阅读说明：本技术 ***特异性抗原的检测试剂与试剂盒 (Detection reagent and kit for prostate specific antigen ) 是由 邹振 贺丽蓓 杨荣华 齐鹏 杨华 罗梓凌 于 2019-05-10 设计创作，主要内容包括：通过对人血清白蛋白(HSA)与荧光染料分子相互作用的研究,我们发现HSA能够使DNA嵌入性染料ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium iodine)-9H-carbazol-9-yl)]butanoate(EBCB)的荧光显著增强。基于此,本发明公开了一种前列腺特异性抗原(PSA)的检测试剂与试剂盒,包括以下步骤：利用PSA核酸适配体序列和三链DNA的组成原理构建三链分子信标(TMS),并通过Au-S键修饰在金纳米颗粒表面,加入双功能荧光染料EBCB嵌入分子信标得到检测探针。当PSA存在时,荧光快速产生。加入dansylamide(DNSA)后,荧光进一步放大。该发明试剂和试剂盒以内源HSA作为荧光信号放大器,工作范围可调,检出限较低,具有较好的检测效果。此外,该体系对血清中其他生物标志物有较好的选择性,不需要外来工具酶或纳米催化体系,具有成本效益,具有原始创新性和应用前景。(Through studies on the interaction of Human Serum Albumin (HSA) and a fluorescent dye molecule, HSA is found to be capable of remarkably enhancing the fluorescence of DNA intercalating dye ethyl-4- [3,6-bis (1-methyl-4-vinylpyridium iodide) -9H-carbazol-9-yl) ] butanoate (EBCB). Based on the above, the invention discloses a detection reagent and a kit for Prostate Specific Antigen (PSA), which comprises the following steps: constructing a three-chain molecular beacon (TMS) by utilizing the composition principle of a PSA nucleic acid aptamer sequence and three-chain DNA, modifying the surface of the gold nanoparticle by an Au-S bond, and adding a bifunctional fluorescent dye EBCB (ethylene-bis-beta-cyclodextrin) to embed the molecular beacon to obtain the detection probe. Fluorescence is rapidly generated when PSA is present. After addition of Dansylamide (DNSA), the fluorescence was further amplified. The reagent and the kit of the invention use endogenous HSA as a fluorescence signal amplifier, have adjustable working range, lower detection limit and better detection effect. In addition, the system has better selectivity on other biomarkers in serum, does not need an external tool enzyme or a nano catalytic system, has cost benefit, and has original innovation and application prospect.)

1. A detection reagent and a kit for prostate specific antigen in serum are characterized in that:

a three-chain molecular beacon is constructed by utilizing the composition principle of a PSA nucleic acid aptamer sequence and three-chain DNA, the surface of a gold nanoparticle is modified by an Au-S bond, a bifunctional fluorescent dye EBCB is added to embed the molecular beacon to obtain a detection probe, and when PSA exists, fluorescence is rapidly generated, so that high-sensitivity detection of prostate specific antigen is realized.

2. The method for analyzing prostate specific antigen according to claim 1, wherein the TMS modified with thiol is added to PBS buffer solution containing AuNPs to form TMS modified AuNPs, EBCB is added to the above solution, the mixture is shaken and mixed to make EBCB and DNA fully react, the supernatant is centrifuged to form TMS nanoprobe for PSA, DNSA is added, and finally the sample to be detected is added; collecting fluorescence of 500 nm-750 nm by using 475 nm as an excitation light source, and performing qualitative and quantitative detection on PSA according to fluorescence intensity.

3. The method of claim 1, wherein HSA is an intrinsic component in blood and serves as a signal amplifier in the method.

4. The method of claim 1, wherein the dye EBCB is a bifunctional dye capable of binding to both DNA and HSA to amplify the fluorescent signal.

5. The method for analyzing prostate specific antigen according to claim 1, wherein the HSA-EBCB complex further enhances fluorescence after DNSA addition, and the optimal concentration is 10 μ M.

Technical Field

The invention belongs to the field of analysis of prostate specific antigens, and particularly relates to an analysis method for detecting prostate specific antigens by using human serum albumin as a signal amplifier.

Background

Prostate cancer is a common malignancy of the male genitourinary system, the most common malignancy in men in developed countries in europe and america, and the mortality rate is usually ranked second among male cancers. In recent years, the tumor with the fastest rise of morbidity and mortality in China becomes the tumor with the fastest rise of morbidity and mortality, and seriously threatens the health of old men. Prostate cancer usually has a hidden onset and a slow growth, and has no obvious symptoms in the early stage, while clinical symptoms are usually present in the later stage. According to clinical diagnosis research on the prostate cancer, PSA is a specific marker of the prostate cancer, so that the development of a high-sensitivity and high-specificity PSA detection method is of great significance in clinical diagnosis.

PSA serves as a clear serum biomarker for early diagnosis of prostate cancer and monitoring of disease recurrence after treatment, but the clinical detection limit of commercially available ELISA tests used clinically is relatively low (0.1 ng/mL). Therefore, in order to improve the detection method of PSA and improve the detection sensitivity, a series of detection methods based on optical, electrochemical, colorimetric methods and biological barcodes are developed. For example, Zhouhong et al (a prostate specific antigen detection reagent and kit, Chinese patent, publication No. CN 107367616A): and amplifying the detection signal of the PSA by using the signal amplification reaction liquid acted by the nano magnetic beads co-labeled by the first PSA antibody, the second PSA antibody and the splicing enzyme. Xiaojianhe et al (a kit for detecting prostate specific antigen PSA and its application, chinese patent, publication No. CN 106932570A): the detection result is amplified in equal proportion by combining gold particles and the antibody by adopting an antigen-antibody combination principle, and the PSA is quantitatively detected by using a full-automatic biochemical analyzer. For example, Xia et al Pd-Ir nanoparticles encapsulated with gold vesicles are used as peroxidase mimics and used as signal amplification technology for PSA colorimetric assay. Chad A, Mirkin and the like take gold nanoparticles as nano-enzyme to prepare a biological barcode detection probe for the detection of prostate specific antigen PSA signal amplification. However, these methods have disadvantages such as signal amplification process requiring a tool enzyme or a nanocatalysis system, complex detection steps, high cost and long time consumption. Therefore, there is a need for a more rapid, efficient and sensitive method for detecting PSA.

Human Serum Albumin (HSA) is the most abundant carrier protein in plasma, with a normal concentration of about 40 mg/ml (~ 0.6.6 mM), accounting for about 60% of the total plasma protein, due to its high concentration, it binds to most exogenous and endogenous substances non-specifically in blood, causing bias in performing detection assays, even false positive results, and is therefore often considered an interferent.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects of the prior amplification technology and provide the prostate specific antigen analysis method which does not need an external tool enzyme or a nano catalytic system and has a simple analysis tool.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method of analyzing prostate specific antigen, comprising the steps of:

constructing a three-chain molecular beacon by using the composition principle of a PSA nucleic acid aptamer sequence and three-chain DNA, modifying the surface of the gold nanoparticle by an Au-S bond, and adding a bifunctional fluorescent dye EBCB (ethylene-bis-beta-cyclodextrin) to embed the molecular beacon to obtain the detection probe. When PSA exists, fluorescence is generated rapidly, and after DNSA is added, the fluorescence is further amplified, so that high-sensitivity detection of the prostate specific antigen is realized.

Preferably, the method for analyzing the prostate specific antigen comprises the steps of adding the TMS modified by sulfydryl into PBS buffer solution containing AuNPs to form TMS modified AuNPs, adding EBCB into the solution, shaking and uniformly mixing the solution to enable the EBCB and DNA to fully act, centrifuging the solution to take down clear liquid to form a TMS nano probe for PSA, then adding DNSA, and finally adding a sample to be detected; collecting fluorescence of 500 nm-750 nm by using 475 nm as an excitation light source, and performing qualitative and quantitative detection on PSA according to fluorescence intensity.

In the method for analyzing prostate specific antigen, preferably, the optimal complementary logarithm of the stem portion of TMS is 8 pairs of bases.

In the method for analyzing the prostate specific antigen, preferably, the molar ratio of the TMS to the AuNPs solution is 108: 1; the concentration of the TMS solution is 1-250 nM, and the concentration of the EBCB dye is 5-500 mu M.

In the method for analyzing a prostate-specific antigen, HSA is preferably a component inherent in blood and serves as a signal amplifier in the method.

In the method for analyzing prostate specific antigen, the EBCB dye is preferably synthesized in the laboratory.

In the method for analyzing a prostate-specific antigen described above, the TMS sequence is preferably purchased from bio-engineering (shanghai) ltd.

In the method for analyzing the prostate specific antigen, preferably, the molar ratio of the EBCB dye to the TMS-AuNPs solution is 10: 1; the concentration of the TMS-AuNPs solution is 1-250 nM, and the concentration of the EBCB dye is 5-500 mu M.

In the method for analyzing prostate-specific antigen described above, the optimal concentration of DNSA is preferably 10 μ M.

In the method for analyzing prostate specific antigen, the dye EBCB is preferably a bifunctional dye which can be combined with DNA and can be specifically combined with HSA to amplify fluorescent signals.

Compared with the prior art, the invention has the advantages that:

1. in the method for analyzing a prostate-specific antigen of the present invention, HSA inherent in blood is used as a signal amplifier for the first time.

2. In the method for analyzing the prostate specific antigen, the dye EBCB is a bifunctional dye which can be combined with DNA and can be specifically combined with HSA to amplify a fluorescent signal.

3. In the method for analyzing a prostate-specific antigen of the present invention, the addition of DNSA to the HSA-EBCB complex can further enhance fluorescence.

4. The prostate specific antigen assay of the present invention can be performed on 96-well PS plates without repeated isolation and washing procedures, making such assays cost-effective. The method has the advantages of wide working range, low detection limit, good detection effect, good selectivity on other biomarkers in serum, cost benefit and capability of meeting application requirements.

Drawings

FIG. 1 shows the screening of DNA binding dyes for PSA detection in this example.

Fig. 2 shows an analysis of the HSA of this example for EBCB signal amplification.

FIG. 3 shows the site investigation of the EBCB of this example into HSA.

Fig. 4 shows signal amplification analysis of HSA of this example for PSA detection.

FIG. 5 shows the fluorescence detection of PSA in PBS buffer by using the TMS nanoprobe of the present embodiment.

Fig. 6 shows the TMS nanoprobe of this example used for the detection of PSA in clinical blood specimens.

FIG. 7 is an abstract drawing (schematic diagram) of the TMS nanoprobe for detecting PSA.

FIG. 8 (Table 1) shows the oligonucleotide sequences used in the TMS nanoprobes of this example.

FIG. 9 (Table 2) shows the TMS nanoprobes and commercial ELISA kits of this example for screening human plasma PSA.

Detailed Description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: the embodiment is implemented on the premise of the technical scheme of the invention, and a detailed implementation manner and a process are given, so that the technical scheme features of the invention are easy to understand, and the protection scope of the invention is not limited at all. All the technical solutions formed by equivalent transformation or equivalent replacement fall within the protection scope of the present invention.