Gold nanorods were modified using organically synthesized TPEND. After the addition of glucose solution, TPEND, as a Raman reporter molecule, was located between the gold nanorods (GNRs) and the chiral glucose molecules. By adjusting its arrangement direction on the GNRs surface, SERS discrimination of D- and L-glucose was achieved: TPEND reacted with D-glucose to activate the AIE effect and enhance fluorescence, while L-glucose had no obvious fluorescence change. Therefore, the SERS and fluorescence responses increased with D and decreased with L. Furthermore, chiral glucose could be qualitatively and quantitatively analyzed based on the intensity of the fluorescence color.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1. A chiral glucose recognition probe material was constructed. This dual-channel sensor utilizes SERS and fluorescence. Gold nanorods, organic molecules, and the analyte form a sandwich-like sensing structure. The gap between the gold nanorods and the glucose molecules generates an extremely strong localized electric field. The presence of "hotspots" ensures high sensitivity and operability even at low concentrations. This method requires no complex pretreatment, offers the advantages of nondestructive testing, and is simple to prepare and readily available for widespread use.

[0046] 2. Based on the composite nanoprobe, SERS and fluorescence dual-mode are used to quickly detect chiral glucose molecules. The detection range is very wide (0-1000 μM), which is more intuitive and has the characteristics of simple operation, rapid analysis and instant detection. It can meet the detection needs of chiral glucose in the biopharmaceutical market; it can be combined with visual colorimetry to compare the sample fluorescence color with the standard color scale to intuitively and quickly determine the sample concentration. The TPEND prepared by the present invention can selectively bind to glucose. Under weak alkaline conditions (pH = 7.5-9), the two undergo a specific reversible complexation reaction to form a cyclic borate complex. This SERS and fluorescence dual-mode sensing probe shows excellent performance in the fingerprint recognition and enantioselective detection of chiral molecules, and has the advantages of wide detection range and rapidity.

[0047] 3. The nanoprobes of this invention exhibit fluorescence, enabling precise identification of glucose enantiomers in mixed systems or complex samples. In the presence of D-glucose, fluorescence is significantly enhanced due to AIE activation; conversely, in the presence of L-glucose, fluorescence is weak. This enables the detection of chiral glucose with similar characteristic peaks, with advantages such as low detection limits and minimal interference.

[0048] 4. The present invention further utilizes machine learning calculations to perform support vector regression analysis on a large amount of sample test data. It has strong generalization capabilities, is resistant to overfitting, and is suitable for small samples. It facilitates big data training of the analytes and can help accurately predict the glucose concentration in actual samples.

[0049] In summary, the SERS / fluorescence method for chiral glucose detection is characterized by ease of operation, high sensitivity, strong anti-interference ability, and a wide range of applications. This method provides important reference value for establishing real-time, rapid, highly selective, and highly sensitive analytical detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 4 is a flow chart of the preparation and monitoring method of the composite probe in Example 1 of the present invention.

[0051] Figure 2This is a transmission electron microscope (TEM) image of the GNRs@TPEND in Example 1 of the present invention.

[0052] Figure 3 3. It is the UV-visible absorption spectra of GNRs, TPEND, and GNRs@TPEND of Example 1 of the present invention.

[0053] Figure 4 This is the SERS quantitative analysis diagram (a) and linear fitting result (b) of detecting D-glucose based on the GNRs@TPEND composite probe in Example 1 of the present invention.

[0054] Figure 5 This is the SERS quantitative analysis diagram (a) and linear fitting result (b) of detecting L-glucose based on the GNRs@TPEND composite probe in Example 1 of the present invention.

[0055] Figure 6 1 is a fluorescence quantitative analysis diagram (a) and a linear fitting result (b) of detecting D-glucose based on the GNRs@TPEND composite probe in Example 1 of the present invention.

[0056] Figure 7 1 is a fluorescence quantitative analysis diagram (a) and a linear fitting result (b) of detecting L-glucose based on the GNRs@TPEND composite probe in Example 1 of the present invention.

[0057] Figure 8 This is a calibration curve of the D-glucose concentration spiked in urine obtained based on the machine learning SVM-R analysis method in Example 1 of the present invention, and the prediction results using GNRs@TPEND and a single receptor platform (GNRs, Comparative Example 1).

[0058] Figure 9 This is a comparison result of the characteristic peak intensity of the SERS signal of chiral glucose based on GNRs@TPEND and a single receptor platform (TPEND, comparative example 2) in Example 1 of the present invention (a) and a SERS spectrum of chiral glucose detected based on GNRs@TPEND (b). DETAILED DESCRIPTION

[0059] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0060] In the following description, unless otherwise specified, the reagents used may be conventional commercial products, the methods used are conventional means in the art, and matters not mentioned may be prior art.

[0061] A SERS / fluorescence dual-modality method for rapid detection of chiral glucose, such as Figure 1As shown in the figure, the steps are as follows: (1) prepare gold nanorods; (2) prepare organic probe tetraphenylethylene core derivatives; (3) synthesize tetraphenylethylene core derivative-modified gold nanorods (GNRs@TPEND); (4) based on the GNRs@TPEND composite probe, chiral glucose (D-glucose and L-glucose) is detected by surface enhanced Raman spectroscopy (SERS) and fluorescence spectroscopy.

[0062] The specific steps are as follows:

[0063] (1) Gold nanorods (GNRs) were prepared and purified in aqueous solution using a seed-based growth strategy;

[0064] (2) Preparation of TPE core molecular probes with AIE properties;

[0065] (3) The two materials in step (1) and step (2) are composited to synthesize GNRs@TPEND nanoprobes, which are then placed in a 0.1±0.02 mM K3[Fe(CN)6] buffer solution and the pH is adjusted to the optimal experimental state. Then, freshly prepared D-glucose or L-glucose of different concentrations are added to the solution, and chiral recognition reactions are carried out at room temperature. A calibration curve is constructed by SERS spectroscopy and fluorescence detection. The sample to be tested is dropped onto a silicon wafer to measure the SERS spectrum, and the fluorescence detection is recorded using a fluorescence spectrophotometer; by comparing the spectral results with the linear fit, an instant and rapid qualitative and quantitative analysis of chiral glucose is achieved.

[0066] (4) In the detection of actual samples, the human blood glucose and urine glucose concentrations can be detected according to the detection principle in step (3), and the concentration of the analyte can be predicted and analyzed through machine learning calculations.

[0067] In step (1), the average length of the gold nanorods is between 60 and 100 nm; Figure 2 , showing the TPEND organic layer wrapped around the gold nanorods.

[0068] The TPEND in step (2) is an organic molecule with aggregation-induced emission (AIE) properties. By leveraging the unique recognition ability between the TPEND molecule and glucose, a sandwich sensing structure consisting of the organic molecule and the analyte is designed.

[0069] In step (3), the concentration range of the newly prepared D-glucose and L-glucose of different concentrations is 0 to 1000 μM, and the concentration of the nanoprobe is 1±0.2 mg / mL; after the sample to be tested is mixed with the nanoprobe and reacted for 10 to 15 minutes, its SERS and fluorescence spectra are obtained, and linear fitting is performed based on the spectral data, thereby achieving instant and rapid qualitative and quantitative analysis of chiral glucose.

[0070] In step (4), human blood and urine samples require certain pre-processing, which can be done using existing processing methods. The support vector regression (SVM-R) model is used as the calculation and prediction analysis method.

[0071] This method enables instant and rapid qualitative and quantitative analysis of chiral glucose by comparing the intensity of the SERS / fluorescence characteristic peak with the fluorescence intensity under ultraviolet light. This method offers low cost, simple instrumentation, rapid analysis, high sensitivity, low sample volume, and a wide range of applications, enabling instant and rapid detection of chiral glucose.

[0072] More specific:

[0073] (1) Preparation and purification of gold nanorods in aqueous solution using a seed growth strategy

[0074] Preparation of gold nanoseed solution: At room temperature (25-28°C), prepare 9.75±0.25mL of 0.1±0.05mol / L hexadecylammonium bromide aqueous solution, stir evenly until transparent, add 0.25±0.05mL of 0.01±0.002mol / L tetrachloroauric acid aqueous solution dropwise, wait until it is evenly dispersed in the solution, and quickly add 0.6±0.02mL of freshly prepared 0.01±0.002mol / L NaBH4 solution at 0±3°C. The mixed solution changes from light yellow to brownish yellow, stir evenly for 3-5min, and stand at room temperature for 2-3h before use.

[0075] Preparation of gold nanorod solution: At room temperature, prepare 10±0.05mL of 0.1±0.002mol / L hexadecyltrimethylammonium bromide aqueous solution, then add 0.5±0.05mL of 0.01±0.002mol / L tetrachloroauric acid aqueous solution, mix well, then add 0.1±0.05mL of 0.01±0.002mol / L AgNO3 and 0.2±0.05mL of 1M HCl, stir well, add 80±1μL of 0.1±0.002mol / L ascorbic acid, the solution changes from dark yellow to colorless, add 10-15μL of the above-prepared gold seed solution, stir evenly for 3-5min, and let stand at room temperature for 5-8h.

[0076] Purification of gold nanorod solution: The prepared gold nanorod solution is centrifuged at 7500-8500 rpm for 5-8 minutes to remove excess solutes in the solution, enrich the sol, wash with pure water, reduce the temperature to 6-10°C, remove the crystallized CTAB, and obtain the gold nanorod material for use.

[0077] (2) Synthesis of organic chemical molecule TPEND

[0078] In a dry 300 mL two-necked round-bottom flask, mix 150 ± 5 mL of anhydrous dioxane and 7.6 ± 0.1 g (~0.08 mol) of potassium acetate and bubble for 30–40 minutes. Under argon, add 5 g (0.008 mol) of TPE-Br4, 5.88 g (0.034 mol) of pinacol diboron, and 200 mg (0.37 mmol) of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride. After degassing with a refrigeration pump three times, reflux at 100–120°C for 68–78 hours (monitor progress with thin-layer chromatography). After cooling, evaporate the dioxane under reduced pressure. Pour the mixture into ice water and stir for 10–15 minutes. Filter, wash the residue with water, and dry it in vacuo. The filter residue was dissolved in chloroform and purified by flash column chromatography using ethyl acetate / petroleum ether (1:10) as eluent. After removing the solvent under reduced pressure, the product was suspended in 200-250 mL of methanol, collected, filtered, washed, and dried to obtain the pure product TPE-Bpin2-2Br.

[0079] In a 50 mL two-necked round-bottom flask, add 30±5 mL of a mixture of THF / water / ethyl acetate (3:1:1, v:v:v) and 6.8 g of NaIO4. Stir under argon at room temperature for 25-35 min, then add 2 g of TPE-Bpin2-2Br. After degassing three times, reflux at 80-90°C for 24-30 h (monitored by thin-layer chromatography until the solution turns clear and yellow). Cool in an ice bath, add 20 mL of a 1 M HCl solution at 0-10°C, and stir vigorously. After incubating at 25-28°C overnight, evaporate the organic solvent in vacuo. Add 150±5 mL of water, stir, collect the white precipitate, wash three times with water and chloroform, and dry in vacuo at room temperature for 24-36 h to obtain a white TPE-B(OH2)2-2Br powder.

[0080] To a 150 mL three-necked flask, add 1 g (1.56 mmol) of TPE-B(OH2)2-2Br and 0.31 g (2.52 mmol) of p-mercaptophenylboronic acid. After three evacuations and nitrogen filling, add 25 mg of tetrakis(triphenylphosphine)palladium. Then, inject 50-70 mL of toluene and 2-5 mL of a 2 mol / L potassium carbonate / potassium fluoride mixture. Stir and react at 85-100°C for 48-60 hours. Terminate the reaction with water. Extract the lower organic phase with dichloromethane, rotary evaporation, and purification by silica gel column chromatography to obtain TPEND as a yellow solid.

[0081] (3) Gold nanorods modified with tetraphenylethylene core derivatives (GNRs@TPEND)

[0082] To further advance the surface modification process, the 40 mL GNRs solution prepared in (1) was centrifuged 2 to 3 times (10,000 to 14,000 rpm, 15 to 20 min) to remove excess impurities. The purified GNRs were redispersed in 40 mL of pure water. Next, 5 mL of TPEND aqueous solution with a concentration of 12.8 ± 0.5 mg / mL was slowly added to the above GNRs solution and stirred continuously at room temperature for 16 to 20 h. Afterwards, the mixed solution was dialyzed in water for 20 to 30 h through a dialysis membrane with a molecular weight cutoff of 8 to 14 kDa to promote the full binding of TPEND to the GNRs surface. Finally, the GNRs@TPEND was purified by centrifugation and redispersed in an aqueous solution for use.

[0083] (4) SERS and fluorescence dual-mode detection of chiral glucose based on GNRs@TPEND composite probe

[0084] The prepared GNRs@TPEND nanoprobes were placed in a 0.1±0.02mM K3[Fe(CN)6] buffer solution and the pH was adjusted to the optimal experimental condition (under weak alkaline conditions, typically pH = 7.5-9). Next, freshly prepared D-glucose or L-glucose was added to the solution, and a chiral recognition reaction was carried out at room temperature. After the reaction, the sample was thoroughly rinsed with deionized water to prepare for subsequent SERS and fluorescence detection. The results showed that different concentrations of chiral glucose produced different SERS signals and fluorescence changes.

[0085] When detecting glucose, 1 mL of glucose solution was mixed with 1 mL of nanoprobe with a concentration of 1±0.2 mg / mL and reacted for 10 minutes, and 5 μL of the mixture was dropped on the surface of the silicon wafer.

[0086] SERS measurement, such as using a portable Raman spectrometer: use 785nm laser, 40mW power and 1-60s integration time to measure SERS spectrum, using 1569±2cm -1 As the characteristic peak for determining chiral glucose signal.

[0087] Fluorescence detection was performed using a fluorescence spectrophotometer to record the fluorescence spectra of D-glucose at different concentrations at an excitation wavelength of 365 nm, and 460±2 nm was used as the characteristic peak for determining the chiral glucose signal.

[0088] Example 1

[0089] (1) Preparation and purification of gold nanorods in aqueous solution using a seed growth strategy

[0090] Prepare the gold nanoparticle seed solution: Prepare 9.75 mL of 0.1 mol / L hexadecylammonium bromide aqueous solution at room temperature (25-28°C). Stir thoroughly until transparent. Add 0.25 mL of 0.01 mol / L tetrachloroauric acid aqueous solution dropwise. Once uniformly dispersed, quickly add 0.6 mL of freshly prepared 0.01 mol / L NaBH₄ solution at 0°C. The mixture will turn from light yellow to brownish yellow. Stir thoroughly for 3 minutes and let stand at room temperature for 2 hours before use. The gold concentration at this point is 0.25 mmol.

[0091] Preparation of gold nanorod solution: At room temperature, prepare 10 mL of 0.1 mol / L hexadecyltrimethylammonium bromide aqueous solution, then add 0.5 mL of 0.01 mol / L tetrachloroauric acid aqueous solution, mix well, then add 0.1 mL of 0.01 mol / LAgNO3 and 0.2 mL of 1 M HCl, stir thoroughly, add 80 μL of 0.1 mol / L ascorbic acid, the solution changes from dark yellow to colorless, add 12 μL of the above-prepared gold seed solution, stir evenly for 3 minutes, and let stand at room temperature for 6 hours.

[0092] Purification of the gold nanorod solution: The prepared gold nanorod solution was centrifuged at 8000 rpm for 5 min to remove excess solutes in the solution, and the sol was enriched and washed with pure water. The temperature was lowered to 10° C. to remove the crystallized CTAB, and the gold nanorod material was obtained for use.

[0093] (2) Synthesis of organic chemical molecule TPEND

[0094] In a dry 300 mL two-necked round-bottom flask, 150 mL of anhydrous dioxane and 7.6 g (0.08 mol) of potassium acetate were mixed and bubbled for 30 minutes. Under argon, 5 g (0.008 mol) of TPE-Br4, 5.88 g (0.034 mol) of pinacol diboron, and 200 mg (0.37 mmol) of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride were added sequentially. After degassing with a refrigeration pump three times, the reaction was refluxed at 110°C for 72 hours (progress monitored by thin-layer chromatography). After cooling, the dioxane was evaporated under reduced pressure, and the mixture was poured into ice water with stirring for 10 minutes. The residue was filtered, washed with water, and dried under vacuum. The residue was dissolved in chloroform and purified by flash column chromatography using ethyl acetate / petroleum ether (1:10) as eluent. After removing the solvent under reduced pressure, the product was suspended in 200 mL of methanol, collected, filtered, washed, and dried to obtain the pure product TPE-Bpin2-2Br (3.9 g, 66%) ( 1 H NMR (600MHz, DMSO-d6)δ=7.91–7.54(m,4H),7.39(d,2H),7.24(d,2H),7.18(m,4H),6.32(m,4H),1.23(s,24H).).

[0095] In a 50 mL double-necked round-bottom flask, 30 mL of a mixture of THF / water / ethyl acetate (3:1:1, v:v:v) and 6.8 g of NaIO4 were added. After stirring at room temperature under argon for 30 min, 2 g of TPE-Bpin2-2Br was added. After three degassings, the mixture was refluxed at 80°C for 24 h (monitored by thin-layer chromatography until the solution turned clear and yellow). After cooling in an ice bath, 20 mL of a 1 M 0-10°C HCl solution (try to control the temperature at low temperature to avoid volatilization of HCl) was added and stirred vigorously. After the mixture was allowed to stand at 25°C overnight, the organic solvent was evaporated in vacuo. 150 mL of water was added and stirred to collect the white precipitate. The precipitate was washed three times with water and chloroform, and dried in vacuo at room temperature for 24 h to obtain a white TPE-B(OH2)2-2Br powder (1.4 g, 89%) ( 1 H NMR (600MHz, DMSO-d6) δ = 6.71 (s, 1H), 6.61 (s, 4H), 6.50 (s, 4H), 6.22 (s, 1H), 6.12 (s, 4H), 6.03 (s, 2H), 1.68 (s, 4H).).

[0096] In a 150 mL three-necked flask, 1 g (1.56 mmol) of TPE-B (OH2) 2-2Br and 0.31 g (2.52 mmol) of p-mercaptophenylboronic acid were added. After three vacuum evacuations and nitrogen filling, 25 mg of tetrakis (triphenylphosphine) palladium was added. 50 mL of toluene and 2 mL of a 2 mol / L potassium carbonate / potassium fluoride mixed solvent were injected. The mixture was stirred at 90°C for 48 h. Water was added to terminate the reaction. The lower organic phase was extracted with dichloromethane, and the mixture was purified by silica gel column chromatography after rotary evaporation to obtain a yellow solid TPEND (0.6 g, 59%) ( 1 H NMR (600MHz, DMSO-d6)δ=7.64–7.52(m,5H),7.52–7.43(m,6H),7.43–7.31(m,6H),7.31–7.05(m,7H),1.20(d,J=1.8Hz,4H),0.80(s,2H).).

[0097] (3) Gold nanorods modified with tetraphenylethylene core derivatives (GNRs@TPEND)

[0098] In order to further promote the surface modification process, the 40mL GNRs solution prepared in (1) was centrifuged twice (12000rpm / 15min) to remove excess impurities. The purified GNRs were dispersed again in 40mL pure water. Then, 5mL of TPEND aqueous solution with a concentration of 12.8mg / mL was slowly added to the above GNRs solution and stirred continuously at room temperature for 16h. After that, the mixed solution was dialyzed in water for 24h through a dialysis membrane with a molecular weight cutoff of 8-14kDa to promote the full combination of TPEND and the GNRs surface. Finally, GNRs@TPEND was purified by centrifugation and redispersed in an aqueous solution for use. The morphology of GNRs@TPEND was characterized by transmission electron microscopy (TEM), as shown in FIG. Figure 2 At the same time, the UV-visible absorption spectrum was measured in the range of 300-750nm. The results are shown in Figure 3 shown.

[0099] (4) SERS and fluorescence dual-mode detection of chiral glucose based on GNRs@TPEND composite probe

[0100] The prepared GNRs@TPEND nanoprobes were placed in a 0.1mM K3[Fe(CN)6] buffer solution, and the pH was adjusted to the optimal experimental condition (under weak alkaline conditions, typically pH = 7.5-9). Next, freshly prepared D-glucose or L-glucose was added to the solution, and a chiral recognition reaction was carried out at room temperature. After the reaction, the sample was thoroughly rinsed with deionized water to prepare for subsequent SERS and fluorescence detection. The results showed that different concentrations of chiral glucose produced different SERS signals and fluorescence changes.

[0101] When detecting glucose, 1 mL of glucose solution with different concentrations (100, 250, 400, 550, 700, 850, 1000 μM) was mixed with 1 mL of 1 mg / mL nanoprobe and reacted for 10 minutes, and 5 μL of the mixture was dropped on the surface of the silicon wafer.

[0102] SERS measurement, such as using a portable Raman spectrometer: use 785nm laser, 40mW power and 10s integration time to measure SERS spectrum, using 1569±2cm -1 As the characteristic peak for determining chiral glucose signal. Figure 4 a. Figure 4 As shown in b, the SERS signal gradually increases with the increase of concentration, and the SERS detection range is 0.8×10 -7 ~10 -2 M. L-glucose test results are as follows Figure 5 a. Figure 5As shown in b, with the increase of concentration, SERS gradually weakened, and the SERS detection range was 0.8×10 -7 ~10 -2 M.

[0103] Fluorescence detection was performed using a fluorescence spectrophotometer to record the fluorescence spectra of different concentrations of D-glucose at an excitation wavelength of 365 nm, and 460 ± 2 nm was used as the characteristic peak for determining the chiral glucose signal. Figure 6 、 Figure 7 As shown in the figure, the fluorescence intensity of D-glucose increases with the concentration, and the L-glucose has the same trend but the enhancement effect is not prominent. The linear range of fluorescence detection is 0~1.0×10 -3 Therefore, this method can achieve simultaneous analysis and detection in different concentration ranges with excellent detection limits.

[0104] Comparative Example 1

[0105] In this comparative example, only GNRs were used as nanoprobes, wherein the preparation method of GNRs and the detection method using the same were consistent with those in Example 1.

[0106] The chiral glucose concentrations were predicted using machine learning based on different substrates for Example 1 and Comparative Example 1:

[0107] Based on urine standard samples, samples spiked with glucose at different concentrations were prepared. Taking the SERS detection results of D-glucose as an example, 800 SERS data were collected based on GNRs@TPEND and single receptor platform GNRs, and a calibration curve (calibration curve) was constructed using support vector machine regression (SVM-R) statistical analysis to compare the quantitative accuracy of glucose of the two substrate materials. Figure 8 As shown, for GNRs@TPEND (composite substrate), the calibration curve shows a near-ideal and good linear relationship, with a prediction coefficient of 0.998. In contrast, the prediction coefficient for glucose using GNRs alone is lower, at 0.964. This indicates that the composite material prepared in this method accurately and selectively recognizes chiral glucose in complex environments. This method provides a technical means for subsequent application in glucose detection in human biological samples, and is expected to achieve non-invasive diabetes screening (necessarily requiring verification in combination with clinical standards).

[0108] Comparative Example 2

[0109] This comparison only uses TPEND as the nanoprobe, wherein the preparation method of TPEND and the detection method using it are consistent with those in Example 1.

[0110] Sensitive and qualitative chiral glucose detection using SERS was performed on different substrates for Example 1 and Comparative Example 2:

[0111] Based on the composite substrate GNRs@TPEND and the single receptor platform TPEND, equal amounts of D / L-glucose were added, and a blank control group was added. The SERS detection results are shown in Figure 2. Figure 9 As shown in a, single TPEND cannot be used as a Raman reporter molecule, and the SERS response signal is weak. The SERS characteristic peak intensity of GNRs@TPEND is high, so the detection performance is better than TPEND. Based on the composite substrate, D / L glucose was added to it, and a blank control group was added. Figure 9 As shown in b, the signal contrast is prominent, indicating that the composite material prepared in this method can accurately and differentially recognize chiral glucose.

[0112] Application Example 1

[0113] L-glucose cannot be found in higher organisms, but can be used as an energy source. Therefore, the GNRs@TPEND composite nanoprobe prepared in Example 1 was used to measure the concentration of blood glucose (D-glucose) in human blood.

[0114] A case-control design was adopted, and a total of 10 subjects were included, including 5 clinically diagnosed diabetic patients (case group) and 5 healthy volunteers (control group). The research protocol was approved and written informed consent was obtained from each participant. Blood was collected from the fingertip of each volunteer using a 5mL blood collection tube containing an anticoagulant (such as heparin, EDTA) to prevent blood coagulation. In order to remove proteins and blood cells to reduce background interference, the blood sample was centrifuged at 3000-4000rpm for 10-15min, and the supernatant (serum) was taken. The pH of the sample was adjusted to neutral (7.0-7.4) for use to avoid acidic or alkaline conditions affecting the stability of the SERS substrate.

[0115] The liquid was extracted and mixed with the nanoprobe at a concentration of 1 mg / mL in a 1:1 ratio for 10 to 15 minutes. A 5 ± 2 μL droplet of the mixture was placed on a silicon wafer. The SERS spectra were measured using a 785 nm laser, 40 mW power, and a 10 s integration time. Fluorescence spectra were recorded using a fluorescence spectrophotometer at an excitation wavelength of 365 nm, demonstrating the GNRs@TPEND's ability to selectively capture glucose molecules in complex media.

[0116] Table 1 shows the results of analyzing D-glucose in Application Example 1 of the present invention using different analytical methods.

[0117] Table 1 Test results of application example 1

[0118]

[0119] As shown in Table 1, the SERS / fluorescence method yields similar results to spiked values, aiming for the same objective signal effect. Furthermore, compared with high-performance liquid chromatography (HPLC), the error in determining D-glucose content in human blood samples is smaller than that obtained by SERS and fluorescence methods, demonstrating the high accuracy of this method. Furthermore, this method can also be used to rapidly determine the quantification of D-glucose content in human blood samples by visual inspection using UV light. Therefore, it can be used for the immediate and rapid detection of D-glucose content in human blood samples, providing support for continuous blood glucose monitoring.

[0120] Application Example 2

[0121] Similar to Application Example 1, the GNRs@TPEND composite nanoprobe synthesized in Example 1 was used to measure the concentration of urine sugar (D-glucose) in human urine.

[0122] A case-control design was used, enrolling 10 subjects, including 5 clinically diagnosed diabetic patients (case group) and 5 healthy volunteers (control group). The study protocol was approved, and written informed consent was obtained from each participant. Morning urine (fasting urine) or random urine samples were collected from volunteers using a sterile urine cup. The urine was collected in a 5 mL disposable vacuum collection tube and refrigerated at 4 ± 2°C (≤ 24 h) to prevent bacterial degradation of glucose. The filtrate was collected by centrifugation using a 3-10 kDa ultrafiltration tube (10,000-14,000 rpm, 15-20 min). The filtrate was adjusted to pH 7.0-7.4 with NaOH / HCl for later use.

[0123] The liquid was extracted and mixed with the nanoprobe at a concentration of 1 mg / mL in a 1:1 ratio for 10 to 15 minutes. A 5 ± 2 μL droplet of the mixture was placed on a silicon wafer. The SERS spectra were measured using a 785 nm laser, 40 mW power, and a 10 s integration time. The fluorescence spectra were recorded using a fluorescence spectrophotometer at an excitation wavelength of 365 nm, demonstrating that GNRs@TPEND can selectively capture glucose molecules in a complex medium.

[0124] Table 2 shows the results of analyzing D-glucose in Application Example 2 of the present invention using different analytical methods.

[0125] Table 2 Test results of application example 2

[0126]

[0127]

[0128] As shown in Table 2, the SERS / fluorescence method yields similar results to the spiked value, aiming to achieve the same objective signal effect. Furthermore, compared with high-performance liquid chromatography (HPLC), the error in determining D-glucose content in human urine samples is smaller than that obtained by SERS and fluorescence methods, demonstrating the high accuracy of this method. Furthermore, this method can also utilize UV ​​light to illuminate the sample for rapid visual determination of quantification, thus enabling rapid and immediate detection of D-glucose content in human urine samples, aiding continuous urine glucose monitoring.

[0129] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A SERS / fluorescence dual-modal composite probe, characterized in that: The composite probe is a gold nanorod modified with a tetraphenylethylene core derivative, wherein the tetraphenylethylene core derivative is a TPE core molecular probe with AIE properties; The tetraphenylethylene core derivative is prepared by reacting tetrabromotetraphenylethylene with biboronic acid pinacol ester, followed by oxidation, and then reacting with p-mercaptophenylboronic acid.

2. A SERS / fluorescence dual-modal composite probe according to claim 1, characterized in that: The average length of the gold nanorods is between 60 and 100 nm.

3. A method for preparing a SERS / fluorescence dual-modal composite probe according to claim 1 or 2, characterized in that: The steps include: S1: Preparation of gold nanorods: Synthesis of gold nanorods by seed growth method; S2: Synthesis of tetraphenylethylene core derivative TPEND: S21: adding tetrabromotetraphenylethylene, bipyralidoborane, and the first palladium catalyst to the first mixed solution to react, and obtaining TPE-Bpin2-2Br through post-treatment; S22: adding TPE-Bpin2-2Br and sodium periodate to the second mixed solution to react, and obtaining TPE-B(OH2)2-2Br through post-treatment; S23: adding TPE-B(OH2)2-2Br, p-mercaptophenylboronic acid, and the second palladium catalyst to the third mixed solution to react, and obtaining TPEND through post-treatment; S3: Modification of gold nanorods with tetraphenylethylene core derivatives: The gold nanorods were dispersed in pure water and TPEND was added, stirred and mixed, dialyzed and centrifuged to obtain the gold nanorods modified with the tetraphenylethylene core derivative.

4. The method for preparing a SERS / fluorescence dual-modal composite probe according to claim 3, characterized in that: Step S1 specifically includes the following steps: S11: preparing a gold nanoparticle seed solution: adding a 0.01±0.002 mol / L aqueous solution of tetrachloroauric acid to a 0.1±0.05 mol / L aqueous solution of cetyl ammonium bromide, adding a 0.01±0.002 mol / L sodium borohydride solution after uniform dispersion, stirring and allowing to stand to obtain a gold nanoparticle seed solution; The volume ratio of tetrachloroauric acid aqueous solution, hexadecylammonium bromide aqueous solution and sodium borohydride solution is 0.25±0.05:9.75±0.25:0.6±0.02; S12: preparing a gold nanorod solution: adding a 0.01±0.002 mol / L tetrachloroauric acid aqueous solution to a 0.1±0.002 mol / L hexadecyltrimethylammonium bromide aqueous solution, mixing well, adding 0.01±0.002 mol / L AgNO3, 1 M HCl, and 0.1±0.002 mol / L ascorbic acid, and then adding the gold nanoseed solution, stirring well, and allowing to stand to obtain a gold nanorod solution; The volume ratio of tetrachloroauric acid aqueous solution, hexadecyltrimethylammonium bromide aqueous solution, AgNO3, HCl, ascorbic acid and gold nanoseed solution is 0.5±0.05mL:10±0.05mL:0.1±0.05mL:0.2±0.05mL:80±1μL:10~15μL; S13: Purifying the gold nanorod solution: centrifuging the gold nanorod solution, collecting the sol and washing it with pure water, cooling it to crystallize hexadecyltrimethylammonium bromide, and obtaining gold nanorods.

5. The method for preparing a SERS / fluorescence dual-modal composite probe according to claim 3, characterized in that: Step S2 specifically includes the following steps: S21: Potassium acetate is added to anhydrous dioxane and mixed and bubbled to obtain a first mixed solution; under a protective atmosphere, tetrabromotetraphenylethylene, pinacol diboron and [1,1-bis(diphenylphosphino)ferrocene] palladium dichloride are added to the first mixed solution, degassed, and refluxed at 100-120° C. for 68-78 hours; after the reaction, anhydrous dioxane is removed by vacuum distillation, ice water is added and stirred, and then filtered, washed with water and vacuum dried in sequence; re-dissolved in chloroform and purified by chromatography, the solvent is removed by vacuum distillation, and the solution is resuspended in methanol, filtered and washed to obtain TPE-Bpin2-2Br; The usage ratio of potassium acetate, anhydrous dioxane, tetrabromotetraphenylethylene, pinacol diboron and [1,1-bis(diphenylphosphino)ferrocene] palladium dichloride is 7.6±0.1g:150±5mL:5g:5.88g:200mg; S22: Sodium periodate is mixed with a mixture of THF / water / ethyl acetate to obtain a second mixed solution. TPE-Bpin2-2Br is added to the second mixed solution under a protective atmosphere. After degassing, the mixture is refluxed at 80-90° C. for 24-30 hours until the solution turns clear yellow. After cooling in an ice bath, 1M HCl solution is added, stirred, and allowed to stand overnight. The organic solvent is evaporated under vacuum, and water is added, stirred, and the precipitate is collected, washed, and dried under vacuum to obtain TPE-B(OH2)2-2Br. The dosage ratio of sodium periodate, mixed solution and TPE-Bpin2-2Br is 6.8 g:30±5 mL:2 g; S23: TPE-B(OH2)2-2Br is mixed with p-mercaptophenylboronic acid, and tetrakis(triphenylphosphine)palladium is added under a protective atmosphere, followed by the addition of a third mixed solution consisting of toluene and a 2 mol / L potassium carbonate / potassium fluoride mixed solvent. The mixture is stirred and reacted at 85-100° C. for 48-60 h. Water is added to terminate the reaction, and dichloromethane is added for extraction. The lower organic phase is separated, rotary evaporated, and purified by chromatography to obtain TPEND. The usage ratio of TPE-B(OH2)2-2Br, p-mercaptophenylboric acid, tetrakis(triphenylphosphine)palladium, toluene and potassium carbonate / potassium fluoride mixed solvent is 1 g: 0.31 g: 25 mg: 50-70 mL: 2-5 mL.

6. The method for preparing a SERS / fluorescence dual-modal composite probe according to claim 3, characterized in that: Step S3 specifically includes the following steps: After gold nanorods were dispersed in pure water, 12.8±0.5 mg / mL TPEND was added, the mixture was stirred and mixed, and dialyzed for 20-30 h using a dialysis membrane with a molecular weight cutoff of 8-14 kDa and centrifuged to obtain gold nanorods modified with tetraphenylethylene core derivatives.

7. Use of the SERS / fluorescence dual-modal composite probe according to claim 1 or 2 in chiral glucose detection.

8. A method for detecting chiral glucose, characterized in that: The steps include: The SERS / fluorescence dual-modal composite probe as described in claim 1 or 2 is added to a K3[Fe(CN)6] buffer solution, the pH is adjusted, and then a glucose solution to be tested is added, and chiral recognition of glucose is performed by SERS and fluorescence detection.

9. The method for detecting chiral glucose according to claim 8, wherein: The concentration of the K3[Fe(CN)6] buffer solution is 0.1±0.02 mM; the concentration of the SERS / fluorescence dual-modal composite probe is 1±0.2 mg / mL; the volume ratio of the SERS / fluorescence dual-modal composite probe to the glucose solution to be tested is 1:1; and / or, The SERS was carried out under the conditions of 785 nm laser, 40 mW power and 1-60 s integration time, using 1569 ± 2 cm -1 As the characteristic peak for determining the chiral glucose signal; the fluorescence detection is carried out at an excitation wavelength of 365nm, and 460±2nm is used as the characteristic peak for determining the chiral glucose signal.

10. The method for detecting chiral glucose according to claim 8, wherein: The detection method quantitatively determines the concentration of chiral glucose in the glucose solution to be tested by a machine learning method, wherein the machine learning method includes support vector regression.

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