SERS (Surface Enhanced Raman Scattering) sensor for detecting excessive lactic acid enantiomers in urine and preparation method of SERS sensor

Abstract

The invention discloses a sensor for rapidly detecting excessive lactic acid enantiomers in urine based on a semiconductor SERS (Surface Enhanced Raman Scattering) technology. The method comprises the following steps: step 1, synthesizing a ZIF-8 nanocrystal precursor; step 2, synthesis of a ZIF-8 / ZnS nano material; and step 3, preparing the ZIF-8 / ZnS / 4-MPY chiral recognition sensor. And 4, detecting the enantiomer excess value of the lactic acid enantiomer in the urine. The sensor for directly detecting excessive lactic acid enantiomers in urine based on the semiconductor SERS technology has the advantages of being low in cost, easy and convenient to operate, sensitive in detection and the like, is particularly suitable for clinical preliminary screening of intestinal diseases, and has wide application prospects. The fitting peak area of shoulder peaks with Raman shifts of 1004 and 1024 cm <-1 > and excessive lactic acid enantiomers in urine show an extremely high linear relationship.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection, and specifically relates to a sensor based on semiconductor surface-enhanced Raman scattering technology, which is a surface-enhanced Raman scattering spectroscopy (SERS) sensor for directly detecting the enantiomeric excess (ee%) of lactic acid in urine. Background Art

[0002] Lactic acid is the most common organic acid in the human body. Due to carbon asymmetry, lactate is typically produced as three isomers: D-lactic acid (D-LA), L-lactic acid (L-LA), and racemic DL-lactic acid (DL-LA). The total lactate concentration in healthy human urine is 0.34 ± 0.13 mmol / mL, of which D-lactic acid (R-lactic acid) accounts for 0.08 ± 0.034 mmol / mL, accounting for 24.42 ± 11% of the total lactate content in urine. L-lactic acid is the predominant form of lactate in the human body, while D-lactic acid is the primary metabolite of intestinal bacteria. D-lactic acid levels are significantly elevated in people with intestinal diseases such as short bowel syndrome, acute appendicitis, and Crohn's disease. Analyzing the relative levels of D-lactic acid and L-lactic acid in patients' urine may provide early warning of intestinal diseases.

[0003] Current methods for detecting lactate in body fluids primarily include enzymatic, electrochemical, and chromatographic methods, which are suitable for detecting lactate enantiomers in samples such as blood, cerebrospinal fluid, and urine. However, these methods suffer from drawbacks such as demanding reagent storage conditions, complex experimental procedures, high costs, and slow analysis speeds. SERS technology, with its exceptional sensitivity and ability to reveal rich molecular information from biological samples, has been continuously researched and developed in recent years. Numerous SERS sensors with biomonitoring capabilities have been developed and are widely used in the field of biomolecular detection. Semiconductor-based SERS technology, particularly using low-cost and easily synthesized semiconductor composites (such as ZIF-8 / ZnS), could have significant application potential in the development of SERS sensors capable of directly detecting lactate enantiomeric excess in urine. Summary of the Invention

[0004] The core objective of the present invention is to develop a sensor based on semiconductor SERS technology to achieve rapid monitoring of lactic acid enantiomeric excess in urine. Specifically, after simply mixing urine with an ethanol solution and filtering it, the sensor can be used to efficiently detect lactic acid enantiomers. In the process of detecting lactic acid enantiomer excess in urine involved in the present invention, it is only necessary to construct a ZIF-8 / ZnS / 4-MPY chiral recognition sensing platform. The constructed SERS sensor is mainly composed of aluminum foil, a centrifuge tube cover and a glass sheet for fixation. The method of using the sensor is simple and easy, and the preparation process of the SERS sensor and the detection step of lactic acid enantiomer excess can be completed within 10 minutes. Its detection principle is to utilize the high-intensity SERS signal generated after the material combines with the lactic acid enantiomer molecules, thereby achieving sensitive detection of lactic acid enantiomer excess.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A sensor for detecting enantiomeric excess of lactic acid in urine based on semiconductor SERS technology, wherein the preparation steps of the sensor are as follows:

[0007] Step 1: Synthesis of ZIF-8 nanocrystal precursor

[0008] Dissolve 330 mg of zinc acetate dihydrate and 985 mg of 2-methylimidazole in 90 mL of anhydrous ethanol. Once the solutes are completely dissolved, add the latter solution to the former with stirring. Allow the mixture to react at room temperature for 24 hours, then wash three times with anhydrous ethanol and dry. The resulting white powder is the ZIF-8 nanocrystal precursor.

[0009] Step 2: Synthesis of ZIF-8 / ZnS nanomaterials

[0010] 25 mg of the prepared ZIF-8 nanocrystals were ultrasonically dispersed in 25 mL of anhydrous ethanol. 250 mg of thioacetamide was then added to the solution, and the mixture was refluxed for 40 minutes. After cooling to room temperature, the mixture was washed three times with anhydrous ethanol. The centrifuged product was dried at 60°C overnight to obtain ZIF-8 / ZnS nanoparticles.

[0011] Step 3. Preparation of ZIF-8 / ZnS / 4-MPY chiral recognition sensor

[0012] (1) Take a 0.5 mg centrifuge tube, cut the centrifuge tube cap and fix it on the surface of the slide, and stick a round aluminum foil with a diameter of 0.66 cm inside the centrifuge tube cap.

[0013] (2) Disperse 2 mg of ZIF-8 / ZnS nanoparticles into 1 mL of a 10 -3The mixture was stirred in a 4-mercaptopyridine (4-MPY) ethanol solution for 2 h.

[0014] (3) The supernatant in the above solution was removed by centrifugation and dispersed in 200 μL of anhydrous ethanol to obtain a ZIF-8 / ZnS / 4-MPY complex. The obtained ZIF-8 / ZnS / 4-MPY complex can be stored for a long time under light-proof and sealed conditions.

[0015] (4) The dispersed liquid of the ZIF-8 / ZnS / 4-MPY complex was dropped onto aluminum foil and air-dried to obtain the ZIF-8 / ZnS / 4-MPY chiral recognition sensor.

[0016] Step 4: Detection of enantiomeric excess of lactate enantiomers in urine

[0017] Urine and ethanol were mixed uniformly in a volume ratio of 1:9 and filtered through a 0.22 μm filter to remove the precipitate. 10 μL of this mixed solution was added dropwise to the SERS sensor prepared in step 3. SERS spectra were obtained under 532 nm laser excitation.

[0018] The beneficial effects of the present invention compared to the prior art are:

[0019] (1) This paper develops a ZIF-8 / ZnS / 4-MPY chiral recognition sensor based on semiconductor SERS, which has a simple and efficient operation method and can be stored in air for a long time;

[0020] (2) The preparation of the ZIF-8 / ZnS / 4-MPY chiral recognition sensor and the detection of enantiomeric excess of lactic acid in urine can be completed within 10 minutes, with a short processing time;

[0021] (3) The detection of enantiomeric excess values ​​of lactic acid enantiomers has a good linear relationship. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scanning electron microscope image of ZIF-8 nanocrystals obtained in Example;

[0023] Figure 2 This is a scanning electron microscope image of ZIF-8 / ZnS nanoparticles obtained in Example;

[0024] Figure 3 : Transmission electron microscope image of ZIF-8 / ZnS nanoparticles obtained in Example;

[0025] Figure 4 This is a photo of the ZIF-8 / ZnS / 4-MPY chiral recognition sensor obtained in Example;

[0026] Figure 5 Schematic diagram of the structure of the ZIF-8 / ZnS / 4-MPY chiral recognition sensor obtained in Example;

[0027] Figure 6 Comparison of SERS signals of the ZIF-8 / ZnS / 4-MPY chiral recognition sensor obtained in the example in artificial urine containing different enantiomeric excesses of lactic acid;

[0028] Figure 7 The ZIF-8 / ZnS / 4-MPY chiral recognition sensor obtained in the embodiment detects lactic acid enantiomers at Raman shifts of 1004 and 1024 cm -1 The linear relationship between the fitted peak area of ​​the shoulder peak at and the enantiomeric excess of lactic acid;

[0029] Figure 8 This is a signal stability test diagram of the ZIF-8 / ZnS / 4-MPY chiral recognition sensor obtained in the embodiment when stored in air for different time periods;

[0030] Figure 9 This is the SERS spectrum of the ZIF-8 / ZnS / 4-MPY chiral recognition sensor obtained in the example when detecting the enantiomeric excess of lactic acid in real urine;

[0031] Figure 10 The least squares method (PLS) diagram of the ZIF-8 / ZnS / 4-MPY chiral recognition sensor obtained in the embodiment and other detection methods for enantiomeric excess of lactic acid in urine; DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments, but the experimental conditions and setting parameters therein should not be regarded as limiting the basic technical solution of the present invention. Any modification or equivalent replacement of the technical solution of the present invention that does not depart from the spirit and content of the technical solution of the present invention should be included in the scope of protection of the present invention.

[0033] Step 1: Synthesis of ZIF-8 nanocrystal precursor

[0034] Accurately weigh 330 mg of zinc acetate dihydrate and 985 mg of 2-methylimidazole and dissolve them in 90 mL of anhydrous ethanol. Once the solutes are completely dissolved, add the latter solution to the former while stirring. Allow the mixture to react at room temperature for 24 hours. Wash the mixture three times with anhydrous ethanol and dry it to obtain a white powder, which is the ZIF-8 nanocrystal precursor.

[0035] Step 2: Synthesis of ZIF-8 / ZnS nanomaterials

[0036] 25 mg of the prepared ZIF-8 nanocrystals were ultrasonically dispersed in 25 mL of anhydrous ethanol. 250 mg of thioacetamide was then added to the solution, and the mixture was refluxed for 40 minutes. After cooling to room temperature, the mixture was washed three times with anhydrous ethanol. The centrifuged product was dried at 60°C overnight to obtain ZIF-8 / ZnS nanoparticles.

[0037] Step 3. Preparation of ZIF-8 / ZnS / 4-MPY chiral recognition sensor

[0038] (1) Take a 0.5 mg centrifuge tube, cut the centrifuge tube cap and fix it on the surface of the slide, and stick a round aluminum foil with a diameter of 0.66 cm inside the centrifuge tube cap.

[0039] (2) Disperse 2 mg of ZIF-8 / ZnS nanoparticles into 1 mL of a 10 -3 The mixture was stirred in a 4-mercaptopyridine (4-MPY) ethanol solution for 2 h.

[0040] (3) The supernatant in the above solution was removed by centrifugation and dispersed in 200 μL of anhydrous ethanol to obtain a ZIF-8 / ZnS / 4-MPY complex. The obtained ZIF-8 / ZnS / 4-MPY complex can be stored for a long time under light-proof and sealed conditions.

[0041] (4) The ethanol dispersion of the ZIF-8 / ZnS / 4-MPY complex was dropped onto aluminum foil and air-dried to obtain the ZIF-8 / ZnS / 4-MPY chiral recognition sensor.

[0042] Step 4: Detection of enantiomeric excess of lactate enantiomers in urine

[0043] Urine and ethanol were mixed uniformly in a volume ratio of 1:9 and filtered through a 0.22 μm filter to remove the precipitate. 10 μL of this mixed solution was added dropwise to the SERS sensor prepared in step 3. SERS spectra were obtained under 532 nm laser excitation.

[0044] Example:

[0045] A sensor for detecting enantiomeric excess of lactic acid in urine based on semiconductor SERS technology is specifically carried out in the following steps:

[0046] Step 1: Synthesis of ZIF-8 nanocrystal precursor

[0047] Accurately weigh 330 mg of zinc acetate dihydrate and 985 mg of 2-methylimidazole and dissolve them in 90 mL of anhydrous ethanol. Once the solutes are completely dissolved, add the latter solution to the former while stirring. Allow the mixture to react at room temperature for 24 hours. Wash the mixture three times with anhydrous ethanol and dry it to obtain a white powder, which is the ZIF-8 nanocrystal precursor. Figure 1 This is a scanning electron microscope photograph of ZIF-8 nanocrystals. It can be seen that the particle size distribution of ZIF-8 nanocrystals is uniform, and a single nanocrystal is a truncated rhombic dodecahedron structure.

[0048] Step 2: Synthesis of ZIF-8 / ZnS nanoparticles

[0049] 25 mg of the prepared ZIF-8 nanocrystals were ultrasonically dispersed in 25 mL of anhydrous ethanol. 250 mg of thioacetamide was then added to the solution, and the mixture was refluxed for 40 minutes. After cooling to room temperature, the mixture was washed three times with anhydrous ethanol. The centrifuged product was dried at 60°C overnight to obtain ZIF-8 / ZnS nanoparticles. Figure 2 This is a scanning electron microscope photo of ZIF-8 / ZnS nanoparticles. ZIF-8 / ZnS nanoparticles perfectly replicate the morphology of ZIF-8. Figure 3 This is a transmission electron microscope image of ZIF-8 / ZnS nanoparticles, showing that ZIF-8 / ZnS nanoparticles have a core-shell structure.

[0050] Step 3. Preparation of ZIF-8 / ZnS / 4-MPY chiral recognition sensor

[0051] Take a 0.5 mg centrifuge tube, cut the centrifuge tube cap and fix it on the surface of the slide, and stick a round aluminum foil with a diameter of 0.66 cm inside the centrifuge tube cap. Disperse 2 mg of ZIF-8 / ZnS nanoparticles into 1 mL of 10 -3 The supernatant was removed by centrifugation and dispersed in 200 μL of anhydrous ethanol to obtain a ZIF-8 / ZnS / 4-MPY complex. The resulting ZIF-8 / ZnS / 4-MPY complex can be stored for a long time under light-proof and sealed conditions. The ethanol dispersion of the ZIF-8 / ZnS / 4-MPY complex was dropped onto aluminum foil and naturally air-dried to obtain a ZIF-8 / ZnS / 4-MPY chiral recognition sensor. Figure 4A photo shows an actual ZIF-8 / ZnS / 4-MPY chiral recognition sensor, which primarily consists of a ZIF-8 / ZnS / 4-MPY sensor chip (white area), a reactor (centrifuge tube cap), and a fixture (glass slide and adhesive tape). The glass slide and adhesive tape serve as the fixation mechanism, while the centrifuge tube cap is where the sample is loaded and where the ZIF-8 / ZnS / 4-MPY complex binds to the chiral molecule, forming the core of the SERS sensor. Figure 5 The schematic structural diagram of the ZIF-8 / ZnS / 4-MPY chiral recognition sensor is shown.

[0052] Step 4: Detection of enantiomeric excess of lactate enantiomers in urine

[0053] Artificial urine solutions with varying enantiomeric excesses of lactic acid (-100, -50, 0, 50, and 100) were prepared using artificial urine as the solvent. Artificial urine and anhydrous ethanol were uniformly mixed in a volume ratio of 1:9 and filtered through a 0.22 μm filter to remove the precipitate. 10 μL of this mixed solution was added dropwise to the SER chiral recognition sensor prepared in step 3. After immersion for 10 minutes, the SERS chiral recognition sensor was excited by a 532 nm laser and acquired SERS spectra. Data acquisition time was 5 s, with one accumulation, and the power was 10 mW. A confocal Raman microscope (RTS2-301-DL) was used, purchased from Beijing Zhuoli Hanguang Instrument Co., Ltd. Figure 6 SERS spectra obtained by the SERS chiral recognition sensor for detecting the enantiomeric excess of different lactic acid enantiomers in artificial urine. Figure 7 For Figure 6 Raman shifts are 1004 and 1024 cm -1 The linear relationship between the enantiomeric excess of lactic acid enantiomers and the peak area ratio was obtained by comparing the fitted peak areas. 2 =0.97901, showing a good linear relationship. The chiral recognition sensor of SERS sensor was stored in air for different time periods to detect L-lactic acid and D-lactic acid. Figure 8 The peak area ratio curve shown shows good stability. The difference index (DI) value is the ratio of the peak areas of L-lactic acid and D-lactic acid detected at the same concentration. A higher ratio indicates better enantiomer recognition performance. Figure 9 The SERS spectrum obtained by the SERS chiral recognition sensor for the detection of enantiomeric excess of lactic acid enantiomers in real urine samples was compared with the standard curve established for the enantiomeric excess of lactic acid in artificial urine to obtain the information (predicted value) of the enantiomeric excess of lactic acid in real urine. By comparing with the currently widely recognized enzymatic method (standard value), the enantiomeric excess of lactic acid in real urine was obtained. Figure 10 As shown in the PLS diagram, the detection results of the two methods show a high degree of consistency.

Claims

1. A rapid detection sensor for enantiomeric excess of lactic acid in urine based on semiconductor SERS technology, the method comprising the following steps: Step 1: Synthesis of ZIF-8 nanocrystal precursor Dissolve 330 mg of zinc acetate dihydrate and 985 mg of 2-methylimidazole in 90 mL of anhydrous ethanol. Once the solutes are completely dissolved, add the latter solution to the former with stirring. Allow the mixture to react at room temperature for 24 hours, then wash three times with anhydrous ethanol and dry. The resulting white powder is the ZIF-8 nanocrystal precursor. Step 2: Synthesis of ZIF-8 / ZnS nanomaterials 25 mg of the prepared ZIF-8 nanocrystals were ultrasonically dispersed in 25 mL of anhydrous ethanol. 250 mg of thioacetamide was then added to the solution, and the mixture was refluxed for 40 minutes. After cooling to room temperature, the mixture was washed three times with anhydrous ethanol. The centrifuged product was dried at 60°C overnight to obtain ZIF-8 / ZnS nanoparticles. Step 3. Preparation of ZIF-8 / ZnS / 4-MPY chiral recognition sensor (1) Take a 0.5 mg centrifuge tube, cut the centrifuge tube cap and fix it on the surface of the slide, and stick a round aluminum foil with a diameter of 0.66 cm inside the centrifuge tube cap. (2) Disperse 2 mg of ZIF-8 / ZnS nanoparticles into 1 mL of a 10 -3 The mixture was stirred in a 4-mercaptopyridine (4-MPY) solution of 1 M for 2 h. (3) The supernatant in the above solution was removed by centrifugation and dispersed in 200 μL of anhydrous ethanol to obtain a ZIF-8 / ZnS / 4-MPY complex. The obtained ZIF-8 / ZnS / 4-MPY complex can be stored for a long time under light-proof and sealed conditions. (4) The ethanol dispersion of the ZIF-8 / ZnS / 4-MPY complex was dropped onto aluminum foil and air-dried to obtain the ZIF-8 / ZnS / 4-MPY chiral recognition sensor. Step 4: Detection of enantiomeric excess of lactate enantiomers in urine Urine and ethanol were mixed uniformly in a volume ratio of 1:9 and filtered through a 0.22 μm filter to remove the precipitate. 10 μL of this mixed solution was added dropwise to the SERS sensor prepared in step 3. SERS spectra were obtained under 532 nm laser excitation.

2. The SERS sensor for rapid detection of enantiomeric excess of lactic acid in urine based on semiconductor SERS technology according to claim 1, characterized in that: The amounts of the substances used in step 1 are as follows: 330 mg of analytical grade zinc acetate dihydrate; 985 mg of analytical grade 2-methylimidazole; 250 mg of analytical grade thioacetamide; and 225 mL of analytical grade anhydrous ethanol.

3. The SERS sensor for rapid detection of enantiomeric excess of lactic acid in urine based on semiconductor SERS technology according to claim 1, characterized in that: Step 2 needs to be prepared under reflux conditions.

4. The SERS sensor for rapid detection of enantiomeric excess of lactic acid in urine based on semiconductor SERS technology according to claim 1, characterized in that: The aluminum foil and centrifuge tube used in step 2 are purchased from offline stores and are easy to obtain.

5. The SERS sensor for rapid detection of enantiomeric excess of lactic acid in urine based on semiconductor SERS technology according to claim 1, characterized in that: The glass sheet used in step 2 only serves to fix the SERS chiral recognition sensor detection chip and can be replaced by other materials.

6. The SERS sensor for rapid detection of enantiomeric excess of lactic acid in urine based on semiconductor SERS technology according to claim 1, characterized in that: In step 2, the ZIF-8 nanocrystals used need to be fully ultrasonically dispersed after adding ethanol solvent.

7. The SERS sensor for rapid detection of enantiomeric excess of lactic acid in urine based on semiconductor SERS technology according to claim 1, characterized in that: In step 4, the urine to be tested needs to completely immerse the chip of the SERS chiral recognition sensor.

8. The SERS sensor for rapid detection of enantiomeric excess of lactic acid in urine based on semiconductor SERS technology according to claim 1, characterized in that: The data collection time in the SERS measurement in step 4 is 5 s, 1 accumulation, and the power is 10 mW.

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