Preparation method of multicolor fluorescent carboxylated carbon nano quantum dot biological probe for detecting hepatocellular cancer

Abstract

The invention relates to the technical field of fluorescent probe preparation, and discloses a preparation method of a multicolor fluorescent carboxylated carbon nano quantum dot biological probe for detecting hepatocellular cancers, and the preparation method comprises the following steps: S1, preparing nanoscale multicolor fluorescent carbon dots; s2, preparation of aminated nano-scale multicolor fluorescent carbon dots; s3, preparation of carboxylated nano-scale multicolor fluorescent carbon dots; s4, preparing an alpha fetoprotein rabbit antibody; s5, confirming alpha fetoprotein rabbit anti-interaction site subunits and constructing related gene libraries and plasmids; and S6, carrying out a biological coupling reaction. According to the method, the sensitivity and specificity of hepatocellular cancer detection can be improved, the reliability of hepatocellular cancer detection can be improved, the detection cost can be reduced, the early diagnosis rate of hepatocellular cancer can be improved, hepatocellular cancer can be detected earlier, the early diagnosis rate of hepatocellular cancer can be improved, and the death rate of hepatocellular cancer can be reduced.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probe preparation, in particular to a method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot biological probe for detecting hepatocellular cancer. Background Art

[0002] Hepatocellular carcinoma is one of the most common cancers in China, and its mortality rate ranks among the top three. Alpha-fetoprotein is one of the diagnostic markers for hepatocellular carcinoma, but its detection has certain false negative and false positive rates, so its detection rate needs to be further improved. Nanomaterials have broad application prospects in the field of biomedical testing, and they have the advantages of high sensitivity, specificity and biocompatibility. Therefore, the use of nanomaterials to construct biological probes for hepatocellular carcinoma detection has become a research hotspot.

[0003] Existing technical solutions: Existing studies have constructed hepatocellular carcinoma detection biological probes by modifying alpha-fetoprotein antibodies with nanomaterials such as gold nanoparticles. However, these methods have problems such as the stability and biocompatibility of gold nanoparticles in biological tissues. In addition, the sensitivity and specificity of these methods need to be further improved. Existing technical problems: The problems existing in the prior art mainly include: ① There are certain limitations in the stability and biocompatibility of gold nanoparticles in biological tissues; ② The existing detection methods have problems with low sensitivity and specificity, and are prone to false positive and false negative results; ③ The existing methods require the use of expensive instruments and equipment for detection, which makes the cost high and is not suitable for large-scale screening and other scenarios. To this end, a method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot biological probe for detecting hepatocellular carcinoma is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot biological probe for detecting hepatocellular carcinoma, so as to solve the problems existing in the prior art raised in the above background technology, mainly including: the stability and biocompatibility of gold nanoparticles in biological tissues have certain limitations; the existing detection methods have the problems of low sensitivity and specificity, and are prone to false positive and false negative results; the existing methods require the use of expensive instruments and equipment for detection, which makes the cost high and is not suitable for large-scale screening and other scenarios.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe for detecting hepatocellular carcinoma, comprising the following steps:

[0006] S1, preparation of nanoscale multicolor fluorescent carbon dots;

[0007] S2, preparation of amination-modified nanoscale multicolor fluorescent carbon dots;

[0008] S3, preparation of carboxylated nanoscale multicolor fluorescent carbon dots;

[0009] S4, alpha-fetoprotein rabbit antibody preparation;

[0010] S5, confirmation of alpha-fetoprotein rabbit anti-interaction site subunits and construction of related gene library and plasmid;

[0011] S6. Bioconjugation reaction.

[0012] Preferably, in S1, nanoscale multicolor fluorescent carbon dots are prepared by biological and material science methods, including multicolor fluorescent properties and nanoscale size characteristics.

[0013] Preferably, in S2, the prepared nanoscale multicolor fluorescent carbon dots are subjected to an amino treatment to facilitate subsequent carboxylation modification.

[0014] Preferably, in S3, the amination-treated nanoscale multicolor fluorescent carbon dots are carboxylated for subsequent biocoupling reaction.

[0015] Preferably, in S4, rabbit antibodies against alpha-fetoprotein are prepared by rabbit immunization technology to obtain its specific binding region subunits, and related gene libraries and plasmids are constructed.

[0016] Preferably, in S5, the amino acid sequence of the alpha-fetoprotein rabbit antibody is obtained by a protein analysis method, the interaction site with the alpha-fetoprotein and the protein domain subunit where the site is located are intercepted, the DNA sequence of the relevant subunit is obtained according to the translation codon, and the relevant gene library and plasmid are constructed.

[0017] Preferably, in S6, the carboxylated nanoscale multicolor fluorescent carbon dots are chemically covalently bonded to the alpha-fetoprotein rabbit anti-specific binding region subunits through bio-coupling technology to form an immunofluorescence bioprobe for detecting hepatocellular carcinoma.

[0018] Compared with the prior art, the present invention adopts the above technical solution and has the following technical effects:

[0019] 1. Improve the sensitivity and specificity of hepatocellular carcinoma detection: The multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe prepared by biological and material science methods has high sensitivity and specificity, which can detect hepatocellular carcinoma more accurately and reduce the false positive and false negative rates.

[0020] 2. Improve the reliability of hepatocellular carcinoma detection: The immunofluorescence bioprobe used in this technical solution has multicolor fluorescence characteristics and can detect multiple markers at the same time, thereby improving the reliability of hepatocellular carcinoma detection.

[0021] 3. Reduce detection costs: The nanomaterials used in this technical solution have good biocompatibility and stability, and do not require the use of expensive instruments and equipment for detection, which can reduce detection costs and is suitable for scenarios such as large-scale screening.

[0022] 4. Improve the early diagnosis rate of hepatocellular carcinoma: This technical solution can detect hepatocellular carcinoma earlier, which helps to improve the early diagnosis rate of hepatocellular carcinoma and reduce its mortality rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 It is a preparation diagram of the amino and carboxyl carbon dots of the present invention;

[0025] Figure 2 For the present invention Figure 2 Middle partial enlarged picture;

[0026] Figure 3 is a fluorescence property diagram of the carbon dots of the present invention;

[0027] Figure 4 The three-dimensional protein structure diagram of alpha-fetoprotein analyzed by cryo-electron microscopy method of the present invention;

[0028] Figure 5 is the secondary structure diagram of the alpha-fetoprotein of the present invention;

[0029] Figure 6 The rabbit anti-AFP protein subunit expression vector diagram is designed using the plasmid backbone Pgex-6p-1 of the present invention;

[0030] Figure 7 This is a diagram of the construction of the immunofluorescence biological probe for detecting hepatocellular carcinoma of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Liver cancer is one of the most common types of cancer in China. Its incidence rate ranks high in China, and its mortality rate has been among the top three in China in recent years. Therefore, early screening and early diagnosis of liver cancer is an important means to prevent the disease from developing to the late stage and causing the patient's painful death. Among liver cancers, hepatocellular carcinoma (HC) is the most common type of liver cancer in China, and the probability of late-stage malignant tumors caused by its occurrence in liver tissue accounts for more than 90%. Among them, alpha-fetoprotein (AFP) is produced in hepatocellular carcinoma cells. The related protein detection indicators of most people in hepatocellular carcinoma patients are significantly increased. In the clinical guidelines and specifications related to liver cancer diagnosis and treatment in China, in order to further improve the detection rate of hepatocellular carcinoma in the early stage, alpha-fetoprotein is used as one of the clinical gold standards for early screening of related cancers.

[0033] The present invention utilizes biological and material science methods to improve the positive detection rate of alpha-fetoprotein, and uses carbon-based materials to prepare nano-multicolor fluorescent carbon dots (NMFCDs). The nano-multicolor fluorescent carbon dots are modified and designed to be carboxylated nano-multicolor fluorescent carbon dots by multi-step reactions. The carboxylated multicolor fluorescent nano-carbon particles are chemically covalently bonded to the specific binding region subunits of the alpha-fetoprotein antibody by bio-coupling technology to form an immunofluorescent biological probe for detecting hepatocellular carcinoma. The specific scheme is as follows:

[0034] Example 1

[0035] The present invention provides a technical solution: a method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot biological probe for detecting hepatocellular carcinoma, comprising the following steps:

[0036] S1. Preparation of nanoscale multicolor fluorescent carbon dots: Preparation of nanoscale multicolor fluorescent carbon dots by biological and material science methods, including multicolor fluorescence properties and nanoscale size characteristics;

[0037] S2, preparing amination-treated nanoscale multicolor fluorescent carbon dots; performing amination treatment on the prepared nanoscale multicolor fluorescent carbon dots for subsequent carboxylation modification;

[0038] S3, preparation of carboxylated nano-scale multicolor fluorescent carbon dots; carboxylation modification of the amination-treated nano-scale multicolor fluorescent carbon dots for subsequent biocoupling reaction;

[0039] S4. Preparation of rabbit antibody against alpha-fetoprotein: rabbit antibody against alpha-fetoprotein was prepared by rabbit immunization technology to obtain its specific binding region subunits, and to construct related gene libraries and plasmids;

[0040] S5. Confirmation of the interaction site subunit of alpha-fetoprotein rabbit antibody and construction of related gene library and plasmid: Obtain the amino acid sequence of alpha-fetoprotein rabbit antibody by protein analysis method, intercept the interaction site with alpha-fetoprotein and the protein domain subunit where the site is located, obtain the DNA sequence of the related subunit according to the translation codon, and construct the related gene library and plasmid;

[0041] S6. Bio-coupling reaction: Through the bio-coupling technology, the carboxylated nano-scale multi-color fluorescent carbon dots are chemically covalently bonded to the alpha-fetoprotein rabbit anti-specific binding region subunit to form an immunofluorescent bio-probe for detecting hepatocellular carcinoma.

[0042] In this technical solution, by chemically covalently bonding carboxylated nanoscale multicolor fluorescent carbon dots to alpha-fetoprotein rabbit anti-specific binding region subunits, an immunofluorescent biological probe for detecting hepatocellular carcinoma can be obtained. This probe has the advantages of high sensitivity, specificity, and biocompatibility. It can be used to improve the detection rate of hepatocellular carcinoma and reduce false positive and false negative results. At the same time, it has low cost and is suitable for scenarios such as large-scale screening.

[0043] Example 2

[0044] This embodiment provides the following solutions:

[0045] An immunofluorescent biological probe for detecting hepatocellular carcinoma, comprising the use of carbon substrates (carbon-rich substrates such as glucosamine, bean dregs, and burned straw) to prepare nanoscale multicolor fluorescent carbon dots, obtaining amino-type nanoscale multicolor fluorescent carbon dot particles through the NHS reaction of the bio-coupling technology, and then obtaining carboxyl-type nanoscale multicolor fluorescent carbon dot particles through the DHC reaction of the bio-coupling technology, and chemically modifying the carboxyl-type nanoscale multicolor fluorescent carbon dots using nitrogen and magnesium elements to improve the efficiency and quantity of multicolor fluorescent light quantum release. Nanoscale multicolor fluorescent carbon dots are obtained through the above multi-step reactions; they serve as an important component of the biological probe. The preparation of amino-type and carboxyl-type carbon dots is as follows: Figure 1 and Figure 2 The fluorescence properties of carbon dots are shown in Figure 3 shown.

[0046] AFP solution was prepared, and rabbits were immunized with the AFP solution to obtain AFP rabbit antibodies. Blood samples were drawn from rabbits 1 week, 1.5 weeks, and 2 weeks after immunization. When a significant increase in antibodies was detected in the serum, blood was drawn and purified to obtain the relevant AFP rabbit antibodies. The specific binding region subunits were detected by protein interaction method, and the interaction sites with AFP and the protein domain subunits where the sites were located were intercepted. The DNA sequences of the relevant subunits were obtained according to the translation codons, and the relevant subunit gene libraries and plasmids were constructed according to the corresponding codons. Appropriate engineering cell lines were selected, and the relevant rabbit antibody specific binding region subunits were obtained by bioengineering methods. The three-dimensional protein structure of AFP resolved by cryo-electron microscopy is shown in the figure. Figure 4 As shown. Figure 5 As shown in Figure 1, the secondary structure of alpha-fetoprotein consists of 592 amino acid residues. Figure 6 As shown, the rabbit anti-AFP protein subunit expression vector was designed using the plasmid backbone Pgex-6p-1.

[0047] The relevant properties of the strains and plasmids are as follows:

[0048]

[0049] The genome encoding rabbit anti-AFP protein was cloned into the vector pGEX-6p-1 through gene editing technology and expressed in Escherichia coli BL21 (DE3). Its protein structure consisted of a fusion protein with a GST tag at one end, which was convenient for screening and verification.

[0050] The cells were grown in LB medium (10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract) for 48 h, with an incubator speed of 200 rpm and a culture temperature of 37°C.

[0051] The immunofluorescence bioprobe for detecting hepatocellular carcinoma was constructed, and the chemical covalent bond was achieved through bioconjugation technology. The specific steps are: chemically covalently bond the carboxylated nanoscale multicolor fluorescent carbon dots with the alpha-fetoprotein rabbit anti-specific binding region subunit through the EDC method, and through a two-step synthesis, an immunofluorescence bioprobe for detecting hepatocellular carcinoma was formed. Figure 7 , diagram of the construction of immunofluorescent bioprobe for detecting hepatocellular carcinoma.

[0052] In this embodiment, the nanoscale multicolor fluorescent carbon dots have multicolor fluorescent properties and nanoscale size characteristics, the alpha-fetoprotein rabbit anti-subunit has specificity and biocompatibility, and the immunofluorescence biological probe after chemical covalent bond binding has the advantages of high sensitivity, specificity and biocompatibility, which can be used to improve the detection rate of hepatocellular carcinoma and reduce false positive and false negative results. At the same time, it has low cost and is suitable for scenarios such as large-scale screening.

[0053] Application prospects of this technology:

[0054] Application prospects: This invention uses biological and material science technologies to construct a multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe for detecting hepatocellular carcinoma, which has the advantages of high sensitivity, specificity and biocompatibility. This bioprobe can be used for the detection of alpha-fetoprotein, which can improve the positive detection rate of hepatocellular carcinoma and is expected to become an important means for early diagnosis and screening of hepatocellular carcinoma.

[0055] Prospects for technical applications: The biological probe of the present invention can be applied to the field of biomedical detection technology, especially in the field of early diagnosis and screening of hepatocellular carcinoma. In addition, the biological probe design can also be applied to the design of probes for the detection and diagnosis of other cancers, such as breast cancer, lung cancer, etc. At the same time, compared with other detection methods, the use of carbon-based materials has a controllable and low preparation cost. The required nanoscale carbon dots can be prepared by processing and reusing carbon-containing waste materials, and the detection probes are constructed using biological methods. During the post-processing of the prepared products, because they are all organic and non-polar carbon materials, the post-processing mode of the product is simple, which is one of the important technical means in the field of medical testing that responds to the current international and domestic carbon neutrality and environmental protection requirements.

[0056] Market demand: Liver cancer is one of the single cancers with the highest number of cases in China, and hepatocellular carcinoma is the main type of liver cancer. Early diagnosis and screening of liver cancer is an important means to prevent the disease from developing to the late stage and causing the patient's painful death. The liver cancer detection methods currently available on the market have problems with low sensitivity and specificity, are prone to false positive and false negative results, and are costly, making them unsuitable for large-scale screening and other scenarios. Therefore, the biological probe of the present invention can fill the market gap, improve the accuracy and efficiency of liver cancer detection, and has broad market prospects and development space.

[0057] In summary, compared with the prior art, the beneficial effects of this technical solution are as follows:

[0058] 1. Improve the sensitivity and specificity of hepatocellular carcinoma detection: The multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe prepared by biological and material science methods has high sensitivity and specificity, which can detect hepatocellular carcinoma more accurately and reduce the false positive and false negative rates.

[0059] 2. Improve the reliability of hepatocellular carcinoma detection: The immunofluorescence bioprobe used in this technical solution has multicolor fluorescence characteristics and can detect multiple markers at the same time, thereby improving the reliability of hepatocellular carcinoma detection.

[0060] 3. Reduce detection costs: The nanomaterials used in this technical solution have good biocompatibility and stability, and do not require the use of expensive instruments and equipment for detection, which can reduce detection costs and is suitable for scenarios such as large-scale screening.

[0061] 4. Improve the early diagnosis rate of hepatocellular carcinoma: This technical solution can detect hepatocellular carcinoma earlier, which helps to improve the early diagnosis rate of hepatocellular carcinoma and reduce its mortality rate.

[0062] Compared with the prior art, the advantages of this technical solution are:

[0063] 1. The biological probe prepared by the nanomaterials used in this technical solution has better biocompatibility and stability, can be better applied in biological tissues, and avoids the limitations of gold nanoparticles in biological tissues in the prior art.

[0064] 2. The biological probe used in this technical solution has multicolor fluorescence characteristics and can detect multiple markers at the same time, thereby improving the detection reliability of hepatocellular carcinoma and avoiding the problems of low sensitivity and specificity in the prior art.

[0065] 3. This technical solution has lower detection costs and is suitable for scenarios such as large-scale screening, avoiding the limitations of using expensive instruments and equipment in existing technologies.

[0066] 4. This technical solution can detect hepatocellular carcinoma earlier, which helps to improve the early diagnosis rate of hepatocellular carcinoma and reduce its mortality rate.

[0067] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe for detecting hepatocellular carcinoma, characterized in that: The following steps are involved: S1, preparation of nanoscale multicolor fluorescent carbon dots; S2, preparation of amination-modified nanoscale multicolor fluorescent carbon dots; S3, preparation of carboxylated nanoscale multicolor fluorescent carbon dots; S4, alpha-fetoprotein rabbit antibody preparation; S5, confirmation of alpha-fetoprotein rabbit anti-interaction site subunits and construction of related gene library and plasmid; S6. Bioconjugation reaction.

2. The method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe for detecting hepatocellular carcinoma according to claim 1, characterized in that: In S1, nanoscale multicolor fluorescent carbon dots were prepared through biological and material science methods, including multicolor fluorescence properties and nanoscale size characteristics.

3. The method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe for detecting hepatocellular carcinoma according to claim 2, characterized in that: In S2, the prepared nanoscale multicolor fluorescent carbon dots are subjected to an amino treatment for subsequent carboxylation modification.

4. The method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe for detecting hepatocellular carcinoma according to claim 3, characterized in that: In S3, the amination-treated nanoscale multicolor fluorescent carbon dots are carboxylated for subsequent bioconjugation reactions.

5. The method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe for detecting hepatocellular carcinoma according to claim 4, characterized in that: In S4, rabbit antibodies against alpha-fetoprotein were prepared by rabbit immunization technology to obtain its specific binding region subunits, and related gene libraries and plasmids were constructed.

6. The method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe for detecting hepatocellular carcinoma according to claim 5, characterized in that: In S5, the amino acid sequence of rabbit antibody against alpha-fetoprotein was obtained by protein analysis method, the interaction site with alpha-fetoprotein and the protein domain subunit where the site was located were intercepted, the DNA sequence of the relevant subunit was obtained according to the translation codon, and the relevant gene library and plasmid were constructed.

7. The method for preparing a multicolor fluorescent carboxylated carbon nano-quantum dot bioprobe for detecting hepatocellular carcinoma according to claim 6, characterized in that: In S6, carboxylated nanoscale multicolor fluorescent carbon dots were chemically covalently bonded to alpha-fetoprotein rabbit anti-specific binding region subunits through bioconjugation technology to form an immunofluorescent bioprobe for the detection of hepatocellular carcinoma.

Images