A method for detecting β-galactosidase based on surface enhanced Raman spectroscopy

Abstract

The present invention develops a method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy technology. β-galactosidase is a marker enzyme for identifying senescent cells. The technical scheme includes: 1) preparing nano-Venus, 2) preparing a two-dimensional array of monolayer, self-assembled nano-Venus and labeled molecules of para-mercaptobenzoic acid, 3) preparing a label, and 4) combining the two-dimensional array with the label through lactose to form a functionalized gold plate as a detection platform. When β-galactosidase acts on the substrate, the two-dimensional array can be separated from the label by decomposing the β-galactosidic bond in lactose, and the data spectrum is collected by means of a laser Raman spectrometer, and the new signal 1437cm generated by PATP on the label when irradiated with laser ‑1 As the marker peak, the ultrasensitive detection of serum β-galactosidase content was achieved. When detecting the activity of the enzyme, the method ‑3 ‑0.5U·mL ‑1 and 0.5-100 U·mL ‑1 The detection limit can reach 4.79×10 ‑5 U·mL ‑1 .

Description

Technical Field

[0001] The invention belongs to the technical field of detection, and specifically relates to a method for detecting beta-galactosidase based on surface enhanced Raman technology. Background Art

[0002] β-Galactosidase (SA-β-gal) is an important exoglycosidase located in membrane-enclosed lysosomes. It can hydrolyze the β-glycosidic bonds between galactose and other organic residues. It is presented in the extracellular environment and is a marker for identifying senescent cells. It is one of the earliest described biomarkers and an important biomarker for monitoring the aging process and studying aging. At present, in the field of cosmetic efficacy evaluation, a fibroblast model can be established for anti-aging evaluation. The anti-aging ability of the cosmetic can be evaluated by evaluating the ability of cosmetics to inhibit the activity of β-galactosidase produced by fibroblasts. However, the commonly used methods currently have more or less defects, such as large errors, long staining time, low accuracy and high cost. At the same time, the national standards and group standards for β-galactosidase have not yet been formulated. Therefore, it is of great significance to develop methods for detecting β-galactosidase.

[0003] There are many detection methods, such as colorimetry, fluorescence, and near-infrared fluorescence (NIRF), each of which has its advantages and disadvantages. Colorimetry is simple, however, this method has problems such as low precision, long staining time, and low detection sensitivity. Fluorescence detection is easily affected by the body's own fluorescence, making it difficult to use for biological imaging; fluorescence imaging in the near-infrared region can effectively reduce background interference and show good imaging quality, but its application is limited due to the low resistance to photobleaching of its organic fluorescent dyes, short imaging luminescence time, and poor water solubility. Surface-enhanced Raman scattering (SERS) is a useful analytical technique for detecting trace amounts of biochemical analytes. Single-molecule recognition has been demonstrated by SERS due to the strong proton enhancement of noble metal nanostructures. By adsorbing the analyte molecules on the rough surface of nanometal materials, the Raman signal can be enhanced to 106 to 1015 times. Compared with traditional detection methods, it has the advantages of sensitivity, rapidity, and good reproducibility.

[0004] Electromagnetic enhancement is the main cause of Raman signal enhancement, and its effect is related to the type and state of the metal substrate and the surface structure. Gold nanostars (Au NSs) are multi-branched nanoparticles with a thorny structure. Due to their excellent SERS enhancement effect, it is a rising star in biomedical applications. The construction of a two-dimensional (2D) substrate as a new hybrid nanostructure has better uniformity, repeatability and stability compared with a single nanoparticle. In addition, the sandwich structure formed based on the 2D structure maintains good uniformity and stability, and can also generate more hot spots, further improving the SERS enhancement effect.

[0005] Patented fluorescent probe for rapid detection of β-galactosidase and its preparation method and application (CN113527389A) specifically discloses a method for detecting β-galactosidase with a fluorescent probe. The method is difficult to prepare and its linear range is only 0-0.03U·mL-1. Summary of the invention

[0006] In order to solve the shortcomings of the above detection methods, this experiment aims to construct a more economical, faster and lower detection limit detection method for SA-β-gal. This method proposes a SERS-based platform, that is, a two-dimensional array of Au NSs constructed by Au NSs and glass plates. First, the multi-branched structure of Au NSs can produce stronger signals and improve the sensitivity of detection. At the same time, the construction of this two-dimensional array can effectively solve the problems of low precision and weak uniformity; the two-dimensional array is modified with PMBA and attached to β-lactose, and connected with Au NSs modified with PMBA and PATP to form a sandwich structure. In addition, the label is connected to the substrate to generate more hotspots, further improving the SERS intensity and greatly improving the sensitivity of detection. The reagents used in the detection substrate constructed by this method are all common and inexpensive chemical reagents, which reduces the detection cost, and does not require long-term staining. It only needs to be soaked with the test sample for 1 hour for testing, which is easy to operate, solving the problems of the current method that is either time-consuming or difficult to operate. It is a more economical, faster and more convenient detection method.

[0007] When SA-β-gal acts on the substrate of the sandwich structure, it can break the β-lactose used to connect the label and the array plate, and the peak of the N=N bond (1437cm -1 ) to determine the activity of the enzyme. At the same time, this method has a high degree of specificity and has no obvious induction to hyaluronidase, alkaline protease, tyrosinase, and α-amylase. The applicable detection range is 0.5-100U·mL -1 , far exceeding the SA-β-gal content in human serum.

[0008] The β-galactosidase is abbreviated as SA-β-gal; the gold nanostar is abbreviated as Au NSs; p-mercaptoaniline is abbreviated as PATP; p-mercaptophenylboronic acid is abbreviated as PMBA; 3-aminopropyltrimethoxysilane is abbreviated as APTMS; sodium citrate is abbreviated as Nact; ascorbic acid is abbreviated as AA; β-lactose is abbreviated as β-lactose; and chloroauric acid is abbreviated as HAuCl4.

[0009] The object of the present invention is achieved through the following solutions:

[0010] 1. Preparation of Au NSs

[0011] 1) Synthesis of gold seeds: 96 mL ultrapure water was heated to boiling and stirred continuously. 4 mL of 1% HAuCl4 aqueous solution was added. The solution turned yellow. 1 min later, 15 mL of 1% sodium citrate aqueous solution was quickly added. After a few seconds, the solution turned black. After 3-4 min, the solution gradually turned wine red. After stirring for 6-7 minutes, heating was stopped and stirring was continued until the temperature reached room temperature.

[0012] 2) Add 200 μL of 1% HAuCl4 aqueous solution to 19.4 mL of water, stir continuously, add 200 μL of gold seeds, and after 2-3 min, add 200 μL of AgNO3 (3 mM) and 100 μL of ascorbic acid solution (100 mM) dropwise. Then, the solution turns blue, stir continuously for 10 min, centrifuge at 4000 rpm for 15 min, terminate the reaction and resuspend to obtain nano-gold stars (Au NSs), and then store at 4°C.

[0013] 2. Preparation of Au NSs-PMBA arrays

[0014] 1) Cut the glass into square pieces of about 0.5 cm × 0.5 cm, and then clean them in ultrasonic cleaning machine with ultrapure water and anhydrous ethanol for 15 minutes, and then dry them in air. After that, immerse these glass pieces in piranha solution (H2O2 / H2SO4, 3:7, v / v) at 90°C for 2 hours to further remove organic matter in the chip, wash the above glass pieces with a large amount of methanol and ultrapure water in turn, and then immerse them in methanol solution of organosilane (APTMS / CH3OH, 1:4, v / v) for 24 hours, rinse the glass pieces with methanol and store them, and wash them with ultrapure water before use.

[0015] 2) The Au NSs concentrated and resuspended in ultrapure water were immersed in the above glass slide for 24 hours, and then washed with ultrapure water to complete the derivatization.

[0016] 3) The Au NSs array was first immersed in a 1 mM PMBA ethanol solution for 24 hours and washed with ethanol to remove unbound PMBA molecules to obtain an Au NSs-PMBA array.

[0017] 3. Preparation of Au NSs / PATP-PMBA: First, under strong magnetic stirring, 100 μL of a mixture of PATP (1 mM) and PMBA (0.5 mM) was quickly added to 100 mL of Au NSs colloidal solution for 1 hour. Then, the solution was centrifuged at 3500 rpm for 10 minutes and washed twice with 1% sodium citrate aqueous solution. Finally, the solution was redispersed in ultrapure water to obtain Au NSs / PATP-PMBA (2:1) colloid.

[0018] 4. Preparation of functionalized substrates with sandwich structures

[0019] 1) Assembly of Au NSs-PMBA array and β-lactose: The Au NSs-PMBA array was incubated with β-lactose solution to achieve adsorption equilibrium, and then rinsed with ultrapure water and dried for later use.

[0020] 2) The prepared labeled Au NSs / PATP-PMBA and the Au NSs-PMBA array incubated with β-lactose were soaked for 1 hour and washed with ultrapure water to remove unbound labels. Before measurement, the sample was dried in air.

[0021] 5. Detection of SA-β-gal activity

[0022] 1) Draw the SA-β-gal working curve: soak the fully assembled sandwich functionalized substrate at 25°C in 0.5-100 U·mL -1 The samples were placed in a SA-β-gal solution for 1 h, washed with ultrapure water, and the data were collected by laser Raman spectrometry. The new signal (1437 cm -1 ) as the characteristic peak intensity to achieve ultrasensitive detection of SA-β-gal activity in serum.

[0023] 2) Select 1437cm -1 The peak intensity at 10 -3 -0.5U·mL -1 When the working curve is Y = -605.63X + 14131.62, it is found that the linearity is good (R 2 =0.9955); 0.5-100U·mL -1 The working curve is Y = -3569.97X + 13245.29, and it is found that the linearity is good (R 2 =0.9945), which shows that the detection method of SA-β-gal of the invention has the characteristics of high sensitivity, good specificity and rapid detection.

[0024] Beneficial effects of the present invention:

[0025] 1) The successful construction of the sandwich model is based on the fact that the two pairs of ortho-dihydroxy groups of β-lactose can react with the boronic acid groups in the substrate and PMBA in the label to form boronate covalent bonds. And because SA-β-gal can specifically decompose the β-glycosidic bond in β-lactose, when the enzyme acts on the substrate of the sandwich structure, it can separate the label from the substrate and select the signal intensity (1437cm-1 ) became an indicator of the enzyme activity, realizing the quantitative detection of the enzyme. -3 -0.5U·mL -1 and 0.5-100 U·mL -1 When Y=-605.63X+14131.62, R 2 =0.9955 and Y = -3569.97X + 13245.29, R 2 =0.9945, with high accuracy and detection range far exceeding the SA-β-gal content in human serum.

[0026] 2) Since the arrangement of the Au NSs array is very regular and uniform, the Raman signal of the PMBA molecules modified by the substrate is also evenly distributed. The RSD is 16.00% after detection and calculation, which is lower than 20%. After forming a sandwich substrate, the label is also evenly distributed on the substrate. After detection and calculation, its RSD is 13.10%, which further improves the sensitivity and accuracy of the substrate for detecting SA-β-gal.

[0027] 3) This method is highly resistant to interference and has no obvious induction to substances such as hyaluronidase, alkaline protease, tyrosinase, α-amylase, etc.

[0028] 4) The multi-branched structure of Au NSs can generate stronger signals and improve the sensitivity of detection. At the same time, the two-dimensional array constructed in the scheme can break through the simple one-dimensional detection structure and improve the detection accuracy and sensitivity from the spatial configuration of the two-dimensional uniform array, effectively solving the problems of many interference items of SA-β-gal in serum, low detection accuracy, and weak uniformity. The two-dimensional array is modified with PMBA and attached to β-lactose, and connected with Au NSs modified with PMBA and PATP to form a sandwich structure. In the sandwich structure, the label is connected to the substrate, generating more hot spots, further improving the SERS intensity, and greatly improving the sensitivity of detection.

[0029] 5) The substrate constructed based on the specificity of SA-β-gal to lactose has high accuracy and strong anti-interference ability for the detection of SA-β-gal in serum. It can also be applied to more scenarios of high-precision detection of SA-β-gal in biological internal and even external environments.

[0030] 6) The reagents used in the detection substrate constructed by this scheme are all common and inexpensive chemical reagents, which reduces the detection cost and does not require long-term staining. It only needs to be soaked with the test sample for 1 hour for testing. It is easy to operate and solves the problem that the current methods are either time-consuming or difficult to operate. It is a more economical, faster and more convenient detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Preparation process of sandwich structure substrate for detecting β-galactosidase.

[0032] Figure 2 is the RSD value of the substrate before the modified label in Example 1 of the present invention.

[0033] Figure 3 is the RSD value of the substrate after the label is modified in Example 1 of the present invention.

[0034] Figure 4 This is the SERS spectrum of different SA-β-gal activities in Example 1 of the present invention.

[0035] Figure 5 The activity and characteristic peak of SA-β-gal (1437 cm -1 )’s linear relationship between the intensities.

[0036] Figure 6 The Raman characteristic peak (1437 cm) is obtained when the Au NSs-PMBA array is incubated with different concentrations of lactose and the substrate is then connected with the label to form a sandwich structure. -1 )’s strength.

[0037] Figure 7 100 U·mL -1 SA-β-gal, hyaluronidase, tyrosinase, alkaline protease and 0.001 g mL -1 After the α-amylase was incubated with the sandwich structure substrate, the Raman characteristic peak (1437 cm -1 ) is a bar graph of SERS intensity at .

[0038] Figure 8 The SA-β-gal activity levels were detected after the sera of normal subjects and gastric cancer patients were co-incubated with the sandwich structure substrate. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] In order to better understand the present invention, the present invention is further described below by way of examples. The examples are only used to explain the present invention and do not constitute any limitation to the present invention.

[0041] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0042] Example 1 (see Figure 1 )

[0043] 1. Preparation of Au NSs

[0044] 1) Synthesis of gold seeds: 96 mL ultrapure water was heated to boiling and stirred continuously. 4 mL of 1% HAuCl4 aqueous solution was added. The solution turned yellow. 1 min later, 15 mL of 1% sodium citrate aqueous solution was quickly added. After a few seconds, the solution turned black. After 3-4 min, the solution gradually turned wine red. After stirring for 6-7 minutes, heating was stopped and stirring was continued until the temperature reached room temperature.

[0045] 2) Add 200 μL of 1% HAuCl4 aqueous solution to 19.4 mL of water, stir continuously, add 200 μL of gold seeds, and after 2-3 minutes, add 200 μL of AgNO3 (3 mM) and 100 μL of ascorbic acid solution (100 mM) dropwise. Then, the solution turns blue, stir continuously for 10 minutes, centrifuge at 4000 rpm for 15 minutes, terminate the reaction, resuspend, and then store at 4°C.

[0046] 2. Preparation of Au NSs-PMBA arrays

[0047] 1) Cut the glass into square pieces of about 0.5 cm × 0.5 cm, and then clean them in ultrasonic cleaning machine with ultrapure water and anhydrous ethanol for 15 minutes, and then dry them in air. After that, immerse these glass pieces in piranha solution (H2O2 / H2SO4, 3:7, v / v) at 90°C for 2 hours to further remove organic matter in the chip, wash the above glass pieces with a large amount of methanol and ultrapure water in turn, and then immerse them in methanol solution of organosilane (APTMS / CH3OH, 1:4, v / v) for 24 hours, rinse the glass pieces with methanol and store them, and wash them with ultrapure water before use.

[0048] 2) The Au NSs concentrated and resuspended in ultrapure water were immersed in the above glass slide for 24 hours, and then washed with ultrapure water to complete the derivatization.

[0049] 3) The Au NSs array was first immersed in a 1 mM PMBA ethanol solution for 24 h and washed with ethanol to remove unbound PMBA molecules.

[0050] 3. Preparation of Au NSs / PATP-PMBA: First, under strong magnetic stirring, a mixture of 100 μL of PATP (1 mM) and PMBA (0.5 mM) was quickly added to 100 mL of Au NSs colloidal solution for 1 hour, then centrifuged at 3500 rpm for 10 minutes, washed twice with 1% sodium citrate aqueous solution, and finally, redispersed in ultrapure water to obtain Au NSs / PATP-PMBA colloid.

[0051] 4. Preparation of functionalized substrates with sandwich structures

[0052] 1) Assembly of Au NSs-PMBA array and β-lactose: The Au NSs-PMBA array was incubated with β-lactose solution to achieve adsorption equilibrium, and then rinsed with ultrapure water and dried for later use.

[0053] 2) The labeled Au NSs / PATP-PMBA and the Au NSs-PMBA arrays linked with β-lactose were soaked for 1 hour and washed with ultrapure water to remove unbound labels. The samples were dried in air before measurement.

[0054] 5. Uniformity detection of functionalized substrate before and after

[0055] 1) Uniformity test of substrate before label modification: randomly select a 20μm×20μm area and scan 121 points at 1073cm -1 The Raman peak at is the signature peak. Figure 2 It can be concluded that the RSD of the corresponding Raman intensity is 16.00%.

[0056] 2) Uniformity test of substrate after label modification: randomly select a 20μm×20μm area and scan 121 points at 1437cm -1 The Raman peak at is the signature peak. Figure 3 It can be concluded that the RSD of the corresponding Raman intensity is 13.10%.

[0057] 6. Detection of SA-β-gal activity

[0058] 1) Draw the SA-β-gal working curve: soak the fully assembled sandwich functionalized substrate at 25°C in 0.5-100 U·mL -1 The samples were placed in a SA-β-gal solution for 1 h, washed with ultrapure water, and the data were collected by laser Raman spectrometry. The new signal (1437 cm -1 ) as the characteristic peak intensity to achieve ultrasensitive detection of SA-β-gal activity in serum.

[0059] 2) Select 1437cm-1 The peak intensity at 10 -3 -0.5U·mL -1 When the working curve is Y = -605.63X + 14131.62, it is found that the linearity is good (R 2 =0.9955); 0.5-100U·mL -1 The working curve is Y = -3569.97X + 13245.29, and it is found that the linearity is good (R 2 =0.9945), which shows that the detection method of SA-β-gal of the invention has the characteristics of high sensitivity, good specificity and rapid detection.

[0060] Example 2 Exploring the Optimal β-lactose Concentration

[0061] 1. The same experimental steps as in Example 1 were used to prepare label Au NSs / PATP-PMBA and Au NSs-PMBA arrays.

[0062] 2. Au NSs-PMBA array was modified with 0-75 mM β-lactose for 3 h.

[0063] 3. The Au NSs-PMBA array was modified with 0-75 mM β-lactose for 3 h, rinsed with ultrapure water, and then incubated with labeled AuNSs / PATP-PMBA for 1 h.

[0064] 4. After rinsing the substrate with ultrapure water, collect data spectra with the aid of a laser Raman spectrometer, and evaluate the optimal β-lactose concentration based on the intensity of the Raman signature peak at 1437 cm-1. Figure 6 It can be seen that the best results were obtained with the sandwich structure substrate modified with 50 mM β-lactose.

[0065] Example 3 Specificity Experiment

[0066] 1. Prepare a functionalized substrate with a sandwich structure using the same experimental steps as in Example 1.

[0067] 2. The functionalized substrate was heated at 25°C with 100 U·mL -1 SA-β-gal, hyaluronidase, tyrosinase, alkaline protease and 0.001 g mL -1 Incubate with α-amylase for 1 h.

[0068] 3. Rinse the substrate with ultrapure water and collect data spectra using a laser Raman spectrometer. Figure 7 It can be seen that only SA-β-gal has a strong affinity to the substrate Raman signature peak (1437 cm -1) intensity was greatly reduced, indicating that this method has a high specificity for detecting SA-β-gal.

[0069] Example 4 Detection of complex component samples

[0070] 1. Prepare a functionalized substrate with a sandwich structure using the same experimental steps as in Example 1.

[0071] 2. The functionalized substrate was incubated with the serum of normal subjects and gastric cancer patients (diluted 50 times) at 25°C for 1 h.

[0072] 3. Rinse the substrate with ultrapure water and collect data spectra with the aid of a laser Raman spectrometer. It was found that the substrate still has good performance in the serum with complex components. Figure 8 It can be seen that the substrate can clearly distinguish the SA-β-gal activity levels between normal people and cancer patients, and p*<0.05, that is, it has good variability, indicating that this method has the ability to analyze SA-β-gal in complex samples.

[0073] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy, characterized in that: The following steps are involved: (1) Preparation of Au NSs: Au NSs solution was obtained using the seed-mediated method; (2) Preparation of Au NSs arrays; (3) Preparation of Au NSs-PMBA arrays; (4) Preparation of Au NSs / PATP-PMBA; (5) Substrate assembly and functionalization: The Au NSs-PMBA array was modified with β-lactose; the Au NSs / PATP-PMBA was then assembled onto the Au NSs-PMBA array modified with β-lactose to form a sandwich structure for incubation with β-galactosidase, which was then washed with ultrapure water; the data spectrum was collected using a laser Raman spectrometer to obtain a working curve, with the new signal 1437 cm generated by the PATP on the label irradiated by laser as the wavelength. -1 As the marker peak, ultrasensitive detection of serum β-galactosidase content is achieved.

2. The method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The method for preparing the nano-Volume stars is as follows: first, 96 mL of ultrapure water is heated to boiling and continuously stirred, 4 mL of 1% HAuCl4 aqueous solution is added, the solution turns yellow, 15 mL of 1% sodium citrate aqueous solution is quickly added after 1 minute, the solution turns black after a few seconds, and the solution gradually turns wine red after 3-4 minutes, and the heating is stopped after stirring for 6-7 minutes, and the stirring is continued to room temperature to obtain the gold seeds; then, 200 μL of 1% HAuCl4 aqueous solution is added to 19.4 mL of water, and the stirring is continued, and 200 μL of gold seeds are added, and 200 μL of 3 mM AgNO3 and 100 μL of 100 mM ascorbic acid solution are added dropwise after 2-3 minutes, and the solution turns blue, and the stirring is continued for 10 minutes, and the solution is centrifuged at 4000 rpm for 15 minutes, the reaction is terminated, and the nano-Volume stars are resuspended and stored at 4°C.

3. The method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The preparation method of the Au NSs array is as follows: first, cut a glass sheet into square sheets of about 0.5 cm×0.5 cm, wash them in ultrasonic cleaning machine with ultrapure water and anhydrous ethanol for 15 minutes, and after drying in air, immerse the glass sheets in a piranha solution with a volume ratio of 3:7, soak them at 90°C for 2 hours, wash the glass sheets with a large amount of methanol and ultrapure water, and then immerse them in a methanol solution of organosilane with a volume ratio of 1:4 for 24 hours, and rinse the glass sheets with methanol to obtain an amino glass plate; then, concentrate and resuspend the nano-gold stars obtained in step (1) into an opaque solution, and soak them in the amino glass plate for 24 hours, and rinse them with ultrapure water and store them.

4. The method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The preparation method of the Au NSs-PMBA array is as follows: the Au NSs array prepared in step (2) is first immersed in a 1 mM PMBA ethanol solution for 24 hours, and then washed with ethanol to remove unbound PMBA molecules.

5. The method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The preparation method of the Au NSs-PMBA array modified with β-lactose is as follows: the Au NSs-PMBA array prepared in step (3) is incubated with β-lactose to reach adsorption equilibrium, and then rinsed with ultrapure water and dried for later use.

6. The method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The preparation method of Au NSs / PATP-PMBA is as follows: After the nano-Venus prepared in step (1) is concentrated and resuspended, 100 μL of a mixture of 1 mM PATP and 0.5 mM PMBA is quickly added to 100 mL of AuNSs colloidal solution under strong magnetic stirring for 1 hour, then centrifuged at 3500 rpm for 10 minutes, washed twice with 1% sodium citrate aqueous solution, and finally, redispersed in ultrapure water to obtain Au NSs / PATP-PMBA colloid.

7. The method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The sandwich structure preparation method is as follows: the Au NSs / PATP-PMBA prepared in step (4) and the Au NSs-PMBA array connected by β-lactose in step (5) are soaked for 1 hour, washed with ultrapure water, and the sample is dried in air before measurement.

8. The method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The preparation method for incubation with β-galactosidase is as follows: the sandwich structure prepared in step (5) and β-galactosidase solutions with different activities are incubated at 25° C. for 1 hour, and the sample is rinsed with ultrapure water and dried.

9. The method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: In step (5), the concentration of β-lactose is 0-75 mM, the reaction time is 3 h, and the activity of β-galactosidase is 10 -3 -100U·mL -1 , incubation time was 1h, and incubation temperature was 25℃.

10. The method for detecting β-galactosidase in serum based on surface enhanced Raman spectroscopy according to claim 1, characterized in that: The working curve in step (5) is selected at 1437cm -1 Draw a working curve between the peak intensity Y at the position and the SA-β-gal activity X and calculate the R 2 , in 10 -3 -0.5U·mL -1 and 0.5-100 U·mL -1 When Y=-605.63X+14131.62, R 2 =0.9955 and Y = -3569.97X + 13245.29, R 2 =0.9945.

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