A non-interference SERS probe and its preparation method and application

Abstract

The present invention discloses a non-interfering SERS probe, a preparation method and an application thereof, which relate to the technical field of surface-enhanced Raman spectroscopy detection. The Raman characteristic peak of the SERS probe of the present invention is in the range of 1800 cm-1 - 2500 cm-1 of the Raman silent region; the SERS probe includes a colloidal gold structure, a silver shell disposed on the surface of the colloidal gold structure, and a plurality of Raman reporter molecules phenylacetylene connected to the silver shell. The outer side of the phenylacetylene is connected with a polydopamine layer formed by self-polymerization of dopamine, and the outer side of the polydopamine layer is connected with a procalcitonin antibody. The probe of the present invention has good stability and reproducibility and strong optical response; it can solve the problem that the SERS probe is easily optically interfered by endogenous biomolecules in the fingerprint region (300 - 1800 cm-1), resulting in a reduction in the accuracy of quantitative analysis, and has good application prospects in the field of single / multiple index quantitative analysis.

Description

Technical Field

[0001] The invention belongs to the technical field of surface enhanced Raman spectroscopy detection, and particularly relates to a non-interference SERS probe, a preparation method of the non-interference SERS probe, and an application of the non-interference SERS. Background Art

[0002] In 1993, French pediatrician Assicot observed that the PCT content in the serum of patients with severe bacterial infection was significantly increased, which was positively correlated with the progression of the disease. Therefore, PCT was proposed as a marker for sepsis. At present, the mainstream detection method commonly used for PCT is immunoassay. However, since PCT in complex biological samples can be as low as picogram or nanogram levels, and there are high-abundance interfering species in the sample matrix, the equipment is large, the operation is complex, and there are many requirements for the reaction environment, which limits the application of immunoassays in bedside diagnosis.

[0003] Surface enhanced Raman scattering (SERS), as an ultra-sensitive vibrational spectroscopy technique, has been applied to many analyses in the past decade, especially in biochemistry and life sciences. SERS overcomes the disadvantage of weak Raman spectroscopy signals. It usually uses the principle of interaction between the local electromagnetic field of precious metal nanoparticles (gold, silver, copper, platinum, etc.) and surface adsorbed molecules to greatly enhance the Raman signal of the substance being measured and directly obtain its fingerprint spectrum. With the continuous maturity of SERS technology, the development of highly sensitive and highly stable Raman-active labels is the key to its application in the detection of ultra-trace substances.

[0004] In order to obtain the best SERS tags, researchers have explored various metal nanoparticles and nanocluster-based SERS substrates. However, another important factor, Raman reporter molecules, has not been widely studied. Researchers prefer to use ready-made SERS tags. However, for complex biological samples, blindly selecting Raman reporter molecules is inappropriate because endogenous interfering molecules or environmental media may overlap with the Raman bands of the reporter molecules, thereby having a greater impact on the accuracy of quantitative analysis. Especially in the screening of major diseases or multi-indicator detection, highly sensitive, non-interfering SERS tags are urgently needed.

[0005] Compounds containing alkynes have a peak at 2120 cm -1 There are Raman characteristic peaks, while most interfering substances are in the Raman silent region (1800cm -1 -2500cm -1) has no Raman response, so how to use this characteristic of alkyne compounds and apply it to SERS detection is a meaningful research direction. Phenylacetylene is a compound containing a carbon-carbon triple bond, in which the π electrons are sensitive to the surface environment. In particular, the stretching vibration peak of the carbon-carbon triple bond is very sensitive to the electronic structure of the bimetallic nanosubstrate. Depending on the ratio of the two metals, the characteristic peak is at 1980cm -1 -2200cm -1 There are significant differences within the range.

[0006] The emergence of portable Raman spectrometers has made it possible to apply SERS technology to major disease screening or clinical diagnosis. This also means that more stable and sensitive SERS tags are needed as a basis. Therefore, it is necessary to synthesize new non-interference high-sensitivity SERS tags with high signal intensity, good stability and biosafety in a relatively simple way. Summary of the invention

[0007] The purpose of the present invention is to provide a non-interference SERS probe with good stability and reproducibility; it can solve the problem of SERS probe in the fingerprint region (300-1800cm -1 ) is susceptible to optical interference from endogenous biomolecules, which leads to reduced accuracy of quantitative analysis.

[0008] The present invention also aims to provide a preparation method, which uses simple instruments, has low reagent costs and mild reaction conditions.

[0009] The object of the present invention is to provide an application of a non-interference SERS probe, which is applied to single-index / multi-index detection of low-abundance disease markers and detection of major diseases.

[0010] In order to solve the above technical problems, the present invention is achieved through the following technical solutions:

[0011] The present invention is a method for preparing a non-interference SERS probe, comprising the following steps:

[0012] Step 1, heat the chloroauric acid solution to boiling, add trisodium citrate solution, keep boiling and continue magnetic stirring to obtain colloidal gold;

[0013] Stp2, add ascorbic acid solution to colloidal gold, add silver nitrate solution dropwise under magnetic stirring, wait until the colloid color changes to earthy yellow, continue stirring for 6 minutes, then centrifuge, and redissolve the precipitate in ultrapure water to obtain the core-shell SERS enhanced substrate Au@Ag;

[0014] Stp3, under magnetic stirring, add phenylacetylene solution to the prepared Au@Ag, then add a mixed solution of Tris-HCl buffer solution and dopamine, stir magnetically for 2-5 minutes, stop stirring and centrifuge when the solution turns dark green, and re-dissolve in the original volume to obtain the SERS tag Au@Ag@PDA-EB;

[0015] Stp4, through the dopamine quinone on the surface of polydopamine, the capture antibody of the analyte is modified on the shell surface of Au@Ag@PDA-EB to synthesize a SERS probe with high specific recognition of the analyte.

[0016] Furthermore, in the Stp1, specifically: 100 mL of 0.01% chloroauric acid solution was prepared, heated to boiling, and then 5 mL of 1% trisodium citrate solution was added, and the mixture was kept boiling and magnetically stirred for 15 minutes to obtain a wine-red Au NPs colloid with a particle size of about 18.3±1.5 nm.

[0017] Furthermore, in the Stp2, 1 mL of 0.1 M ascorbic acid solution was added to 10 mL of colloidal gold, and 1 mL of 10 mM silver nitrate solution was added dropwise under magnetic stirring, and the colloid color changed to khaki, and then centrifuged after stirring for 6 min, and the precipitate was redissolved in 10 mL of ultrapure water to obtain a core-shell SERS enhanced substrate Au@Ag;

[0018] In Stp3, 1 mL of 1 mM phenylacetylene solution was added dropwise to the Au@Ag obtained in Stp2, and then 10 mL of a mixed solution was added, wherein the mixed solution was a mixture of Tris-HCl buffer solution and dopamine;

[0019] Wherein, the phenylacetylene solution is obtained by dissolving phenylacetylene in ethanol.

[0020] Furthermore, in Stp1-Stp3, the speed of the magnetic stirring is 300-500 rpm; in Stp2 and Stp3, the speed of the centrifugation is 7000-7800 rpm and the time is 10-15 min.

[0021] A non-interference SERS probe is prepared by the preparation method as described above.

[0022] Furthermore, the Raman characteristic peak of the SERS probe is in the Raman silent region of 1800 cm -1 -2500cm -1The invention relates to a colloidal gold structure, a silver shell arranged on the surface of the colloidal gold structure, a plurality of Raman reporter molecules phenylacetylene connected to the silver shell, a polydopamine layer formed by self-polymerization of dopamine connected to the outside of the phenylacetylene, and a procalcitonin antibody connected to the outside of the polydopamine layer.

[0023] Among them, the connection method of phenylacetylene is to coat it on the surface of SERS substrate through self-polymerization of dopamine; the modification method of procalcitonin antibody is to bond it with dopamine quinone of polydopamine layer.

[0024] Application of a non-interference SERS probe in the detection of disease marker procalcitonin in human serum.

[0025] The specific application methods include the following:

[0026] S1. Testing of PCT standards:

[0027] S11. Prepare PCT standard solutions of different concentrations in fetal bovine serum; the concentrations of the PCT standard solutions are 10, 50, 100, 500, 1000, 5000, 10000, and 50000 pg / mL, respectively;

[0028] S12. Take 1 mL of the standard solution, add 0.3 mg of the functionalized magnetic nanomaterial, incubate at 25°C in a shaker for 30 min, and selectively capture PCT using the boric acid affinity. After washing three times with PBS buffer at pH 7.4, add 0.5 mL of SERS probe and incubate at 37°C in a shaker for 6 min to form a "sandwich" structure through antigen and antibody immune binding.

[0029] S13, after completing the detection with the portable Raman spectrometer, establishing a standard curve, and obtaining a linear equation of the standard curve according to the standard curve;

[0030] The shaking speed in the above steps was 300 rpm, and a blank group was prepared under the same conditions;

[0031] The SERS detection described in the above steps is performed at a power of 200 mW and an integration time of 500 ms, and the average Raman intensity is recorded 3 times. All values ​​are the average of 5 repeated measurements, and baseline correction is performed;

[0032] S2. Serum sample testing:

[0033] S21, take serum samples and divide them into 1 mL / portion, add different amounts of 500 ng / mL PCT solution, mix thoroughly for 30 min, and prepare spiked serum sample solutions of different concentrations;

[0034] The spiked serum sample solutions were divided into 8 groups, and the PCT concentrations in each group of serum samples were 0, 0.05, 0.5, 0.8, 2.0, 5.0, 10.0, and 25.0 ng / mL;

[0035] S22. Take 1 mL of spiked serum sample solution, add 0.3 mg of functionalized magnetic nanomaterials, incubate at 25°C for 30 min in a shaker, and selectively capture PCT using the boric acid affinity. After washing three times with PBS buffer at pH 7.4, add 0.5 mL of SERS probe and incubate at 37°C for 6 min in a shaker to form a "sandwich" structure through antigen-antibody immune binding.

[0036] S23. Use a Raman spectrometer to detect the SERS signal intensity of the serum sample solution, and calculate the concentration value of PCT in the spiked serum sample according to the linear equation of the standard curve.

[0037] Furthermore, the synthesis of the functionalized magnetic nanomaterial mainly includes the following steps:

[0038] S31. Preparation of Fe3O4 nanoparticles: Dissolve FeCl3·6H2O, 1.5g polyethylene glycol and 4.8g anhydrous sodium acetate in 60mL ethylene glycol and stir magnetically until completely dissolved; seal the solution in a PTFE-lined autoclave and place it in an oven at T=180℃ for 8h to complete the reaction; take it out and wash it with water and ethanol three times respectively, and vacuum dry it overnight;

[0039] Preparation of S32 and Fe3O4@SiO2 nanoparticles: Fe3O4NPs were ultrasonically dispersed in an ethanol-water solution, 2.8mL NH3·H2O and 0.3mL TEOS were added, magnetically stirred for 90min, washed with water and ethanol three times respectively, and dried under vacuum; the volume ratio of ethanol to water in the ethanol-water solution was 4:1;

[0040] S33, amino functionalization (AMNPs): Fe3O4@SiO2 was ultrasonically dispersed in 150 mL of ethanol, and 5 mL of APTES was added, and refluxed in a water bath at 80 °C for 12 h; washed with water and ethanol three times respectively, and dried in vacuum;

[0041] S34, polyethyleneimine modified magnetic nanoparticles: The amino-functionalized magnetic material was ultrasonically dispersed in 50 mL of glutaraldehyde-anhydrous methanol solution, wherein the volume ratio of glutaraldehyde to anhydrous methanol was 1:9, and magnetic stirring was performed for 10 h; after washing with anhydrous methanol for 4 times, it was further dispersed in 50 mL of anhydrous methanol containing polyethyleneimine, and magnetic stirring was performed for 12 h. Sodium cyanoborohydride was added and stirring was continued for 12 h; PEI-AMNPs were collected by magnet, washed with water and ethanol for 3 times respectively, and vacuum dried;

[0042] S35. Boric acid functionalized dendritic magnetic material: Ultrasonic dispersion of polyethyleneimine modified magnetic nanoparticles in 50 mL of anhydrous methanol containing 4-formylphenylboronic acid and sodium cyanoborohydride, magnetic stirring for 24 h, wash the obtained magnetic material with water and ethanol three times respectively, vacuum dry to obtain FPBA-PEI-AMNPs dendritic molecules assisted with boric acid functionalized magnetic nanoparticles, which are sealed for later use.

[0043] In the synthesis process of the functionalized magnetic nanomaterial, the rotation speed of the magnetic stirring is 300-500 rpm.

[0044] At the same time, under the same system and detection environment, different proteins 10 ng / mL PCT, 25 ng / mL AFP, 4 mg / mL TrF and 0.1 mg / mL BSA were added to 1 mL of serum for SERS detection to investigate the specificity of the system for PCT.

[0045] The present invention has the following beneficial effects:

[0046] 1. The non-interference SERS probe provided by the present invention has good stability and reproducibility; it can solve the problem of SERS probe in the fingerprint region (300-1800cm -1 ) is susceptible to optical interference from endogenous biomolecules, which leads to reduced accuracy of quantitative analysis.

[0047] 2. The preparation method of the present invention uses simple instruments, low reagent costs, and mild reaction conditions; and the SERS probe can be applied to single-index / multi-index detection of low-abundance disease markers and detection of major diseases.

[0048] 3. The SERS tag prepared by the present invention can directly modify the antibody of the object to be detected on the shell through the dopamine quinone on the surface of polydopamine to synthesize a SERS probe with high specificity for the object to be detected, without the need to form an active ester by using the carboxyl group of the antibody and N-hydroxysuccinimide in the presence of a cross-linking agent carbodiimide as in the past. This can not only save preparation time, but also reduce detection costs.

[0049] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0051] Figure 1 A in the middle is the TEM image of Au NPs;

[0052] Figure 1 B is the TEM image of Au@Ag@PDA-EB;

[0053] Figure 1 C in the middle is the particle size distribution diagram of Au@Ag@PDA-EB;

[0054] Figure 1 D in the middle is the electron microscope scanning image of Au@Ag@PDA-EB;

[0055] Figure 1 E in the figure is the EDS image of Au@Ag@PDA-EB;

[0056] Figure 1 F in the figure is the UV spectrum of Au@Ag@PDA-EB;

[0057] Figure 1 G in the middle is the Raman characterization image of each synthesis stage of the SERS probe;

[0058] Figure 1 H in the figure is the zeta potential measurement diagram of each synthesis stage of the SERS probe;

[0059] Figure 2 A is the TEM image of Fe3O4;

[0060] Figure 2 B is the TEM image of Fe3O4@SiO2-PEI-FPBA;

[0061] Figure 2 C in the middle is the SEM image of Fe3O4;

[0062] Figure 2 D is the SEM image of Fe3O4@SiO2-PEI-FPBA;

[0063] Figure 3 A in the middle is the FT-IR image of functionalized magnetic nanoparticles;

[0064] Figure 3 B is the XRD pattern of functionalized magnetic nanoparticles;

[0065] Figure 3 Middle C is the VSM image of functionalized magnetic nanoparticles;

[0066] Figure 3 D in the middle is the zeta potential diagram of functionalized magnetic nanoparticles;

[0067] Figure 3 Middle E is the XPS spectrum of functionalized magnetic nanoparticles;

[0068] Figure 4 A in the middle is the (A) SERS spectrum of the synthesized SERS probe combined with functionalized magnetic nanoparticles for detecting 10pg / mL-50ng / mL PCT;

[0069] Figure 4 Middle B is the standard curve of this strategy used to detect 10pg / mL-50ng / mL PCT;

[0070] Figure 4 Middle C represents the specific recognition of different interfering glycoproteins by this strategy;

[0071] Figure 4 D in the middle is the SEM image of the "sandwich" structure;

[0072] Figure 5 Diagram of the detection strategy of SERS combined with dendrimer boronic acid affinity magnetic nanoparticles for the disease marker PCT. DETAILED DESCRIPTION

[0073] Embodiment 1, a method for preparing a non-interference SERS probe, comprising the following steps:

[0074] Stp1, prepare 100 mL of 0.01% chloroauric acid solution, heat to boiling, add 5 mL of 1% trisodium citrate solution, keep boiling and magnetically stir for 15 min; obtain wine-red Au NPs colloid with a particle size of about 18.3±1.5 nm;

[0075] Stp2, add 1 mL of 0.1 M ascorbic acid solution to 10 mL of colloidal gold, add 1 mL of 10 mM silver nitrate solution dropwise under magnetic stirring, wait until the colloid color changes to earthy yellow, continue stirring for 6 min, centrifuge, and redissolve the precipitate in 10 mL of ultrapure water to obtain the core-shell SERS enhanced substrate Au@Ag;

[0076] Stp3, under magnetic stirring, add phenylacetylene solution to the prepared Au@Ag, then add a mixed solution of Tris-HCl buffer solution and dopamine, stir magnetically for 2-5 minutes, stop stirring and centrifuge when the solution turns dark green, and re-dissolve in the original volume to obtain the SERS tag Au@Ag@PDA-EB;

[0077] Stp4, through the dopamine quinone on the surface of polydopamine, the capture antibody of the analyte is modified on the shell surface of Au@Ag@PDA-EB, and a SERS probe with high specific recognition characteristics for the analyte is synthesized.

[0078] Furthermore, in Stp1, specifically: 100 mL of 0.01% chloroauric acid solution was prepared, heated to boiling, and then 5 mL of 1% trisodium citrate solution was added, and the mixture was kept boiling and magnetically stirred for 15 minutes to obtain a wine-red Au NPs colloid with a particle size of about 18.3±1.5 nm.

[0079] Furthermore, in Stp2, 1 mL of 0.1 M ascorbic acid solution was added to 10 mL of colloidal gold, and 1 mL of 10 mM silver nitrate solution was added dropwise under magnetic stirring. When the colloid color turned into khaki, stirring was continued for 6 min and then centrifuged. The precipitate was redissolved in 10 mL of ultrapure water to obtain the core-shell SERS enhanced substrate Au@Ag.

[0080] In Stp3, 1 mL of 1 mM phenylacetylene solution was added dropwise to the Au@Ag obtained in Stp2, and then 10 mL of a mixed solution was added, wherein the mixed solution was a mixture of Tris-HCl buffer solution and dopamine;

[0081] The phenylacetylene solution is obtained by dissolving phenylacetylene in ethanol.

[0082] In Stp1-Stp3, the number of revolutions of magnetic stirring was 400 rpm; in Stp2 and Stp3, the number of revolutions of centrifugation was 7500 rpm and the time was 12 in.

[0083] Example 2: A non-interference SERS probe is prepared by the preparation method of a non-interference SERS probe provided in Example 1 above.

[0084] The SERS probe was obtained by reducing HAuCl4 with trisodium citrate to obtain a size of about 18.3±1.5nm ( Figure 1 C), uniform and stable Au NPs as plasma cores ( Figure 1 A). Figure 1 B is the TEM image of Au@Ag@PDA-EB, which shows a distinct core-shell structure and a relatively uniform size with an average particle size of about 37.5 nm ( Figure 1 C), the thickness of the silver shell is about 9 nm, but there is no obvious PDA layer;

[0085] Figure 1 The EDS graph of E only shows Au and Ag due to the low N content. Figure 1 The Au@Ag@PDA-EB SEM-mapping of D clearly shows the distribution of characteristic elements Au, Ag, N and O, proving the successful modification of the gold core and silver shell as well as the EB carried by PDA.

[0086] The UV-Vis spectra of different nanoparticles were measured and the results are shown in Figure 2. Figure 1 As shown in Figure F, the characteristic absorption peak of Au NPs is at 521nm. After being coated with Ag, the absorption peak of Au NPs disappears, and the characteristic absorption peak of silver appears at 412nm. The colloid also changes from wine red to gray, indicating that the surface of the gold core is covered by a thicker silver layer.

[0087] The absorbance of the SERS tag formed after PDA coating was slightly reduced and a slight red shift occurred, which preliminarily proved the successful preparation of Au@Ag@PDA-EB. Figure 1 Raman characterization of G, the label is at 1987cm -1 An obvious characteristic peak appears at the center, proving the successful synthesis of the SERS probe.

[0088] In addition, the zeta potential of nanoparticles at different stages was measured ( Figure 1 H), the average zeta potential of AuNPs is -24.533mv. After the surface is coated with a silver shell, the zeta potential decreases. After further coating with PDA, the zeta potential increases significantly, which is caused by the protonation of -NH2. After adding PCT-Ab, the average zeta potential of Au@Ag@PDA-EB-Ab rises to -1.06mV. Combined with the above other characterizations, the successful synthesis of the SERS probe is once again proved. The Raman characteristic peak of the SERS probe is in the Raman silent zone of 1800cm -1 -2500cm -1 The invention relates to a colloidal gold structure, a silver shell arranged on the surface of the colloidal gold structure, a plurality of Raman reporter molecules phenylacetylene connected to the silver shell, a polydopamine layer formed by self-polymerization of dopamine connected to the outside of the phenylacetylene, and a procalcitonin antibody connected to the outside of the polydopamine layer.

[0089] Among them, the connection method of phenylacetylene is to coat it on the surface of SERS substrate through self-polymerization of dopamine; the modification method of procalcitonin antibody is to bond it with dopamine quinone of polydopamine layer.

[0090] Example 3, application of a non-interference SERS probe in the detection of procalcitonin, a disease marker, in human serum; the specific application method includes the following:

[0091] S1. Testing of PCT standards:

[0092] S11. Prepare PCT standard solutions of different concentrations in fetal bovine serum; the concentrations of the PCT standard solutions are 10, 50, 100, 500, 1000, 5000, 10000, and 50000 pg / mL, respectively;

[0093] S12. Take 2 mL of the standard solution, add 0.3 mg of the functionalized magnetic nanomaterial, and incubate at 25°C in a shaker for 30 min; wash three times with PBS buffer at pH 7.4, add 0.5 mL of SERS probe, and incubate at 37°C in a shaker for 6 min to allow the antigen and antibody to specifically bind.

[0094] S13, after completing the detection with the Raman spectrometer, establishing a standard curve, and obtaining a linear equation of the standard curve according to the standard curve;

[0095] The shaking speed in the above steps was 300 rpm. A blank group was prepared under the same conditions.

[0096] The SERS detection in the above steps was performed at a power of 200 mW and an integration time of 500 ms, and the average Raman intensity was recorded 3 times. All values ​​were the average of 5 repeated measurements, and baseline correction was performed.

[0097] S2. Serum sample testing:

[0098] S21, take serum samples and divide them into 1 mL portions, add different amounts of 500 ng / mL CT solution, mix thoroughly for 30 min, and prepare spiked serum samples of different concentrations;

[0099] The spiked serum sample solutions were divided into 8 groups, and the PCT concentrations in each group of serum samples were 0, 0.05, 0.5, 0.8, 2.0, 5.0, 10.0, and 25.0 ng / mL;

[0100] S22. Take 1 mL of spiked serum sample solution, add 0.3 mg of functionalized magnetic nanomaterials, incubate at 25°C for 30 min in a shaker, and selectively capture PCT using the boric acid affinity. After washing three times with pH 7.4 PBS buffer, add 0.5 mL of SERS probe and incubate at 37°C for 6 min in a shaker to form a "sandwich" structure through antigen-antibody immune binding.

[0101] S23. Use a Raman spectrometer to detect the SERS signal intensity of the serum sample solution, and calculate the concentration value of PCT in the spiked serum sample according to the linear equation of the standard curve.

[0102] The constructed “sandwich” system was used to detect the SERS spectra of PCT standard samples with different concentration gradients. Figure 4 As shown in A, a single antibody detection of glycoprotein PCT was constructed on the magnetic material through boronate affinity. As the PCT concentration increased, the 1987 cm -1 The SERS signal of the characteristic peak at 1987cm -1 The SERS signal intensity (I1987 ) and the corresponding logarithmic value of PCT concentration lg C (pg / mL) to make a working curve ( Figure 4 B), and the linear regression equation (I 1987 =1949.8349lgC-1051.6626, R 2 =0.9948), and the LOD was obtained to be 4.44 pg / mL; Figure 4 The SEM image of the D sandwich structure also confirms the establishment of this method.

[0103] At the same time, under the same system and detection environment, different proteins 10 ng / mL PCT, 25 ng / mL AFP, 4 mg / mL TrF and 0.1 mg / mL BSA were added to 1 mL of serum for SERS detection to investigate the specificity of the system for PCT.

[0104] At the same time, the same method was used to detect different proteins IL-6, AFP, BSA and TrF to test the specificity of the system. Figure 4 C It can be clearly observed that compared with various interfering glycoproteins, this system has a high specific recognition for PCT. The Raman intensity of BSA used in the preparation of SERS probes is weak in this system, and its influence can be ignored.

[0105] SERS and ELISA kits were used to detect and recover PCT in human serum samples (n=3), and the linear regression equation (I 1987 =1949.8349lgC-1051.6626, R 2 =0.9948), calculate the measured value of this method, as shown in the following table:

[0106]

[0107] In order to verify the feasibility of the constructed sensing strategy for PCT detection in human serum. First, the present method was used to perform SERS detection on spiked serum samples with different PCT concentrations (0, 0.05, 0.5, 0.8, 2.0, 5.0, 10.0 and 25.0 ng / mL). Then, the PCT concentrations of pure serum sample C0 and spiked samples C1-C7 were calculated according to the linear equation of the standard solution. The above table summarizes the detection results of this method and the gold standard ELISA method, with a detection range of 0.05-25.0 ng / mL (n=3) and a PCT recovery rate of 94.00%-108.00%.

[0108] The results showed that there was no significant difference in specificity and accuracy between the proposed functional magnetic material-portable Raman spectroscopy and ELISA methods. Moreover, this method reduced the detection cost and shortened the analysis time, and the linear detection range (10pg / mL-50ng / mL) was much better than that of ELISA kits. These results strongly confirmed the potential prospects of this method for clinical PCT-related disease screening.

[0109] Example 4, the synthesis of the functionalized magnetic nanomaterial of Example 3 mainly includes the following steps:

[0110] S31. Preparation of Fe3O4 nanoparticles: Dissolve FeCl3·6H2O, 1.5g polyethylene glycol and 4.8g anhydrous sodium acetate in 60mL ethylene glycol and stir magnetically until completely dissolved; seal the solution in a PTFE-lined autoclave and place it in an oven at T=180℃ for 8h to complete the reaction; take it out and wash it with water and ethanol three times respectively, and vacuum dry it overnight;

[0111] Preparation of S32 and Fe3O4@SiO2 nanoparticles: Fe3O4 NPs were ultrasonically dispersed in an ethanol-water solution, 2.8 mL NH3·H2O and 0.3 mL TEOS were added, magnetically stirred for 90 min, washed with water and ethanol three times, respectively, and dried under vacuum; the volume ratio of ethanol to water in the ethanol-water solution was 4:1;

[0112] S33, amino functionalization (AMNPs): Fe3O4@SiO2 was ultrasonically dispersed in 150 mL of ethanol, and 5 mL of APTES was added, and refluxed in a water bath at 80 °C for 12 h; washed with water and ethanol three times respectively, and dried in vacuum;

[0113] S34, polyethyleneimine modified magnetic nanoparticles: The amino-functionalized magnetic material was ultrasonically dispersed in 50 mL of glutaraldehyde-anhydrous methanol solution, wherein the volume ratio of glutaraldehyde to anhydrous methanol was 1:9, and magnetic stirring was performed for 10 h; after washing with anhydrous methanol for 4 times, it was further dispersed in 50 mL of anhydrous methanol containing polyethyleneimine, and magnetic stirring was performed for 12 h. Sodium cyanoborohydride was added and stirring was continued for 12 h; PEI-AMNPs were collected by magnet, washed with water and ethanol for 3 times respectively, and vacuum dried;

[0114] S35. Boric acid functionalized dendritic magnetic material: Ultrasonic dispersion of polyethyleneimine modified magnetic nanoparticles in 50 mL of anhydrous methanol containing 4-formylphenylboronic acid and sodium cyanoborohydride, magnetic stirring for 24 h, wash the obtained magnetic material with water and ethanol three times respectively, vacuum dry to obtain FPBA-PEI-AMNPs dendritic molecules assisted with boric acid functionalized magnetic nanoparticles, which are sealed for later use.

[0115] During the synthesis of the functionalized magnetic nanomaterials, the magnetic stirring speed is 300-500 rpm.

[0116] The morphology and size of Fe3O4 and functionalized magnetic nanoparticles were characterized by TEM and SEM. Figure 2 It can be clearly seen in the AD series that compared with Fe3O4, the spherical surface of functionalized magnetic nanoparticles ( Figure 2 D) has obvious branches distributed on the surface of the sphere, which preliminarily confirms the successful preparation of Fe3O4@SiO2-PEI-FPBA.

[0117] FT-IR was used to investigate the surface groups of functionalized magnetic nanoparticles ( Figure 3 A), in the spectrum, Fe3O4 at 581cm -1 The characteristic peak at 1076cm is attributed to the stretching vibration of Fe-O. -1 The broad band at 1645 cm corresponds to the asymmetric stretching vibration of Si-O-Si. -1 and 3309cm -1 The peak at 2846 cm is attributed to the stretching and bending vibrations of NH. -1 and 2912cm -1 The characteristic peak at 1645 cm is attributed to the stretching vibration of CH. -1 and 3309cm -1 The absorption enhancement at 1455 cm corresponds to the stretching and bending vibrations of NH in the dendritic PEI. -1 The characteristic peak at is attributed to the adsorption of BO. FT-IR results show that boric acid functionalized magnetic nanoparticles were successfully prepared.

[0118] The XRD patterns of functionalized magnetic nanoparticles are shown in Figure 3 B. XRD shows characteristic diffraction peaks of Fe3O4 at (440), (511), (422), (400), (311) and (220). After functionalizing Fe3O4 with SiO2 and boric acid, the characteristic diffraction peaks of Fe3O4 did not disappear, and no other impurity peaks appeared, indicating that the crystal structure was not destroyed during the functionalization process.

[0119] The hysteresis loop of the magnetic material was measured using VSM, proving that its magnetic properties are good ( Figure 3 C). In addition, when an external magnetic field is used for adsorption, the enrichment and separation process can be completed within 10 seconds, which will greatly save the pretreatment time before detection.

[0120] In order to further study the changes in elemental composition and counts after each step of modification of the magnetic material, we used XPS. Figure 3 As shown in E, the full spectrum contains peaks of Fe, N, C, B and Si elements. The XPS spectrum of B1s has a peak at 190.67eV ( Figure 3 E2), indicating that FPBA was successfully modified on the surface of the magnetic material. Compared with the N content modified with APTES alone, the addition of PEI increased the N1S count at 398.76 eV by 5-6 times ( Figure 3 E1). The XPS and FT-IR results are consistent, proving the successful preparation of Fe3O4@SiO2-PEI-FPBA.

[0121] In addition, the Zeta potential of magnetic materials at different modification stages was measured ( Figure 3 D), the change of the material's Zeta potential not only reflects its successful preparation, but also Figure 1 H and 3D also indicate that the formation of the sandwich structure is the result of boronic acid affinity and immune binding, rather than electrostatic adsorption.

[0122] like Figure 5 , using the synthesized new type of highly sensitive and non-interfering SERS probe, a non-interfering and high-precision biosensor based on SERS combined with dendrimer-assisted boronic acid functionalized MNPs was constructed for the detection of glycoprotein disease marker PCT in human serum. This sensing strategy is a sandwich structure based on "non-interfering SERS probe-target glycoprotein-dendrimer-assisted boronic acid functionalized MNPs". First, 4-formylphenylboronic acid (FPBA) with a pKa of 7.3 (selected with reference to the human blood environment pH = 7.35-7.45) was selected to selectively capture glycoproteins in biological samples, and the superparamagnetism of Fe3O4 was used to separate it from the complex matrix. Secondly, the successfully synthesized Raman silent zone (1800-2500cm) was added -1 ) A highly sensitive and specific SERS probe with characteristic peaks specifically captures the target glycoprotein PCT on the functionalized magnetic material through antigen-antibody immune binding. Finally, the "sandwich" structure formed after incubation is collected and separated by a magnet, and the Raman signal is measured using a portable Raman spectrometer to complete the quantitative analysis of PCT in human serum. The successful construction of this strategy is intended to provide a widely applicable method for the high-sensitivity detection of glycoprotein disease markers in the future, and is also expected to provide a new method and experimental reference for the early diagnosis, clinical treatment, prognosis assessment and POCT (point-of-care diagnosis) of major diseases.

[0123] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0124] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing a non-interference SERS probe, characterized in that: The steps include: Step 1, heat the chloroauric acid solution to boiling, add trisodium citrate solution, keep boiling and continue magnetic stirring to obtain colloidal gold; Stp2, add ascorbic acid solution to colloidal gold, add silver nitrate solution dropwise under magnetic stirring, wait until the colloid color changes to earthy yellow, continue stirring for 6 minutes, then centrifuge, and redissolve the precipitate in ultrapure water to obtain the core-shell SERS enhanced substrate Au@Ag; Stp3, under magnetic stirring, add phenylacetylene solution to the prepared Au@Ag, then add a mixed solution of Tris-HCl buffer solution and dopamine, stir magnetically for 2-5 minutes, stop stirring and centrifuge when the solution turns dark green, and re-dissolve in the original volume to obtain the SERS tag Au@Ag@PDA-EB; Stp4, through the dopamine quinone on the surface of polydopamine, the capture antibody of the analyte is modified on the shell surface of Au@Ag@PDA-EB to synthesize a SERS probe with high specificity for the analyte; The Raman characteristic peak of the SERS probe is in the Raman silent zone of 1800 cm -1 -2500cm -1 The SERS probe comprises a colloidal gold structure, a silver shell arranged on the surface of the colloidal gold structure, a plurality of Raman reporter molecules phenylacetylene are connected to the silver shell, a polydopamine layer formed by self-polymerization of dopamine is connected to the outside of the phenylacetylene, and a procalcitonin antibody is connected to the outside of the polydopamine layer. The application includes the following: S1. Testing of PCT standards: S11, preparing PCT standard solutions of different concentrations in fetal bovine serum; S12, take 1 mL of the standard solution, add 0.3 mg of the functionalized magnetic nanomaterial, and incubate at 25°C for 30 min in a shaker; wash three times with PBS buffer at pH = 7.4, add 0.5 mL of SERS probe, and incubate at 37°C for 6 min in a shaker to allow the antigen and antibody to immunobind; S13, after completing the detection with the portable Raman spectrometer, establishing a standard curve, and obtaining a linear equation of the standard curve according to the standard curve; S2. Serum sample testing: S21, taking serum samples and dividing them into 1 mL portions, adding PCT standard solutions of different concentrations thereto, and mixing them thoroughly for 30 minutes to prepare serum sample solutions of different concentrations; S22, take 1 mL of spiked serum sample solution of different concentrations, add 0.3 mg of functionalized magnetic nanomaterials, and incubate in a shaker at 25°C for 30 min; after washing three times with PBS buffer at pH = 7.4, add 0.5 mL of SERS probe and incubate in a shaker at 37°C for 10 min to allow antigen and antibody to immunologically bind; S23, using a portable Raman spectrometer to detect the SERS signal intensity of the serum sample solution, and calculating the concentration value of PCT in the spiked serum sample according to the linear equation of the standard curve; The synthesis of the functionalized magnetic nanomaterial mainly includes the following steps: S31. Preparation of Fe3O4 nanoparticles: FeCl3·6H2O, 1.5 g polyethylene glycol and 4.8 g anhydrous sodium acetate were dissolved in 60 mL ethylene glycol and stirred magnetically until completely dissolved; the solution was sealed in a PTFE-lined high-pressure reactor and placed in an oven to complete the reaction; after taking out, it was washed with water and ethanol three times respectively, and vacuum dried overnight; S32, preparation of Fe3O4@SiO2 nanoparticles: Fe3O4NPs were ultrasonically dispersed in an ethanol-water solution, 2.8mL NH3·H2O and 0.3mL LTEOS were added, magnetically stirred for 90min, washed with water and ethanol three times, respectively, and dried in vacuum; S33, amino functionalization: ultrasonically disperse Fe3O4@SiO2 in 150 mL ethanol, add 5 mL APTES, reflux in a water bath; wash with water and ethanol three times respectively, and dry in vacuum; S34, polyethyleneimine modified magnetic nanoparticles: The amino-functionalized magnetic material was ultrasonically dispersed in 50 mL of glutaraldehyde-anhydrous methanol solution and magnetically stirred for 10 h; after washing with anhydrous methanol for 4 times, it was further dispersed in 50 mL of anhydrous methanol containing polyethyleneimine and magnetically stirred for 12 h, sodium cyanoborohydride was added and stirred for 12 h; PEI-AMNPs were collected by magnet, washed with water and ethanol for 3 times respectively, and vacuum dried; S35, boric acid functionalized dendritic magnetic material: polyethyleneimine modified magnetic nanoparticles were ultrasonically dispersed in 50 mL of anhydrous methanol containing 4-formylphenylboronic acid and sodium cyanoborohydride, and magnetically stirred for 24 h. The obtained magnetic material was washed with water and ethanol three times, respectively, and vacuum dried to obtain FPBA-PEI-AMNPs dendritic molecules assisted in boric acid functionalized magnetic nanoparticles for sealing and standby use; Using the synthesized non-interference SERS probe, a non-interference high-precision biosensor based on SERS combined with dendrimer-assisted boronic acid functionalized MNPs was constructed for the detection of glycoprotein disease marker PCT in human serum. The sensing strategy is a sandwich structure based on "non-interference SERS probe-target glycoprotein-dendrimer-assisted boronic acid functionalized MNPs". First, 4-formylphenylboronic acid with a pKa of 7.3 was selected to selectively capture glycoproteins in biological samples, and the superparamagnetic property of Fe3O4 was used to separate them from the complex matrix. Secondly, a highly sensitive and specific SERS probe with a characteristic peak in the Raman silent region that had been successfully synthesized was added to specifically capture the target glycoprotein PCT on the functionalized magnetic material through antigen-antibody immune binding. Finally, the "sandwich" structure formed after incubation was collected and separated by a magnet, and the Raman signal was measured using a portable Raman spectrometer to complete the quantitative analysis of PCT in human serum.

2. The method for preparing a non-interference SERS probe according to claim 1, characterized in that: In the Stp1, specifically: prepare 100 mL of 0.01% chloroauric acid solution, heat to boiling, add 5 mL of 1% trisodium citrate solution, keep boiling and magnetically stir for 15 minutes, and obtain wine-red AuNPs colloid with a particle size of 18.3±1.5 nm.

3. The method for preparing a non-interference SERS probe according to claim 1, characterized in that: In the Stp2, specifically, 1 mL of 0.1 M ascorbic acid solution was added to 10 mL of colloidal gold, and 1 mL of 10 mM silver nitrate solution was added dropwise under magnetic stirring, and the colloid color changed to khaki, and then the mixture was stirred for 6 min and centrifuged, and the precipitate was redissolved in 10 mL of ultrapure water to obtain a core-shell SERS enhanced substrate Au@Ag; In Stp3, 1 mL of 1 mM phenylacetylene solution was added dropwise to the Au@Ag obtained in Stp2, and then 10 mL of a mixed solution was added, wherein the mixed solution was a mixture of Tris-HCl buffer solution and dopamine; Wherein, the phenylacetylene solution is obtained by dissolving phenylacetylene in ethanol.

4. The method for preparing a non-interference SERS probe according to claim 1, characterized in that: In Stp1-Stp3, the speed of the magnetic stirring is 300-500 rpm; in Stp2 and Stp3, the speed of the centrifugation is 7000-7800 rpm and the time is 10-15 min.

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