阅读说明：本技术 一种信号放大的荧光检测体系、荧光生物传感器及其用途 (Signal amplification fluorescence detection system, fluorescence biosensor and application thereof ) 是由 周洁 许改霞 于 2020-05-11 设计创作，主要内容包括：本发明公开了一种信号放大的荧光检测体系,包括识别单元,标记有荧光分子,用于识别阿尔兹海默症的生物标志物分子,且与生物标志物分子结合形成生物复合体；荧光淬灭单元,吸附识别单元,使荧光分子的荧光淬灭；荧光淬灭单元与生物复合体解吸附,使荧光分子的荧光恢复；信号放大单元,用于降解生物复合体中的识别单元。以上述荧光检测体系对阿尔兹海默症的生物标志物分子进行检测,具有快速、简单、灵敏、高效的优势；同时,上述体系能够放大荧光分子信号,降低体系的检出限,具有高灵敏度。本发明还公开了一种荧光生物传感器,包括上述的荧光检测体系,能够为阿尔兹海默症的诊断与筛查提供有效信息。(The invention discloses a signal amplification fluorescent detection system, which comprises an identification unit, a signal amplification unit and a signal amplification unit, wherein the identification unit is marked with fluorescent molecules and used for identifying biomarker molecules of Alzheimer's disease and combining with the biomarker molecules to form a biological complex; a fluorescence quenching unit that adsorbs the recognition unit and quenches fluorescence of the fluorescent molecule; desorbing the fluorescence quenching unit and the biological complex to recover the fluorescence of the fluorescent molecules; and the signal amplification unit is used for degrading the identification unit in the biological complex. The fluorescence detection system is used for detecting the biomarker molecules of the Alzheimer's disease, and has the advantages of rapidness, simplicity, sensitivity and high efficiency; meanwhile, the system can amplify fluorescent molecular signals, reduces the detection limit of the system and has high sensitivity. The invention also discloses a fluorescence biosensor which comprises the fluorescence detection system and can provide effective information for the diagnosis and screening of the Alzheimer disease.)

1. A signal amplifying fluorescent detection system comprising:

a recognition unit labeled with a fluorescent molecule for recognizing a biomarker molecule for alzheimer's disease, and binding to the biomarker molecule to form a biocomplex;

a fluorescence quenching unit that adsorbs the recognition unit and quenches fluorescence of the fluorescent molecule; desorbing the fluorescence quenching unit and the biological complex to recover the fluorescence of the fluorescent molecule;

a signal amplification unit for degrading the recognition unit in the biocomplex.

2. The fluorescence detection system of claim 1,

the recognition unit is a single-stranded DNA probe marked by a fluorescent molecule, the biomarker molecule is miRNA, and the signal amplification unit is DNase I.

3. The fluorescence detection system according to claim 2, wherein the single-stranded DNA probe has a nucleotide sequence shown in any one of SEQ ID No.1 to SEQ ID No.3, and the miRNA has a nucleotide sequence shown in any one of SEQ ID No.4 to SEQ ID No. 6.

4. The fluorescence detection system according to any one of claims 1 to 3, wherein the recognition unit comprises: an avidin-labeled fluorescent molecule, and a biotin-labeled single-stranded DNA molecule, said avidin being coupled to said biotin;

preferably, the fluorescent molecule is a fluorescent quantum dot;

preferably, the avidin-labeled fluorescent quantum dots are coupled with the biotin-labeled single-stranded DNA molecules in a molar ratio of 1: 4.

5. The fluorescence detection system according to any one of claims 1 to 4, wherein the fluorescence quenching unit is selected from any one of graphene and graphene oxide;

preferably, the fluorescence quenching unit is graphene oxide;

preferably, the concentration of the graphene oxide is 100 μ g/mL.

6. The fluorescence detection system according to any one of claims 1 to 5, wherein the volume ratio of the recognition unit to the fluorescence quenching unit is 10: 1.

7. A fluorescent biosensor comprising the fluorescent detection system of any one of claims 1 to 6.

8. Use of the fluorescent detection system of any one of claims 1 to 6, or the fluorescent biosensor of claim 7, in the manufacture of a product for diagnosing alzheimer's disease.

9. A kit for diagnosing alzheimer's disease, comprising the fluorescent detection system of any one of claims 1-6, or the fluorescent biosensor of claim 9.

Technical Field

The invention relates to the field of biomolecule detection, in particular to a signal amplification fluorescence detection system, a fluorescence biosensor and application thereof.

Background

Alzheimer's Disease (AD) is an irreversible neurodegenerative disease primarily associated with age. The Alzheimer disease affects the cognition and short-term memory function of patients, and as the disease progresses, the daily living ability of the patients is gradually affected, and finally the self-care ability of life is lost. In epidemiology, the incidence of alzheimer's disease is multiplied with age. With the increasing trend of aging in the world, alzheimer's disease has become the most serious cause of disease and death following tumor and cardiovascular diseases.

Because the incubation period of the Alzheimer disease is long and can reach about 10-20 years, a lot of patients miss precious time of early treatment because the Alzheimer disease is not found in time. The disease, if it can be discovered at an earlier stage and given effective treatment, will significantly improve the health and quality of life of the patient and greatly increase the survival rate of the patient. Therefore, the early diagnosis has very important research significance and application value for the treatment and prevention of the Alzheimer's disease.

The existing disease process diagnosis methods include the MMSE scoring test based on questionnaire and neuron imaging detection aiming at amyloid beta, and some invasive methods such as analysis of related biomolecules (a β molecules, tau proteins, etc.) in cerebrospinal fluid (CSF) to help diagnosis of AD, and it has been considered that the diagnosis accuracy of AD by biomarkers in cerebrospinal fluid is better, but there is a distinct disadvantage: the cost is too high, and compared with the acquisition process of biological tissues related to peripheral blood, the acquisition process is difficult; the patient is more painful to obtain cerebrospinal fluid, and sequela may be left. Compared with the detection of the biomarker in cerebrospinal fluid, the peripheral blood of the AD patient is easy to obtain, and larger physical and economic burden can not be brought to the patient. Therefore, detecting changes in biomarkers in peripheral blood of AD patients has important clinical application value for AD diagnosis.

MicroRNA (miRNA) is a non-coding single-stranded RNA molecule with the length of about 22 nucleotides coded by endogenous genes, and participates in processes of regulating and controlling the expression of transcribed genes, regulating the growth, development and apoptosis of organisms and the like in animals and plants. In the field of medical research, miRNA participates in the regulation of the occurrence and development processes of various diseases, and meanwhile, miRNA is expected to become a new biological marker for disease diagnosis based on the regulation mechanism of miRNA. The detection of the AD specific serum miRNA can provide a powerful basis for AD diagnosis. However, in human serum, the content of miRNA is low, the existing high-sensitivity miRNA detection technologies (Q-PCR, microarray, high-throughput sequencing technology, etc.) mostly rely on large-scale precision detection equipment, the detection cost is high, the detection steps are cumbersome, the quantitative accuracy is limited, and the popularization is poor.

Therefore, how to realize the rapid, simple and sensitive quantitative detection of the serum specific miRNA of the AD patient has important significance for the early screening and diagnosis of the Alzheimer disease.

Disclosure of Invention

Therefore, the invention aims to overcome the defects of high detection cost, complicated detection steps and limited quantitative accuracy in the disease diagnosis of the Alzheimer's disease in the prior art.

Therefore, the invention provides the following technical scheme:

in a first aspect, the present invention provides a signal amplification fluorescent detection system comprising:

a recognition unit labeled with a fluorescent molecule for recognizing a biomarker molecule for alzheimer's disease, and binding to the biomarker molecule to form a biocomplex;

a fluorescence quenching unit that adsorbs the recognition unit and quenches fluorescence of the fluorescent molecule; desorbing the fluorescence quenching unit and the biological complex to recover the fluorescence of the fluorescent molecule;

a signal amplification unit for degrading the recognition unit in the biocomplex.

Alternatively, the above-described fluorescence detection system,

the recognition unit is a single-stranded DNA probe marked by a fluorescent molecule, the biomarker molecule is miRNA, and the signal amplification unit is DNase I.

Further optionally, in the above fluorescence detection system, the single-stranded DNA probe has a nucleotide sequence shown in any one of SEQ ID No.1 to SEQ ID No.3, and the miRNA has a nucleotide sequence shown in any one of SEQ ID No.4 to SEQ ID No. 6.

Optionally, in the above fluorescence detection system, the recognition unit comprises: an avidin-labeled fluorescent molecule, and a biotin-labeled single-stranded DNA molecule, said avidin being coupled to said biotin;

preferably, the fluorescent molecule is a fluorescent quantum dot;

preferably, the avidin-labeled fluorescent quantum dots are coupled with the biotin-labeled single-stranded DNA molecules in a molar ratio of 1: 4.

Optionally, in the above fluorescence detection system, the fluorescence quenching unit is selected from any one of graphene and graphene oxide;

preferably, the fluorescence quenching unit is graphene oxide;

preferably, the concentration of the graphene oxide is 100 μ g/mL.

Optionally, in the above fluorescence detection system, the volume ratio of the recognition unit to the fluorescence quenching unit is 10: 1.

In a second aspect, the present invention provides a fluorescent biosensor comprising the above-described fluorescence detection system.

In a third aspect, the present invention provides the use of the above-mentioned fluorescence detection system, or a fluorescence biosensor, in the manufacture of a product for diagnosing alzheimer's disease.

In a fourth aspect, the present invention provides a kit for diagnosing alzheimer's disease, which comprises the above-described fluorescence detection system, or a fluorescence biosensor.

The technical scheme of the invention has the following advantages:

1. the present invention provides a fluorescence detection system comprising: the identification unit is marked with fluorescent molecules, is used for identifying biomarker molecules of the Alzheimer's disease, and is combined with the biomarker molecules to form a biological complex; the fluorescence quenching unit adsorbs the fluorescent probe to quench the fluorescence of the fluorescent molecule; desorbing the fluorescence quenching unit and the biological complex to recover the fluorescence of the fluorescent molecules; and the signal amplification unit is used for degrading the identification unit in the biological complex.

In the above fluorescence detection system to identifyThe unit-labeled fluorescent molecule is used as a fluorescence donor, the fluorescence quenching unit is used as a fluorescence donor, and when the fluorescence quenching unit adsorbs the recognition unit, the distance between the fluorescence quenching unit and the recognition unit is close enough, and fluorescence resonance energy transfer occurs (theresonance energy transfer, FRET), the energy of the fluorescent donor is transferred to the fluorescent acceptor, causing fluorescence quenching of the fluorescent molecule of the recognition unit. After a target detection object containing the biomarker molecules of the Alzheimer's disease is added into the system, the recognition unit recognizes and combines the biomarker molecules of the Alzheimer's disease to form a biological complex. As the fluorescence quenching unit desorbs with the biocomplex, the fluorescence of the fluorescent molecule in the biocomplex is restored after the biocomplex leaves the fluorescence quenching unit. Quantitative detection of target biomarker molecules related to the Alzheimer's disease can be realized by analyzing the enhancement degree of fluorescence intensity in a fluorescence detection system.

The signal amplification unit in the fluorescence detection system is capable of specifically recognizing the biological complex and degrading the recognition unit in the biological complex. After the identification unit is degraded, the fluorescent molecules and the biomarker molecules are released, the biomarker molecules can continue to be combined with the identification unit adsorbed on the fluorescent quenching unit to form a biological complex after being released, so that more identification units are degraded, the fluorescent molecules can be continuously enriched in a detection system by utilizing the cyclic reaction, the amplification effect of a fluorescent signal is achieved, and the fluorescent detection system has high sensitivity on the detection of the target biomarker molecules.

The fluorescence detection system is used for detecting the biomarker molecules of the Alzheimer's disease, a large instrument or complex reaction steps are not needed, and the fluorescence detection system is only added into a target detection object, so that the rapid detection of the biomarker molecules of the Alzheimer's disease can be realized, and the advantages of rapidness, simplicity, sensitivity and high efficiency are achieved. Meanwhile, the fluorescence detection system can realize the amplification effect of the detection fluorescence signal of the biomarker molecules, has high detection sensitivity, and effectively improves the accuracy of the detection of the biomarker molecules of the Alzheimer's disease. The kit provides effective clinical information for early diagnosis and screening of the Alzheimer's disease, is beneficial to early discovery and early treatment of diseases, and effectively improves the survival quality and the survival rate of patients with the Alzheimer's disease.

2. In the fluorescence detection system provided by the invention, the fluorescence quenching unit is selected from any one of Graphene and Graphene Oxide (GO). The graphene and the graphene oxide have large-size two-dimensional aromatic planar structures, can be used as good platforms to adsorb specific biomolecules, can be used as energy receptors to quench fluorescence of various organic dyes and quantum dots due to large-area conjugated structures, and are wide-adaptability fluorescence quenchers. Compared with the traditional quencher, the graphene material has higher quenching efficiency, so that the FRET sensor has the remarkable advantages of low background, high signal-to-noise ratio, multiple detection and the like.

According to the fluorescence detection system provided by the invention, the graphene oxide is specifically selected as the fluorescence quenching unit, and the graphene oxide has the advantages of good water dispersibility, excellent biocompatibility, high stability, low toxicity, low cost, easiness in surface modification, easiness in large-scale production, long-distance quenching effect, wide absorption spectrum range, capability of absorbing almost all blue and violet light and good quenching effect on the fluorescence of most fluorescent dyes and quantum dots.

3. In the fluorescence detection system provided by the invention, a fluorescent probe is a single-stranded DNA probe with any one of the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.3, and a biomarker molecule is miRNA with any one of the nucleotide sequences shown in SEQ ID NO.4-SEQ ID NO. 6. According to the invention, single-stranded DNA (ssDNA) is used as a probe, a strong pi-pi acting force exists between a graphene oxide sheet layer and a base of the single-stranded DNA, and the single-stranded DNA probe can be adsorbed to the surface of the graphene oxide, so that fluorescence quenching occurs on a fluorescent molecule marked on the probe. And when the fluorescent detection system is added with a target detection object, the single-stranded DNA molecule of the fluorescent probe is hybridized with the miRNA complementary to the single-stranded DNA molecule to obtain the double-stranded DNA. The graphene oxide has weak adsorption force on double-stranded DNA molecules, the double-stranded DNA is desorbed from the graphene oxide, and fluorescence of fluorescent molecules is recovered, so that the detection of the target biomarker molecules is completed. According to the invention, researches show that the miRNA molecules with the nucleotide sequences shown in SEQ ID NO.4-SEQ ID NO.6 have larger expression difference between a diseased group and a healthy group of the Alzheimer's disease, and provide a biomarker molecule with strong specificity and high accuracy for the disease diagnosis of the Alzheimer's disease. The single-stranded DNA probe with the nucleotide sequences of SEQ ID NO.1-SEQ ID NO.3 is complementarily combined with the miRNA molecules to realize high-sensitivity detection of the miRNA molecule expression level, and further realize noninvasive early screening and diagnosis of Alzheimer's disease.

4. The present invention provides the fluorescence detection system, wherein the identification unit includes: an avidin-labeled fluorescent molecule, and a biotin-labeled single-stranded DNA molecule, the avidin being coupled to the biotin. The identification unit is a single-stranded DNA probe marked by fluorescent molecules constructed based on a biotin-avidin system, and the biotin-avidin system is beneficial to increasing the number of fluorescent molecules marked by each single-stranded DNA molecule, realizing cascade amplification of fluorescent signals, further improving the detection sensitivity of a fluorescent detection system and reducing the detection limit.

Furthermore, the invention provides a molar ratio of coupling of the avidin-labeled fluorescent quantum dots and the biotin-labeled single-stranded DNA molecules in the fluorescence detection system and a volume ratio of the recognition unit to the fluorescence quenching unit, wherein the fluorescence detection system has a linear range of 10-1000nM for miRNA molecule detection related to Alzheimer's disease and has better linearity; the detection limit of the fluorescence detection system is 17nM, with a low detection limit and high sensitivity.

5. The fluorescence biosensor provided by the invention comprises the fluorescence detection system, can realize rapid detection of biomarker molecules of Alzheimer's disease, has the advantages of rapidness, simplicity, sensitivity and high efficiency, has accurate detection result, and is beneficial to early screening and diagnosis of Alzheimer's disease.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the change of fluorescence intensity with miRNA concentration after changing the graphene oxide concentration in the fluorescence detection system in Experimental example 1 of the present invention;

FIG. 2 is a graph showing the change of fluorescence intensity depending on the concentration of miRNA after the concentration of DNase I in the fluorescence detection system is changed in Experimental example 1 of the present invention;

FIG. 3 is a graph showing the linear detection results of the fluorescence detection system for miRNA detection at different concentrations in Experimental example 2.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

All reagents used in the following examples were of analytical grade. The graphene oxide solution is purchased from Nanjing Xiancheng nanotechnology Co. CdSe/ZnS quantum dot labeling streptavidin labels were purchased from Wuhan Jia Quantum dot development technologies, Inc. DEPC water was purchased from Beijing Soilebao Tech. Tris, NaCl, KCl, CaCl₂And MgCl₂All purchased from Shanghai Aladdin Biotechnology, Inc. DNase I (DnaseI) was purchased from Beijing Quanjin Biotechnology, Inc. The preparation method of the detection buffer solution comprises the following steps: first, a Tris-HCl solution containing 10mM Tris, 100mM NaCl, 20mM KCl and 10mM MgCl was prepared in an ultra-clean environment using DEPC water₂And 100. mu.M CaCl₂. The solution was adjusted to pH 7.5 with HCl.