CN111624179A - Fluorescence detection system, fluorescence biosensor and application thereof - Google Patents
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Abstract
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.
Description
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).
Example 1
The present embodiment provides a fluorescence detection system comprising the following components:
1. the first fluorescent probe is a single-stranded DNA probe pDNA1 of which the 5 'end is marked with fluorescent molecule 6-FAM, the nucleotide sequence of pDNA1 is shown in SEQ ID NO.1, and the first fluorescent probe can recognize and combine with a biomarker molecule miRNA1 of Alzheimer's disease.
2. And the second fluorescent probe is a single-stranded DNA probe pDNA2 marked with a fluorescent molecule ROX at the 5 'end, the nucleotide sequence of pDNA2 is shown as SEQ ID NO.3, and the second fluorescent probe can recognize and combine with a biomarker molecule miRNA2 of Alzheimer's disease.
3. And the third fluorescent probe is a single-stranded DNA probe pDNA3 with a 5 'end labeled with a fluorescent molecule Cy5, the nucleotide sequence of the pDNA3 is shown in SEQ ID NO.3, and the third fluorescent probe can recognize and combine with a biomarker molecule miRNA3 of Alzheimer's disease.
4. The fluorescence quencher is graphene oxide.
The fluorescent detection system is prepared by the following steps: the first, second, and third fluorescent probes were configured using DEPC water-treated PBS, and 100 μ l of solution I containing 50nM pDNA1, 50nM pDNA2, and 100nM pDNA3 was taken, 10 μ l of solution II containing 200 μ g/mL graphene oxide was added to solution I, and incubated at room temperature for 5 minutes, to obtain a fluorescent detection system.
After the first fluorescent probe, the second fluorescent probe and the third fluorescent probe in the fluorescent detection system are incubated, the first fluorescent probe, the second fluorescent probe and the third fluorescent probe are adsorbed on the sheet structure of the graphene oxide. The graphene oxide is used as a fluorescence quencher, has no requirement on the emission spectrum of fluorescent molecules, and can quench the fluorescence of various fluorescent dyes. Therefore, when the single-stranded DNA probe is adsorbed on graphene oxide, fluorescence resonance energy transfer occurs, and the fluorescent molecule labeled on the single-stranded DNA probe undergoes fluorescence quenching. After the fluorescent detection system is added into a target detection system, the single-stranded DNA molecules of the first fluorescent probe, the second fluorescent probe and the third fluorescent probe can be combined with miRNA molecules, and the miRNA molecules are subjected to complementary hybridization to form a double-stranded DNA molecule. The graphene oxide has weak adsorption force on double-stranded DNA molecules, and after the double-stranded DNA molecules are desorbed from the graphene oxide, the fluorescence of the fluorescent molecules is recovered. Quantitative detection of the biomarker molecule miRNA can be realized by detecting the enhancement degree of the fluorescence intensity before and after the target detection system is added. Because the fluorescent molecules 6-FAM, ROX and 6-Cy5 marked on each fluorescent probe have different fluorescence emission spectra, the fluorescence detection system can simultaneously collect the fluorescence of three different emission spectra, thereby simultaneously realizing the quantitative detection of three miRNA molecules. The fluorescent detection system provided by the embodiment can realize noninvasive diagnosis of the Alzheimer's disease by specifically identifying and detecting the biomarker molecules of the Alzheimer's disease, does not need to use large instruments or complicated detection steps in the diagnosis process, and has the advantages of rapidness, simplicity, sensitivity and high efficiency. The three fluorescent probes are combined with target miRNA molecules, so that the specificity is strong, the sensitivity is high, the fluorescent detection system has better accuracy when being used for diagnosing the Alzheimer's disease, effective clinical information is provided for early diagnosis and screening of the Alzheimer's disease, early discovery and early treatment of diseases are facilitated, the survival quality of patients with the Alzheimer's disease is effectively improved, and the survival rate of the patients is improved.
Example 2
The present embodiment provides a fluorescence biosensor comprising the fluorescence detection system of embodiment 2. The fluorescent biosensor can realize the rapid detection of the biomarker molecules of the Alzheimer's disease, has the advantages of rapidness, simplicity, sensitivity, high efficiency and accuracy, and can be used for disease screening and diagnosis of the Alzheimer's disease.
Experimental example 1
1. Purpose of the experiment: the specificity and sensitivity of binding of the first, second and third fluorescent probes to the target biomarker molecules in example 1 was determined.
2. Experimental procedures and results:
the fluorescence emission spectra of the first fluorescent probe in the wavelength range of 510nm to 640nm (excitation wavelength: 490nm), the first fluorescent probe in the wavelength range of 600nm to 740nm (excitation wavelength: 580nm) and the third fluorescent probe in the wavelength range of 670nm to 790nm (excitation wavelength: 640nm) are respectively detected before the three fluorescent probes are incubated with graphene oxide, after the three fluorescent probes are incubated with graphene oxide, and after the three fluorescent probes are incubated with graphene oxide and miRNA simultaneously, and the results are shown in FIG. 1.
FIG. 1A shows the change in fluorescence emission spectra of the first fluorescent probe pDNA1 in three cases: in the absence of GO, pDNA1 exhibited a strong fluorescence emission spectrum with an emission peak at λ 520 nm. Fluorescence intensity decreased much when GO was added to the solution, indicating that pDNA1 adsorbed on the surface of the BPNSs and fluorescence of the QDs quenched. However, when complementary target miRNA1 was added to the solution and hybridized to pDNA1, the DNA duplex was dissociated from GO and the fluorescence of the QDs was significantly restored to the level before GO was added. The fluorescence spectra of the other two pdnas showed the same properties. Fig. 1B shows the change of the fluorescence emission spectrum of the second fluorescent probe pDNA2 in three cases, and fig. 1C shows the change of the fluorescence emission spectrum of the third fluorescent probe pDNA3 in three cases, and as can be seen from fig. 1B and 1C, the second fluorescent probe pDNA2 and the third fluorescent probe pDNA3 exhibit the same properties as the first fluorescent probe pDNA 1.
Experimental example 2
1. Purpose of the experiment: detection of Linear detection Range of the fluorescent detection System in example 1
2. The experimental process comprises the following steps:
1) fluorescence detection was performed in a completely black 96-well plate using a multifunctional microplate reader Varioka Flash (Thermo Scientific, USA). First, 100. mu.l of a solution containing 50nM pDNA1, 50nM pDNA2 and 100nM pDNA3 was added to the well plate. Next, 10. mu.l GO at a concentration of 200. mu.g/mL was added to the solution. After 5 min incubation at room temperature, 100. mu.L of different concentrations of miRNA1(1000nM, 500nM, 200nM, 100nM, 50nM, 20nM, 10nM and 0nM) or miRNA2(1000nM, 500nM, 200nM, 100nM, 50nM, 20nM, 10nM and 0nM) or miRNA3(1000nM, 500nM, 200nM, 100nM, 50nM, 20nM, 10nM and 0nM) were added to the solution and incubated for 20 min. For the control group, 100. mu.LPBS was added. At the same time, the utility of the detection system was determined using four spiked samples (30nM miRNA1, 70nM miRNA2), (30nM miRNA1, 125nM miRNA3), (70nM miRNA1, 125nM miRNA3) and (30nM miRNA1, 70nM miRNA2, 125nM miRNA3), respectively. At the end of each step, the fluorescence emission spectra of each well were recorded in the 510nm-640nm band (excitation: 490nm), the 600nm-740nm band (excitation: 580nm) and the 670nm-790nm band (excitation: 640 nm). During the detection, the fluorescence emission intensities at 520nm, 610nm and 670nm were recorded every five minutes. The detection results are shown in fig. 2-4.
2) And (3) measuring the fluorescence intensity of the wells with the labeled samples at lambda of 520nm, and substituting the fluorescence intensities of 610nm and 660nm into a calibration equation to obtain the concentrations of miRNA1, miRNA2 and miRNA 3. Table 1 shows the results of the measurement of the spiked samples.
3. The experimental results are as follows:
fig. 2 shows fluorescence spectra measured after incubation of pDNA1-GO mixtures with different concentrations of target miRNA1(0nM-1000nM), and fig. 2A shows that the fluorescence emission intensity of FAM increases with increasing target miRNA concentration, indicating the recovery of fluorescence by target miRNA 1. Fig. 2B shows the fluorescence intensity change Δ F1(Δ F1 ═ F1-F01, F01 is the fluorescence intensity of the pDNA1-GO mixture at a wavelength λ ═ 520nm, F1 is the fluorescence intensity measured at a wavelength λ ═ 520nm after incubation of a concentration of target miRNA1 with the pDNA1-GO mixture for 20 minutes) plotted against the target miRNA1 concentration. As can be seen from fig. 2B, Δ F is linearly related to the concentration of the target miRNA1 in the concentration range of 10nM to 200nM, and the linear equation is Δ F0.023 × CmiRNA1+2.07(nM, r2 0.9962).
Fig. 3 shows fluorescence spectra measured after incubation of pDNA2-GO mixtures with different concentrations of the target miRNA2(0nM-1000nM), and fig. 3A shows that the fluorescence emission intensity of ROX increases with increasing target miRNA concentration, indicating the recovery of fluorescence by the target miRNA 2. Fig. 3B shows the fluorescence intensity change Δ F2(Δ F2 ═ F2-F02, F02 is the fluorescence intensity of the pDNA2-GO mixture at a wavelength λ ═ 610nm, F2 is the fluorescence intensity measured at a wavelength λ ═ 610nm after incubation of a concentration of target miRNA2 with the pDNA2-GO mixture for 20 minutes) plotted against the target miRNA2 concentration. As shown in fig. 3B, Δ F2 is linearly related to the target miRNA concentration in the range of 20nM to 500nM, and the linear equation is Δ F0.019 × CmiRNA1+0.67(nM, r2 0.991).
Fig. 4 shows fluorescence spectra measured after incubation of pDNA3-GO mixtures with different concentrations of target miRNA3(0nM-1000nM), and fig. 4A shows that the fluorescence emission intensity of 6-Cy5 increases with increasing target miRNA concentration, indicating the recovery of fluorescence by target miRNA 3. Fig. 4B shows a calibration curve of the change in fluorescence intensity Δ F3(Δ F3 ═ F3-F03, F03 is the fluorescence intensity of the pDNA3-GO mixture at a wavelength λ ═ 660nm, and F3 is the fluorescence intensity measured at a wavelength λ ═ 660nm after incubation of a concentration of the target miRNA3 with the pDNA3-GO mixture for 20 minutes) versus the concentration of the target miRNA 3. As shown in fig. 4B, Δ F3 is linearly related to the concentration of miRNA3 in the concentration range of 20nM-200nM, and the linear equation is Δ F0.016 × CmiRNA1-0.41(nM, r2 × 0.995).
As can be seen from FIGS. 2 to 4, the linear detection range of the fluorescence detection system for miRNA1 is 10nM-200nM, the linear detection range for miRNA2 is 20nM-500nM, the linear detection range for miRNA3 is 20nM-200nM, and the linearity is good.
TABLE 1 miRNA spiked sample detection results
As can be seen from table 1, the detection accuracy of the fluorescence detection system in example 2 for miRNA1, miRNA2, and miRNA3 is between 80% and 120%, which indicates that the fluorescence detection system has higher accuracy for detecting the biomarker molecules of alzheimer's disease.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
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Claims (8)
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.
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