CN118603958A - A Raman analysis method for ALS, APOC3, C3, and FCN2 proteins and its biological application - Google Patents

Abstract

The present invention provides a Raman analysis method and biological application for ALS, APOC3, C3, and FCN2 proteins. First, chemical synthesis is used to prepare rod-shaped gold nanoparticles (gold nanorods) as Raman enhanced substrate materials for SERS probes. Secondly, the surface of the gold nanorods is modified to adjust the hydrophilicity and hydrophobicity of the surface, and a gold nanorod dispersion with stable structure and good dispersibility in different blood samples (blood, plasma, serum) is constructed. Subsequently, a variety of chemical molecules containing thiol, amino, carboxyl and other groups are used as Raman signal molecules, and are co-modified with antibodies of ALS, APOC3, C3 and FCN2 to prepare SERS probes of related proteins. The analysis chip has the characteristics of high sensitivity, good selectivity, wide linear range, simple analysis operation, fast detection speed, and high result accuracy. It can realize rapid, accurate, and sensitive analysis and detection of ALS, APOC3, C3, and FCN2 proteins in blood samples within 10 minutes, providing an indicator quantitative basis for early warning of clinical related disease risks.

Description

A Raman analysis method and biological application for ALS, APOC3, C3, and FCN2 proteins

Technical Field

The present invention relates to the technical field of biological detection, and in particular to a Raman analysis method and biological application for ALS, APOC3, C3 and FCN2 proteins.

Background Art

In modern medical research and clinical diagnosis, the analysis of disease-related proteins plays a vital role. As a key medium for intracellular and extracellular communication, proteins are not only involved in all aspects of life activities, but are also directly related to the occurrence, development and prognosis of a variety of diseases. By accurately analyzing these proteins, it is possible to diagnose the disease in its early stages, assess the severity of the disease, monitor the effectiveness of treatment, and predict the development trend of the disease, providing patients with personalized treatment plans. In addition, these analyses help reveal the molecular mechanisms of the disease and promote the development of new drugs and the advancement of medical research.

However, the current analysis of disease-related proteins faces several technical difficulties. First, the expression level of proteins and their distribution in the body are extremely complex, and different proteins may show different expression patterns in different tissues, different cell types, and even different disease states. This requires that the analysis method must have a high degree of sensitivity and specificity to distinguish and detect trace amounts of proteins and their variants. Secondly, the structural diversity and modification complexity of proteins also pose challenges to analysis. Proteins can be modified in a variety of ways, such as phosphorylation and glycosylation, which affect their function and activity. Therefore, they need to be accurately identified and distinguished during analysis, which brings new challenges to the accuracy of clinical protein analysis. In addition, the pathogenesis and development mechanisms of diseases are complex, and there are multiple interactions and networks between proteins. In disease diagnosis, it is often difficult to accurately diagnose and predict the disease by analyzing only a single type of protein. It is necessary to construct a method for quantitative analysis and detection of multiple different disease-related proteins.

In order to overcome these difficulties, researchers and clinicians have developed a series of high-throughput and high-sensitivity technologies, such as mass spectrometry, protein chips, and immunoassays. These technologies make large-scale screening and in-depth analysis of disease-related proteins possible, but they also place higher demands on experimental design, sample processing, data analysis, and interpretation. Therefore, despite the challenges, sensitive, rapid, and accurate analysis of disease-related proteins is of irreplaceable importance for improving the diagnosis, treatment, and prevention of diseases. Therefore, further improving the accuracy and efficiency of disease-related protein analysis will bring revolutionary changes to clinical medicine.

Raman spectroscopy is a method of spectral quantitative analysis that uses the Raman spectral signal of the probe molecule and establishes it based on the signal intensity and position information. Its fast analysis response speed and narrow half-peak width make it very suitable for constructing multi-channel, rapid analysis probes to achieve high-throughput rapid analysis of specific biomarkers. In addition, surface enhanced Raman spectroscopy (SERS) is an ultra-sensitive analysis and detection technology with an enhancement factor of up to 10 ¹⁰ or more. Therefore, SERS-based analysis technology usually has extremely high sensitivity and rapid response, and can achieve the analysis and detection of a variety of trace substances in a variety of complex environments, providing a new technical means for clinical high-sensitivity and high-accuracy analysis and detection of proteins related to a variety of diseases.

ALS protein is a recombinant protein of the acid-labile subunit of insulin-like growth factor, which exists in human serum, plasma and related liquid samples.

APOC3 protein is a protein synthesized by the liver and mainly exists in plasma. Its function is to regulate lipoprotein metabolism, especially in blood lipoprotein metabolism. By observing its effect on the lipoprotein content in the blood, its regulatory effect on lipoprotein metabolism can be evaluated.

C3 protein is a serum protein synthesized by the liver and is also an acute phase reaction protein. It has the highest content among all the components of the complement system. Determination of C3 content can reflect the total complement level in serum and is used to evaluate complement activation. It has an auxiliary diagnosis effect for immune diseases such as active lupus erythematosus and rheumatoid arthritis, and can also be used to evaluate the course of disease and efficacy.

FCN2 is a protein with immune function and a member of the oligomeric hemagglutinin family. Its main function is to regulate the body's immune response and participate in the recognition and elimination of pathogens.

Summary of the invention

The present invention is based on the above research and constructs a highly sensitive and accurate Raman analysis method for multiple disease-related proteins ALS, APOC3, C3, and FCN2, which has the advantages of fast analysis speed, high detection sensitivity, high accuracy, and multi-channel analysis. It can achieve highly sensitive and accurate analysis and detection of human ALS protein (Insulin-like growth factor-binding proteincomplex acid labile subunit, ALS), human APOC3 protein (Apolipoprotein C-III, APOC3), human C3 protein (Complement C3, C3) and human FCN2 protein (Ficolin-2, FCN2) protein.

The technical solution of the present invention is summarized as follows: First, gold nanoparticles (gold nanorods) with a rod-like structure are prepared as the Raman enhancement substrate material of the SERS probe by means of chemical synthesis. Secondly, the surface of the gold nanorods is further modified to adjust the hydrophilicity and hydrophobicity of the surface of the gold nanorods, and a gold nanorod dispersion with stable structure and good dispersibility in different blood samples (blood, plasma, serum) is constructed. Subsequently, a variety of chemical molecules containing chemically reactive groups such as thiol, amino, and carboxyl groups are used as Raman signal molecules, and are co-modified with antibodies of related proteins ALS, APOC3, C3, and FCN2 to prepare SERS probes with high sensitivity and high accuracy analytical response to related proteins. Finally, the analytical performance and results of the prepared SERS probe were verified using Raman spectroscopy analysis equipment, showing that the SERS probe prepared by the present invention has the characteristics of high sensitivity (detection limit 0.078ng/mL), good selectivity (S/N>16.8), wide linear range (0.5ng/mL-5×10 ⁴ ng/mL), simple analysis and operation, fast detection speed (<10min), and high result accuracy (error <10%). Rapid, accurate and sensitive analysis and detection of ALS, APOC3, C3 and FCN2 proteins in blood samples can be achieved within 10 minutes, providing quantitative indicators for early warning of clinical related disease risks.

The specific technical solutions of the present invention are as follows:

In a first aspect of the present invention, a Raman analysis method for ALS, APOC3, C3, and FCN2 proteins is provided, comprising the following steps:

A. Preparation of SERS-enhanced substrates

(1) Preparation of gold nano seed solution: AuCl ₄ (chloroauric acid) was used as a reaction raw material, and nano-scale HAuCl 4 nanoparticles were prepared as a gold nano seed solution by chemical reduction method.

Specifically, an equal volume of _HAuCl4 solution and a surfactant are mixed, and an iced reducing agent solution with a volume of 3.5% to 17% of the above-mentioned mixed volume and a concentration of 5 to 15 mM is added under stirring, and the solution is waited to change from golden yellow to brown yellow; then the reaction system is placed in a 10 to 50°C water bath environment to react for 1 to 5 minutes, and after taking out, the reaction solution is transferred to a 10 to 50°C environment to obtain a gold seed solution, which is stored for later use.

The preferred process conditions for this step are as follows:

The concentration of HAuCl ₄ is 0.3-0.6 mM; preferably, 0.5 mM.

The volume of HAuCl ₄ is 3-7 mL; preferably, 5 mL.

The surfactant is one of cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), cetylbenzyldimethylammonium chloride (BDAC), and cetylbenzyldimethylammonium bromide (BDAB), preferably cetyltrimethylammonium bromide (CTAB).

The concentration of the surfactant is 0.15-0.35M; preferably, 0.2M.

The volume of the surfactant is 3-7 mL; preferably, 5 mL.

The reducing agent solution is one of sodium borohydride (NaBH ₄ ), hydrazine hydrate, ascorbic acid, sodium ascorbate, and sodium citrate; preferably, it is NaBH ₄ .

The concentration of the reducing agent solution is 5 mM-15 mM; preferably, 10 mM.

The volume of the reducing agent solution is 0.5-1 mL; preferably, it is 0.6M.

The reaction time of the solution is 1 min-5 min; preferably, 2 min.

The reaction temperature of the solution is 10°C-50°C; preferably, 30°C.

The storage temperature of the solution is 10°C-50°C; preferably, 25°C.

(2) Preparation of seed growth solution: adding different volumes of AgNO ₃ solution and HAuCl ₄ solution of equal volume to the surfactant to the surfactant, and adding reducing agent solution to the above solution at the same time, after stirring evenly, adding the gold seed solution thereto to obtain gold nanorods with different aspect ratios; placing the mixed solution in a 10-50° C. water bath for 10-30 min, centrifuging the above solution at 3000-8000 rpm for 3-10 min, washing multiple times, redispersing and storing at a constant temperature of 0-10° C.

The preferred process conditions for this step are as follows:

The volume of surfactant in the growth solution is 3-8 mL; preferably, 5 mL.

The surfactant is one of cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), cetylbenzyldimethylammonium chloride (BDAC), and cetylbenzyldimethylammonium bromide (BDAB), preferably cetyltrimethylammonium bromide (CTAB).

The concentration of the surfactant in the growth solution is 0.15-0.35M; preferably, it is 0.2M.

The concentration of AgNO ₃ solution in the growth solution is 3-6 mM; preferably, 4 mM.

The volume of the AgNO ₃ solution in the growth solution is 0.05-0.3 mL; different volumes of AgNO ₃ solution are added according to the preparation of gold nanorods with different aspect ratios. Preferably, the addition amount of the short aspect ratio is 0.05 mL, and the addition amount of the long aspect ratio is 0.3 mL.

The concentration of HAuCl ₄ solution in the growth solution is 0.5 mM-2 mM; preferably, 1 mM.

The volume of HAuCl ₄ solution in the growth solution is 3 mL-8 mL; preferably, 5 mL.

The reducing agent solution of the growth solution is one of sodium borohydride (NaBH ₄ ), hydrazine hydrate, ascorbic acid, sodium ascorbate, and sodium citrate; preferably, it is ascorbic acid.

The concentration of the reducing agent in the growth solution is 0.05M-0.09M; preferably, it is 0.075M.

The volume of the reducing agent in the growth solution is 50 μL-80 μL; preferably, 80 μL.

The volume of the gold seed solution obtained in step (1) added is 5 μL-20 μL; preferably, 12 μL.

The reaction temperature is 10°C-50°C; preferably, 30°C.

The reaction time is 10 min-30 min; preferably, 20 min.

The centrifugal speed is 3000 rpm-8000 rpm; preferably, it is 5000 rpm.

The centrifugation time is 3 min-10 min; preferably, 5 min.

The washing solvent is one of ultrapure water, methanol and ethanol, preferably ultrapure water.

The number of washing operations is 1-7 times, preferably 3 times.

The storage temperature is 0°C-10°C; preferably, 4°C.

B. Construction of Raman analysis probe

The surface modification of the gold nanorods and the Raman signal molecule and antibody modification methods are used to construct specific Raman spectroscopy probes for ALS, APOC3, C3 and FCN2. The core of the method is (1) to modify the surface of the gold nanorods and adjust the surface hydrophilicity and structural stability of the gold nanorods for body fluid samples such as blood, urine, and tissue fluid to ensure the stability of the surface enhanced Raman spectroscopy properties of the gold nanorods. (2) Based on the structurally stable gold nanorods, the corresponding protein antibodies and Raman signal molecules are further modified on the surface of the gold nanorods to achieve specific recognition and signal output of specific proteins, and to establish SERS analysis probes for multiple proteins.

The specific steps are as follows:

(1) Surface modification of gold nanorods: The gold nanorod solution prepared in step A is added to 5 to 200 times the volume of a surface modification agent with a concentration of 1 to 50 mM, and stirred at a constant temperature for 8 to 15 hours in a water bath at 20 to 40° C.; then centrifuged at 3000 to 8000 rpm for 3 to 10 minutes, and the precipitate is washed several times for later use;

The preferred process conditions are as follows:

The aspect ratio of the gold nanorods is one of 1.2-13, preferably 4.7.

The surface modification active agent of the gold nanorods is one of polyvinyl pyrrolidone (PVP), polyvinyl chloride (PVC), polystyrene (PS), and polyethylene glycol (PEG), preferably polyethylene glycol (PEG).

The molecular weight of the surfactant is one of 100-1000000, preferably 10000.

The modifying group of the surface modification active agent is thiol (-SH), amino (-NH ₂ ), carboxyl (-COOH), preferably, carboxyl (-COOH).

The surfactant concentration is 1 mM-50 mM; preferably, 10 mM.

The volume of the gold nanorod solution added is 100-1000 μL; preferably, 200 μL.

The total volume of the surface modification solution is 5-20 mL; preferably, 10 mL.

The solvent used for surface modification is one of ultrapure water, anhydrous ethanol and anhydrous methanol; preferably, it is ultrapure water.

The reaction time of the surface modification process is 8-15 hours; preferably, 12 hours.

The reaction temperature of the surface modification process is 20-40°C; preferably, 30°C.

The centrifugal speed is 3000 rpm-8000 rpm; preferably, it is 5000 rpm.

The centrifugation time is 3 min-10 min; preferably, 5 min.

The washing solvent is one of ultrapure water, methanol and ethanol, preferably ultrapure water. The washing operation is performed 1 to 7 times, preferably 3 times.

(2) Targeted modification of Raman signal molecules: According to different target analysis proteins, surface-modified gold nanorod solutions were added to the Raman signal molecule solutions of ALS, APOC3, C3, and FCN2 proteins, respectively. The above reaction system was placed in a water bath at 20 to 40°C and stirred at a constant temperature for 8 to 15 hours for targeted modification of Raman signal molecules.

Preferably, the Raman signal molecule is one or more of mercaptobenzoic acid, mercaptoaniline, mercaptophenylacetonitrile, mercaptonaphthalene, mercaptophenylacetylene, mercaptonaphthol and their various derivatives. Depending on the different proteins, preferably, they are mercaptobenzoic acid (ALS), mercaptophenylacetylene (APOC3), mercaptonaphthol (C3), mercaptophenylacetonitrile (FCN2).

The concentration of the Raman signal molecule is 1 mM-50 mM; preferably, 10 mM.

The concentration of the modified gold nanorods is 100-1000 μL; preferably, 200 μL.

The total volume of the Raman signal molecule modified solution is 5-20 mL; preferably, 10 mL.

The solvent used for Raman signal molecule modification is one of ultrapure water, anhydrous ethanol and anhydrous methanol; preferably, it is anhydrous ethanol.

The reaction time of Raman signal molecule modification is 8-15 hours; preferably, 12 hours.

The reaction temperature for Raman signal molecule modification is 20-40°C; preferably, 30°C.

(3) Antibody-targeted molecule modification: After centrifugation of the above solution, the precipitate is redispersed in a solvent with a pH of 3 to 8, and antibodies corresponding to ALS, APOC3, C3, and FCN2 are added thereto in sequence, and then N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mixed coupling agents are added to the system respectively; the above reaction system is placed in a water bath at 20 to 40° C. and stirred at a constant temperature for 8 to 15 hours to perform antibody-targeted molecule modification;

Preferably, the antibody targeting molecule is one of a monoclonal antibody and a polyclonal antibody, preferably a polyclonal antibody.

The antibody targeting molecule is one or more of human, mouse, and rabbit antibodies, preferably, a mouse antibody.

The concentration of the antibody targeting molecule is 10 μM-80 μM; preferably, 60 μM.

The total volume of the antibody targeting molecule modification solution is 5-20 mL; preferably, 10 mL.

The solvent used for antibody targeting molecule modification is one of ultrapure water, anhydrous ethanol, anhydrous methanol, PBS buffer, Tris-HCl buffer, and HAc/NaAc buffer; preferably, it is PBS buffer.

The pH of the solvent used for modification of the antibody targeting molecule is one of 3-8, preferably, pH=5.5.

The dosage ratio of the coupling agent (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS)) used for antibody targeting molecule modification is 5-20 mg/2-8 mg, preferably 10 mg/5 mg.

The reaction time of the antibody-targeted molecule modification is 8-15 hours; preferably, 12 hours.

The reaction temperature of the Raman signal molecule modification is 20-40°C; preferably, 30°C.

(4) The solution is centrifuged and washed under the same conditions to obtain Raman spectroscopy probes modified with antibodies and signal molecules, and then stored at 0-10° C. for future use to obtain ALS probes, APOC3 probes, C3 probes, and FCN2 probes.

Preferably, the centrifugal speed is 3000rpm-8000rpm; preferably, it is 5000rpm.

The centrifugation time is 3 min-10 min; preferably, 5 min.

The washing solvent is one of ultrapure water, methanol and ethanol, preferably ultrapure water.

The washing operation is performed 1-7 times, preferably 3 times.

The storage temperature is 0°C-10°C; preferably, 4°C.

C. Detection

(1) Based on the above experimental method, Raman spectroscopy probes with specific recognition effects were prepared for a variety of different proteins and protein combinations.

(2) The Raman spectroscopy probes prepared in step (1) are mixed with standard laboratory standard simulated serum samples, respectively, and after incubation, the probe analysis performance is tested using a Raman spectrometer.

(3) The Raman spectroscopy probes prepared in step (1) are mixed with standard experimental mouse serum samples respectively, and after incubation, the probe actual sample is tested using a Raman spectrometer and compared with the results of a traditional ELISA detection kit.

Preferably, in step (1), the protein is one or any combination of ALS, APOC3, C3 and FCN2 proteins;

In step (1), in the specific recognition Raman probe, the names of the target protein, probe and signal molecule are respectively:

ALS: ALS-probe: mercaptobenzoic acid;

APOC3: APOC3-probe: mercaptophenylacetylene;

C3: C3-probe: mercaptonaphthol;

FCN2: FCN2-probe: mercaptophenylacetonitrile.

In step (2), the volume of the standard simulated serum sample is 200 μL-800 μL; preferably, 500 μL.

In step (2), the volume of the Raman spectroscopy probe solution is 100 μL-500 μL, preferably 200 μL.

In step (2), the concentration of the Raman spectroscopy probe solution is 0.5-2 μM; preferably, 1 μM.

In step (2), the co-incubation temperature is 30°C-80°C; preferably, 36°C.

In step (2), the mixed incubation time is 1-20 min; preferably, 10 min.

In step (2), the laser wavelength used in the Raman spectrum test is one of 532 nm, 633 nm and 785 nm; preferably, 785 nm.

In step (2), the laser power used in the Raman spectroscopy test is 0.5-5 mW; preferably, 1 mW.

In step (2), the exposure time used in the Raman spectrum test is 0.5-2s; preferably, 1s.

In step (3), the volume of the mouse serum sample is 200 μL-800 μL; preferably, 500 μL.

In step (3), the volume of the Raman spectroscopy probe solution is 100 μL-500 μL, preferably 200 μL.

In step (3), the concentration of the Raman spectroscopy probe solution is 0.5-2 μM; preferably, 1 μM.

In step (3), the co-incubation temperature is 30°C-80°C; preferably, 36°C.

In step (3), the mixed incubation time is 1-20 min; preferably, 10 min.

In step (3), the laser wavelength used in the Raman spectrum test is one of 532 nm, 633 nm and 785 nm; preferably, 633 nm.

In step (3), the laser power used in the Raman spectroscopy test is 0.5-5 mW; preferably, 1 mW.

In step (3), the test exposure time used in the Raman spectroscopy test is 0.5-2s; preferably, 1s.

In the present invention, based on the specific recognition of specific antibodies on different proteins, the Raman enhancement effects of different signal molecules on the surface of the gold nanorod substrate material before and after the reaction and recognition are different, and the specific analysis and detection of the target proteins ALS, APOC3, C3 and FCN2 in the sample can be achieved by corresponding to the Raman signal intensity of the signal molecules.

In a specific embodiment of the present invention, the method for constructing the multi-channel protein Raman analysis probe and the method for detecting ALS, APOC3, C3 and FCN2 proteins include the following steps:

(1) Preparation of SERS-enhanced substrate

Mix 5mL of 0.2M CTAB solution with 5mL of 0.5mM HAuCl ₄ solution, add 0.6mL of 10mM iced NaBH ₄ solution while stirring, and wait for the solution to change from golden yellow to brownish yellow. Then, place the reaction system in a 30℃ water bath environment for 2min. After taking it out, transfer the reaction solution to 25℃ for storage. Prepare another 5mL of 0.2M CTAB solution, add different volumes of 4mMAgNO ₃ solution (short aspect ratio: 0.05mL; long aspect ratio: 0.3mL) and 5mL of 1mM HAuCl ₄ solution. Add 80μL of 0.075M ascorbic acid solution to the above solution, and after stirring evenly, add 12μL of the gold seed solution obtained in step (1). And place it in a 30℃ water bath for 20min. Finally, the solution was centrifuged at 5000 rpm for 5 min, washed three times with ultrapure water, redispersed in ultrapure water and stored at a constant temperature of 4°C.

(2) Construction of Raman analysis probe

200 μL of the gold nanorod solution prepared above was added to a PEG-COOH solution containing 10 mM molecular weight of 10,000 with carboxyl terminal modification to ensure that the total volume of the solution was 10 mL. The above reaction system was stirred at a constant temperature for 12 hours under a 30°C water bath. Then centrifuged at 5000 rpm for 5 minutes, and the precipitate was washed three times with ultrapure water for standby use. Subsequently, according to different proteins, 200 μL of surface-modified gold nanorod solution was added to 10 mL of anhydrous ethanol solution containing 10 mM mercaptobenzoic acid, mercaptophenylacetylene, mercaptonaphthol, and mercaptophenylacetonitrile. The above reaction system was stirred at a constant temperature for 12 hours under a 30°C water bath. Then centrifuge at 5000rpm for 5min, redisperse the precipitate in 10mL PBS buffer at pH=5.5, add 60μM mouse polyclonal antibodies (corresponding to ALS, APOC3, C3, FCN2 in sequence), and then add 10mgN-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 5mg N-hydroxysuccinimide (NHS) to the system. Stir the above reaction system at a constant temperature for 12 hours in a 30℃ water bath. Then centrifuge at 5000rpm for 5min, and wash the precipitate three times with ultrapure water. Obtain Raman spectroscopy probes modified with antibodies and signal molecules, and then store at 4℃ for use. Obtain ALS-probe, APOC3-probe, C3-probe, and FCN2-probe.

(3) Establishment of multi-channel analysis method and verification of analytical application

The multi-channel Raman analysis method and application verification include: first, according to the different target proteins, different target protein antibodies are modified on the surface of modified gold nanorods to prepare specific recognition probes. Then, different concentrations of target proteins are added to 500μL standard simulated serum samples, and mixed with 200μL 1μM corresponding Raman spectroscopy probes. The reaction system is incubated at 36°C for 10 minutes. The reaction system is then transferred to a Raman spectroscopy analysis device for analysis, and the sample Raman spectroscopy signal is collected using the experimental parameters of a wavelength of 785nm, a power of 1mW, and an exposure time of 1s to test the response of each Raman spectroscopy probe to the target protein. Finally, based on real mouse serum samples, 500 μL of mouse serum samples were mixed with 200 μL of 1 μM Raman spectroscopy probe, and the reaction system was incubated at 36°C for 10 min. The Raman spectroscopy signal of the sample was collected using the experimental parameters of wavelength 785 nm, power 1 mW, and exposure time 1 s. The response of the Raman spectroscopy probe to the target protein in mouse serum was tested and compared with the results of a commercial ELISA kit.

In a second aspect, the present invention provides a Raman analysis kit for ALS, APOC3, C3, and FCN2 proteins, comprising the above-mentioned SERS enhanced substrate and Raman analysis probe. The kit may also include raw materials for preparing the above-mentioned SERS enhanced substrate and Raman analysis probe, and the SERS enhanced substrate and Raman analysis probe are prepared according to the above-mentioned method.

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

The present invention firstly prepares gold nanorods with different aspect ratios as Raman enhancement substrates by chemical synthesis based on surface enhanced Raman spectroscopy technology to achieve Raman spectroscopy signal enhancement. On this basis, by surface modification of gold nanorods, the structural stability of gold nanorods in different analytical environments is greatly improved, ensuring the accuracy of analytical data. Further, for different target proteins, a series of Raman analysis probes with characteristic recognition functions are specifically designed, and the analysis distinction and quantitative detection of multiple different target proteins are achieved based on the fingerprint spectrum distinction and peak intensity changes of different Raman signal molecules. Based on the above Raman analysis probes, the present invention realizes multi-channel high-sensitivity detection of ALS, APOC3, C3 and FCN2 proteins in in vitro experiments and biological fluid samples without using amplification technology, with a detection limit of 0.078ng/mL and an average detection time of only 10min. The operational difficulty, detection time cost and economic cost of analytical detection are greatly reduced. It provides a highly sensitive, highly selective, fast and accurate bioanalytical platform for the rapid detection and quantitative analysis of a variety of proteins in biological fluids, and plays a significant positive role in clinical protein analysis, detection and disease screening.

The analytical performance and results of the prepared SERS probe were verified by using Raman spectroscopy analysis equipment, showing that the SERS probe prepared by the present invention has the characteristics of high sensitivity (detection limit 0.078ng/mL), good selectivity (S/N>16.8), wide linear range (0.5ng/mL-5×10 ⁴ ng/mL), simple analysis and operation, fast detection speed (<10min), and high result accuracy (error <10%). Rapid, accurate and sensitive analysis and detection of ALS, APOC3, C3 and FCN2 proteins in blood samples can be achieved within 10 minutes, providing quantitative indicators for early warning of clinical related disease risks.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a SEM image of gold nanorods with different aspect ratios synthesized in Example 1.

FIG. 2 is a graph showing the UV-visible absorption spectra of gold nanorods with different aspect ratios in Example 1.

FIG. 3 is a UV-visible absorption spectrum of the Raman probe constructed based on gold nanorods in Example 2.

FIG. 4 is a SEM image of the Raman probe constructed in Example 3 before and after the reaction with the target protein.

FIG. 5 is a graph showing the selectivity test data of the Raman probe constructed in Example 3 for different target proteins.

FIG. 6 is a linear response relationship diagram of different Raman probes to different proteins ALS, APOC3, C3 and FCN2 in Example 3.

FIG. 7 is a Raman response curve of the Raman probe prepared in Example 3 for specific response detection of different protein combination mixtures.

FIG8 is a comparison of the experimental results of the Raman probe prepared in Example 3 for detecting ALS, APOC3, C3 and FCN2 proteins in mouse serum samples and the ELISA results.

DETAILED DESCRIPTION

The present invention will be described in detail below in conjunction with the embodiments and drawings, but the implementation of the present invention is not limited thereto.

The reagents and raw materials used in the present invention are all commercially available or can be prepared according to literature methods. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

Example 1 Preparation of SERS Enhanced Substrate

Mix 5 mL of 0.2 M CTAB solution with 5 mL of 0.5 mM HAuCl ₄ solution, add 0.6 mL of 10 mM iced NaBH ₄ solution under stirring, and wait for the solution to change from golden yellow to brown yellow. Then, place the reaction system in a 30 ° C water bath environment to react for 2 minutes, take out and transfer the reaction solution to 25 ° C for storage, and prepare a gold seed solution.

Prepare another 5mL of 0.2M CTAB solution, add different volumes of 4mM AgNO ₃ solution (short aspect ratio: 0.05mL; long aspect ratio: 0.3mL) and 5mL 1mM HAuCl ₄ solution. Add 80μL 0.075M ascorbic acid solution to the above solution, stir evenly, add 12μL gold seed solution, and place it in a 30℃ water bath for 20min. Finally, centrifuge the above solution at 5000rpm for 5min, wash it with ultrapure water 3 times, redisperse it in ultrapure water and store it at 4℃.

Figure 1 shows the morphology of the gold nanorods, which are rod-like structures with a length of 37nm and a width of 11nm. The morphology and size are uniform, indicating that the gold nanorods were successfully prepared by chemical synthesis. Figure 2 is the UV-visible absorption spectrum of the prepared gold nanorods. It can be seen from the figure that the gold nanorods have obvious spectral absorption signals at ~510nm and ~780nm, which correspond to the two plasma resonance absorptions of the gold nanorods in the transverse and longitudinal directions, respectively.

Example 2 Construction of Raman Analysis Probe

200 μL of the gold nanorod solution prepared above was added to a PEG-COOH solution containing 10 mM molecular weight of 10,000 with carboxyl terminal modification to ensure that the total volume of the solution was 10 mL. The above reaction system was stirred at a constant temperature for 12 hours under a 30°C water bath. Then centrifuged at 5000 rpm for 5 minutes, and the precipitate was washed three times with ultrapure water for standby use. Subsequently, according to different proteins, 200 μL of surface-modified gold nanorod solution was added to 10 mL of anhydrous ethanol solution containing 10 mM mercaptobenzoic acid, mercaptophenylacetylene, mercaptonaphthol, and mercaptoaniline. The above reaction system was stirred at a constant temperature for 12 hours under a 30°C water bath. Then centrifuge at 5000rpm for 5min, redisperse the precipitate in 10mL PBS buffer at pH=5.5, add 60μM mouse polyclonal antibodies (corresponding to ALS, APOC3, C3, FCN2 in sequence), and then add 10mgN-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 5mg N-hydroxysuccinimide (NHS) to the system. Stir the above reaction system at a constant temperature for 12 hours in a 30℃ water bath. Then centrifuge at 5000rpm for 5min, and wash the precipitate three times with ultrapure water. Obtain Raman spectroscopy probes modified with antibodies and signal molecules, and then store at 4℃ for use. Obtain ALS-probe, APOC3-probe, C3-probe, and FCN2-probe.

FIG3 is a UV-visible absorption spectrum of the Raman analysis probe constructed based on the SERS substrate. It can be seen from the figure that the spectral absorption peaks at about 510 nm and 780 nm have undergone a certain degree of red shift to 515 nm and 785 nm. This red shift phenomenon is mainly attributed to the effect of PEG and antibody modification on the surface plasmon resonance absorption of gold nanorods. In addition, a new spectral absorption peak appears at about 260 nm, which is mainly attributed to the spectral absorption of the antibody protein modified on the substrate surface. The UV-visible absorption spectrum shows the successful preparation of the Raman analysis probe.

Example 3 Raman spectroscopy probes for quantitative analysis of various proteins by Raman spectroscopy

The multi-channel Raman analysis method and application verification include: first, according to the different target proteins, different target protein antibodies are modified on the surface of modified gold nanorods to prepare specific recognition probes. Then, different concentrations of target proteins are added to 500μL standard simulated serum samples, and mixed with 200μL 1μM corresponding Raman spectroscopy probes. The reaction system is incubated at 36°C for 10min. Then, the reaction system is transferred to the Raman spectroscopy analysis equipment for analysis, and the sample Raman spectroscopy signal is collected using the experimental parameters of wavelength 785nm, power 1mW, and exposure time 1s to test the response of each Raman spectroscopy probe to the target protein. Finally, based on the real mouse serum sample, 500μL mouse serum sample is mixed with 1μM 200μL Raman spectroscopy probe, and the reaction system is incubated at 36°C for 10min. The Raman spectral signals of the samples were collected using the experimental parameters of a wavelength of 785 nm, a power of 1 mW, and an exposure time of 1 s. The response of the Raman spectral probe to the target protein in mouse serum was tested and compared with the results of a commercial ELISA kit.

Figure 4 shows the changes in the morphology of the APOC3 selective Raman spectroscopy probe before and after the reaction with the target protein APOC3. As shown in the SEM image, after the Raman spectroscopy probe reacts with the target protein, due to the interaction between the protein and the antibody, the gold nanorods that were independently arranged before the reaction undergo obvious aggregation, which is also the main reason for the enhancement of the Raman spectroscopy signal. Figure 5 shows the analytical selectivity of the four Raman spectroscopy probes constructed by the present invention for their corresponding proteins. The four Raman spectroscopy probes have good selectivity for their corresponding proteins, and the signal-to-noise ratio S/N>16.8. Figure 6 shows the Raman spectroscopy response linear graph of the four Raman spectroscopy probes to the corresponding proteins at different concentrations. It can be seen from the figure that the four Raman spectroscopy probes show a good linear response relationship with the corresponding protein concentration in the concentration range of 0.5ng/mL-5×10 ⁴ ng/mL. Figure 7 shows the Raman spectral response curve of the mixed Raman spectral probe for a sample solution containing a mixture of multiple different target proteins. It can be seen from the figure that different Raman spectral probes show different Raman spectral signals in the Raman spectrum, which are clearly distinguished from each other, indicating that the constructed series of Raman spectral probes have multi-channel detection capabilities for multiple mixture targets.

Finally, in order to verify the analytical detection accuracy of the Raman spectroscopy probe involved in the present invention in actual biological fluid samples, the present invention uses the constructed Raman spectroscopy probe to test the quantitative concentration of four target proteins in mouse serum samples. As shown in Figure 8, by comparing with the standard ELISA method, it is found that the Raman spectroscopy probe constructed by the present invention has extremely high accuracy, and the error between its concentration and the result of the standard ELISA method is <10%, indicating that the probe has extremely high accuracy and reliability.

In summary, the present invention achieves multi-channel high-sensitivity detection of ALS, APOC3, C3 and FCN2 proteins in in vitro experiments and biological fluid samples without using amplification technology, with a detection limit of 0.078ng/mL and an average detection time of only 10min. It greatly reduces the operational difficulty, detection time cost and economic cost of analysis and detection. It provides a highly sensitive, highly selective, rapid and accurate bioanalysis platform for the rapid detection and quantitative analysis of multiple proteins in biological fluids, and plays a significant positive role in clinical analysis and detection of proteins and disease screening.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims (9)

1. A method of raman analysis for ALS, APOC3, C3, FCN2 proteins, comprising the steps of:

A. SERS enhanced substrate preparation

Preparing a gold nano seed solution: mixing 0.3-0.6 mM HAuCl ₄ solution with 0.15-0.35M surfactant in equal volume, adding 3.5-17% of the mixed volume and 5-15 mM iced reducing agent solution under stirring, and waiting for the solution to change from golden yellow to brown yellow; then placing the reaction system in a water bath environment with the temperature of 10-50 ℃ for reaction for 1-5 min, taking out, transferring the reaction solution to the environment with the temperature of 10-50 ℃ to obtain gold seed solution, and preserving for later use;

Seed growth solution preparation: adding 3-6 mM AgNO ₃ solution with different volumes and 0.5-2 mM HAuCl ₄ solution with the same volume as the surfactant into 0.15-0.35M surfactant, adding 0.05-0.09M reducer solution into the solution, and adding gold seed solution into the solution after uniform stirring to obtain gold nanorods with different length-diameter ratios; placing the mixed solution in a water bath with the temperature of 10-50 ℃ for heat preservation for 10-30 min, centrifuging the solution for 3-10 min at 3000-8000 rpm, washing for multiple times, re-dispersing, placing in a constant temperature of 0-10 ℃ for preservation,

B. Raman analysis probe construction

Adding the gold nanorod solution prepared in the step A into a surface modification active agent with the volume of 5-200 times and the concentration of 1-50 mM, and stirring for 8-15 hours at a constant temperature under the water bath condition of 20-40 ℃; centrifuging at 3000-8000 rpm for 3-10 min, and washing the precipitate for several times;

According to different target analysis proteins, respectively adding a surface modified gold nanorod solution into Raman signal molecule solutions of ALS, APOC3, C3 and FCN2 proteins, and carrying out constant-temperature stirring on the reaction system for 8-15 hours under the water bath condition of 20-40 ℃ to carry out Raman signal molecule targeted modification;

After centrifuging the solution, re-dispersing the precipitate in a solvent with the pH value of 3-8, adding antibodies sequentially corresponding to ALS, APOC3, C3 and FCN2, and then respectively adding N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mixed coupling agent into the system; stirring the reaction system for 8-15 hours at constant temperature under the water bath condition of 20-40 ℃ to modify antibody targeting molecules;

Continuously centrifuging the solution under the same condition, washing and precipitating to obtain an antibody and signal molecule modified Raman spectrum probe, then storing the Raman spectrum probe at 0-10 ℃ for standby to obtain an ALS probe, an APOC3 probe, a C3 probe and an FCN2 probe,

C. Detection of

According to different target proteins, different target protein antibodies are modified on the surface of the modified gold nanorod, and a specific recognition Raman spectrum probe is prepared; mixing a probe with the volume of 0.5-2 mu M with a detection sample with the volume of 2-3 times, incubating at the temperature of 30-80 ℃, collecting a Raman spectrum signal of the sample by adopting parameters with the wavelength of 532nm, 633nm or 785nm, the power of 0.5-5 mW and the exposure time of 0.5-2 s, and testing the response condition of the Raman spectrum probe in the detection sample to target proteins.

2. The method for Raman analysis of ALS, APOC3, C3, FCN2 proteins according to claim 1, wherein in the gold nanoparticle solution preparation step of step A,

The surfactant is selected from any one of cetyltrimethylammonium bromide CTAB, cetyltrimethylammonium chloride CTAC, cetylbenzyl dimethyl ammonium chloride BDAC and cetylbenzyl dimethyl ammonium bromide BDAB;

the reducing agent is selected from any one of sodium borohydride NaBH4, hydrazine hydrate, ascorbic acid, sodium ascorbate and sodium citrate,

The reaction temperature in the water bath environment is 30 ℃, the reaction time is 2min, and the preservation temperature is 25 ℃.

3. The method for raman analysis of ALS, APOC3, C3, FCN2 proteins according to claim 1, wherein in the seed growth solution preparation step of step a,

The concentration of the surfactant is 0.2M, and the surfactant is any one selected from cetyltrimethylammonium bromide CTAB, cetyltrimethylammonium chloride CTAC, cetylbenzyl dimethyl ammonium chloride BDAC and cetylbenzyl dimethyl ammonium bromide BDAB;

The concentration of AgNO ₃ solution is 4mM, and the volume ratio of AgNO ₃ solution to HAuCl ₄ solution is 1:100-3:50 according to the preparation of gold nanorods with different length-diameter ratios;

the concentration of the reducer solution is 0.075M, and the reducer solution is any one of sodium borohydride NaBH4, hydrazine hydrate, ascorbic acid, sodium ascorbate and sodium citrate; the volume of the reducer solution is 1/160-1/35 of the volume of the HAuCl ₄ solution, and the volume of the gold seed solution is 1/6-1/7 of the reducer solution;

During the reaction, placing the mixed solution in a water bath at 30 ℃ for heat preservation for 20min, centrifuging the solution at 5000rpm for 5min, washing with a washing solvent for three times, then redispersing in the washing solvent, and placing in a constant temperature at 4 ℃ for preservation;

the washing solvent is one of ultrapure water, methanol and ethanol.

4. The method for Raman analysis of ALS, APOC3, C3, FCN2 proteins according to claim 1, wherein in the step B,

The length-diameter ratio of the gold nanorods is 1.2-13;

the concentration of the surface modifying active agent is 10mM, and the surface modifying active agent is one selected from polyvinylpyrrolidone PVP, polyvinyl chloride PVC, polystyrene PS and polyethylene glycol PEG;

The molecular weight of the surfactant is one of 100-1000000, and the modification group is mercapto, amino or carboxyl;

the solvent used for surface modification is one of ultrapure water, absolute ethyl alcohol and absolute methyl alcohol;

When the surface modification reaction is carried out, the mixed solution is placed in a water bath at 30 ℃ for reaction for 12 hours, and the solution is centrifuged for 5 minutes at 5000rpm and is washed by a washing solvent for three times for standby.

5. The method for Raman analysis of ALS, APOC3, C3, FCN2 proteins according to claim 4,

The length-diameter ratio of the gold nanorods is 4.7;

The surface modifying activator is PEG-COOH solution with molecular weight 10000 and carboxyl terminal modification;

The solvent used for surface modification is ultrapure water;

the washing solvent is selected from one of ultrapure water, methanol and ethanol.

6. The method for Raman analysis of ALS, APOC3, C3, FCN2 proteins according to claim 1, wherein in the step B,

The Raman signal molecules of ALS, APOC3, C3 and FCN2 proteins are respectively mercaptobenzoic acid ALS, mercaptophenylacetylene APOC3, mercaptonaphthol C3 and mercaptophenylacetonitrile FCN2;

The concentration of the Raman signal molecules is 1mM-50mM, and the volume of the surface modified gold nanorod solution is 1/50 of the total volume;

The solvent used for modifying the Raman signal molecules is one of ultrapure water, absolute ethyl alcohol and absolute methyl alcohol; .

The reaction time of the modification of the Raman signal molecules is 12 hours, and the reaction temperature is 30 ℃.

7. The method for Raman analysis of ALS, APOC3, C3, FCN2 proteins according to claim 1, wherein in the step B,

The solvent used for modifying the antibody targeting molecule is one of ultrapure water, absolute ethyl alcohol, absolute methyl alcohol, PBS buffer solution, tris-HCl buffer solution and HAc/NaAc buffer solution, and the pH value is 5.5;

The antibodies of ALS, APOC3, C3 and FCN2 are one of monoclonal antibodies and polyclonal antibodies, and are humanized, murine and rabbit antibodies; the concentration of the antibody is 10 mu M-80 mu M;

in the mixed coupling agent, the dosage ratio of EDC and NHS is 5-20mg/2-8mg;

when the modification reaction is carried out, the reaction system is stirred for 12 hours at constant temperature under the water bath condition of 30 ℃ to carry out the modification of the antibody targeting molecule; after the reaction is completed, the centrifugal speed is 3000rpm-8000rpm, the centrifugal time is 3min-10min, the washing solvent is one of ultrapure water, methanol and ethanol, the washing times are three times, and the storage temperature is 4 ℃.

8. The method for Raman analysis of ALS, APOC3, C3, FCN2 proteins according to claim 1, characterized in that in the step C,

Mixing 1 mu M probe with 2-3 times volume of detection sample, incubating at 36 ℃ for 10min, and collecting sample Raman spectrum signals by adopting parameters of 785nm wavelength, 1mW power and 1s exposure time;

the detection sample is selected from any one of serum, urine or tissue fluid.

9. A raman analysis kit for ALS, APOC3, C3, FCN2 proteins comprising the SERS enhancing substrate of claim 1 and a raman analysis probe.