阅读说明：本技术 荧光检测体系、荧光生物传感器及其用途 (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：本发明公开了一种荧光检测体系,包括：至少两种荧光探针,结合至少两种阿尔兹海默症的生物标志物分子,每种荧光探针标记的荧光分子的发光光谱互不相同；荧光淬灭剂,荧光淬灭剂吸附荧光探针,使荧光分子的荧光淬灭；荧光淬灭剂与结合生物标志物分子的荧光探针解吸附,使荧光分子的荧光恢复。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,无需使用大型仪器或经过复杂的反应步骤,仅需将荧光检测体系加入目标检测物中,即能实现对阿尔兹海默症的生物标志物分子的快速检测,具有快速、简单、灵敏、高效的优势。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 fluorescence detection system, comprising: at least two fluorescent probes which are combined with at least two biomarker molecules of Alzheimer's disease, and the luminescence spectra of the fluorescent molecules marked by each fluorescent probe are different from each other; the fluorescence quenching agent absorbs the fluorescent probe to quench the fluorescence of the fluorescent molecule; the fluorescence quencher desorbs the fluorescent probe bound to the biomarker molecule, and the fluorescence of the fluorescent molecule is recovered. 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. 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 fluorescence detection system, comprising:

at least two fluorescent probes, wherein the at least two fluorescent probes are combined with at least two biomarker molecules of Alzheimer's disease, and the light-emitting spectrum of the fluorescent molecules marked by each fluorescent probe is different from each other;

a fluorescence quencher that adsorbs the fluorescent probe and quenches fluorescence of the fluorescent molecule; the fluorescence quencher desorbs the fluorescent probe bound to the biomarker molecule, restoring the fluorescence of the fluorescent molecule.

2. The fluorescence detection system of claim 1, wherein the fluorescent probe is a single-stranded DNA probe having a nucleotide sequence set forth in any one of SEQ ID No.1 to SEQ ID No.3, and the biomarker molecule is a miRNA having a nucleotide sequence set forth in any one of SEQ ID No.4 to SEQ ID No. 6.

3. The fluorescence detection system of claim 2, wherein the at least two fluorescent probes comprise:

the nucleotide sequence of the first fluorescent probe is shown as SEQ ID NO.1, and the fluorescent molecule marked by the first fluorescent probe is FAM;

the nucleotide sequence of the second fluorescent probe is shown as SEQ ID NO.2, and the fluorescent molecule marked by the second fluorescent probe is ROX;

a nucleotide sequence of the third fluorescent probe is shown as SEQ ID NO.3, and the fluorescent molecule marked by the second fluorescent probe is Cy 5;

preferably, the concentration of the first fluorescent probe is 50nM, the concentration of the second fluorescent probe is 50nM, and the concentration of the third fluorescent probe is 100 nM.

4. The fluorescence detection system according to any one of claims 1 to 3, wherein the fluorescence quencher is any one of graphene and graphene oxide;

preferably, the fluorescence quencher is graphene oxide;

preferably, the concentration of the graphene oxide is 200 μ g/mL.

5. The fluorescence detection system of any of claims 1-4, wherein the volume ratio of the fluorescent probe to the fluorescence quencher is 10: 1.

6. A fluorescent biosensor comprising the fluorescent detection system of any one of claims 1-5.

7. Use of the fluorescent detection system of any one of claims 1 to 5, or the fluorescent biosensor of claim 6, in the manufacture of a product for diagnosing alzheimer's disease.

8. A kit for diagnosing alzheimer's disease, comprising the fluorescent detection system of any one of claims 1-5, or the fluorescent biosensor of claim 6.

Technical Field

The invention relates to the field of biomolecule detection, in particular to a 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 fluorescence detection system comprising:

at least two fluorescent probes, wherein the at least two fluorescent probes are combined with at least two biomarker molecules of Alzheimer's disease, and the light-emitting spectrum of the fluorescent molecules marked by each fluorescent probe is different from each other;

a fluorescence quencher that adsorbs the fluorescent probe and quenches fluorescence of the fluorescent molecule; the fluorescence quencher desorbs the fluorescent probe bound to the biomarker molecule, restoring the fluorescence of the fluorescent molecule.

Optionally, in the above fluorescence detection system, the fluorescent probe is a single-stranded DNA probe having a nucleotide sequence shown in any one of SEQ ID nos. 1 to 3, and the biomarker molecule is miRNA having a nucleotide sequence shown in any one of SEQ ID nos. 4 to 6.

Further optionally, in the above fluorescence detection system, the at least two kinds of fluorescent probes include:

the nucleotide sequence of the first fluorescent probe is shown as SEQ ID NO.1, and the fluorescent molecule marked by the first fluorescent probe is FAM;

the nucleotide sequence of the second fluorescent probe is shown as SEQ ID NO.2, and the fluorescent molecule marked by the second fluorescent probe is ROX;

a nucleotide sequence of the third fluorescent probe is shown as SEQ ID NO.3, and the fluorescent molecule marked by the second fluorescent probe is Cy 5;

preferably, the concentration of the first fluorescent probe is 50nM, the concentration of the second fluorescent probe is 50nM, and the concentration of the third fluorescent probe is 100 nM.

Optionally, in the fluorescence detection system, the fluorescence quencher is any one of graphene and graphene oxide;

preferably, the fluorescence quencher is graphene oxide;

preferably, the concentration of the graphene oxide is 200 μ g/mL.

Optionally, in the above fluorescence detection system, the volume ratio of the fluorescence probe to the fluorescence quencher is 10: 1.

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

The fluorescent detection system or the application of the fluorescent biosensor in preparing products for diagnosing the Alzheimer's disease.

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

The technical scheme of the invention has the following advantages:

1. the present invention provides a fluorescence detection system comprising: the at least two fluorescent probes are combined with at least two biomarker molecules of Alzheimer's disease, and the light-emitting spectrum of each fluorescent probe-marked fluorescent molecule is different from each other; a fluorescence quencher that adsorbs the fluorescent probe and quenches fluorescence of the fluorescent molecule; and desorbing the fluorescence quencher with the fluorescent probe combined with the biomarker molecule to recover the fluorescence of the fluorescent molecule.

In the fluorescence detection system, the fluorescent probe marked with fluorescent molecules is used as a fluorescence donor, the fluorescence quencher is used as a fluorescence acceptor, and after the fluorescence quencher adsorbs the fluorescent probe,the distance between the two is close enough to generate fluorescence resonance energy transfer (resonance energy transfer, FRET), the energy of the fluorescent donor is transferred to the fluorescent acceptor, causing fluorescence quenching of the fluorescent molecule of the fluorescent probe. And after the fluorescent probe is combined with the biomarker molecules of the Alzheimer disease, the compound formed by combining the fluorescent probe and the biomarker molecules is desorbed with the fluorescence quencher, so that the fluorescence of the fluorescent molecules is recovered. After the target detection object is added into the fluorescence detection system, quantitative detection of target biomarker molecules related to the Alzheimer's disease can be realized by analyzing the enhancement degree of fluorescence intensity in the fluorescence detection system. The fluorescence detection system based on fluorescence resonance energy transfer has high sensitivity and selectivity. 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.

By utilizing the fluorescence detection system, effective detection of the Alzheimer disease can be realized only by sampling peripheral blood of an AD patient, the detection process is non-invasive, noninvasive diagnosis of the Alzheimer disease can be realized, and the pain, spirit and economic burden of the patient are effectively relieved. On the other hand, since the pathogenesis of AD is very complex, detection of only one biomarker molecule is not sufficient. In the above-mentioned fluorescent detection system, the fluorescence spectra of the fluorescent molecules labeled with each fluorescent probe are different from each other, and therefore, the fluorescent molecules having a specific emission spectrum are labeled with each fluorescent probe, and the above-mentioned fluorescent probes can be used to detect at least two kinds of biomarker molecules in the same detection system. The accuracy of early diagnosis of AD can be improved by jointly detecting multiple AD biomarker molecular disease markers, effective clinical information is provided for early diagnosis and screening of Alzheimer's disease, early discovery and early treatment of disease are facilitated, the survival quality of patients with Alzheimer's disease is effectively improved, and the survival rate of patients is improved.

2. In the fluorescence detection system provided by the invention, the fluorescence quencher is 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 fluorescence quencher specifically selects graphene oxide, the graphene oxide has good water dispersibility, excellent biocompatibility, high stability, low toxicity, low cost, easy surface modification, easy large-scale production and long-distance quenching effect, the absorption spectrum range is wide, almost all blue and violet light can be absorbed, and the fluorescence detection system has good quenching effect on most of 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 fluorescence detection system further provides the concentrations of the first fluorescent probe, the second fluorescent probe, the third fluorescent probe and the fluorescence quencher, and the volume ratio of the fluorescent probes to the fluorescence quencher. The fluorescent detection system of the substance components can realize the linear detection of the miRNA molecules differentially expressed in the three Alzheimer's diseases, wherein the linear range of the detection of the first fluorescent probe is 10nM-200nM, the linear range of the detection of the second and third fluorescent probes is 20nM-200nM, and the linearity is good. The fluorescence detection system can simultaneously detect various biomarker molecules with different concentrations in a target detection sample, and the detection result is accurate.

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 changes of fluorescence emission spectra of a first fluorescent probe, a second fluorescent probe and a third fluorescent probe before incubation with graphene oxide, after incubation with graphene oxide and miRNA in experimental example 1 of the present invention;

FIG. 2 is a graph showing the results of fluorescence spectrum measurements of a mixture of pDNA1-GO incubated with different concentrations of target miRNA1(0nM to 1000nM) in Experimental example 2 of the present invention;

FIG. 3 is a graph showing the results of fluorescence spectroscopy measurements of pDNA2-GO mixtures incubated with different concentrations of the target miRNA2(0nM-1000 nM);

FIG. 4 is a graph showing the results of fluorescence spectroscopy measurements of pDNA3-GO mixtures incubated with different concentrations of the target miRNA3(0nM-1000 nM).

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. Diethyl pyrocarbonate (DEPC) was purchased from Amresco (USA). Phosphate buffer powder (PBS, 0.01M, ph7.2, 137mM NaCl, 2.7mM KCl) was purchased from Solarbio Science & Technology co., Ltd. (Beijing, China). PBS was first diluted with deionized water and then treated overnight with 0.1% DEPC. Followed by autoclaving the PBS solution for 15-20 minutes to remove DEPC therefrom for subsequent fluorescence experiments. Both DNA and miRNA sequences were synthesized by Shanghai Biotechnology, Inc. (Shanghai).