WO2021114914A1 - 一种基于铋烯纳米片荧光淬灭的生物传感器、miRNA检测试剂盒及应用 - Google Patents

一种基于铋烯纳米片荧光淬灭的生物传感器、miRNA检测试剂盒及应用 Download PDF

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WO2021114914A1
WO2021114914A1 PCT/CN2020/124037 CN2020124037W WO2021114914A1 WO 2021114914 A1 WO2021114914 A1 WO 2021114914A1 CN 2020124037 W CN2020124037 W CN 2020124037W WO 2021114914 A1 WO2021114914 A1 WO 2021114914A1
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bismuthene
nanosheets
mirna
fluorescence
nucleic acid
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French (fr)
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梁维源
张家宜
范涛健
康建龙
黄浩
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深圳瀚光科技有限公司
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    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
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Definitions

  • the present invention requires the priority of the prior application of the application number 201911283041.5 with the title of "a biosensor based on fluorescence quenching of bismuthene nanosheets, miRNA detection kit and application” submitted on December 13, 2019.
  • the content of the application is incorporated into this text by way of introduction.
  • the invention relates to the technical fields of biomedicine and analytical chemistry, in particular to a reagent for clinical diagnosis, and more particularly to a biosensor based on bismuthene nanosheet fluorescence quenching, and including the bismuthene nanosheet fluorescence quenching
  • the miRNA detection kit of the biosensor also relates to the application of the biosensor based on the fluorescence quenching of the bismuthene nanosheets in the detection of miRNA.
  • MicroRNA is a small single-stranded RNA that is expressed endogenously in the body. It is located in the non-coding region of the genome. It does not have an open reading frame (ORF). It is highly conservative, sequential and tissue specific. . miRNA is widely present in various eukaryotic cells, does not encode any protein, and is only 20-24 nt in length. Mature miRNA has a phosphate group at the 5'end and a hydroxyl group at the 3'end, which is formed by Dicer enzyme processing of a 70-90nt single-stranded RNA precursor with a hairpin-like structure.
  • RNA-induced gene silencing complex RNA-induced silencing complex, RISC
  • RISC RNA-induced silencing complex
  • miRNAs play an important role in biological processes such as cell growth, development, differentiation, and death.
  • miRNAs are involved in different processes such as blood cell production, insulin secretion, nervous system composition, and human cancer cell growth. Therefore, it is very necessary to develop effective detection tools to measure miRNA.
  • the current methods for detecting miRNA mainly include Northern blot analysis, microarray analysis and quantitative Real-Time PCR.
  • the disadvantage of Northern blot analysis is that the process of Northern analysis involves a lot of manual operations, which requires high technical requirements for inspectors, and only one miRNA probe is hybridized with one Northern blot at a time. Therefore, it is not suitable for large-scale analysis. Clinical screening test.
  • microarray analysis it needs enough RNA samples, it is impossible to distinguish miRNAs with small differences, the detection resolution is low, and it is difficult to meet the clinical detection requirements.
  • Real-time quantitative PCR requires RNA reverse transcription and subsequent amplification, and can only perform relative quantitative or qualitative analysis, which is difficult to meet the requirements of clinical absolute quantitative detection.
  • 2D Nanosheets have attracted widespread attention in biomedicine, electronics, environmental and energy applications.
  • 2D materials for biosensor applications are graphene, black phosphorus (BP), two-dimensional transition metal sulfides (TMDs), group III chalcogenides, etc.
  • the VA elements are called "photonogen", which opens a new undeveloped area for the application of single-element olefins.
  • 2D materials in the VA family such as BP, arsenic and antimony
  • their application in the field of biosensing is still a challenge.
  • bismuth As the heaviest atomic weight in the VA family with strong spin-orbit coupling, bismuth has excellent metallic and topological properties, such as fast carrier migration, excellent biocompatibility, strong light-material interaction, chemical and Thermal stability, which shows great application prospects in biosensors.
  • the key to detecting miRNA with fluorescent probes lies in the mechanism of fluorescence quenching, including Resonance energy transfer (FRET) and charge transfer (CT).
  • FRET Resonance energy transfer
  • CT charge transfer
  • the present invention provides a biosensor based on bismuthene nanosheet fluorescence quenching, and the present invention also provides a miRNA detection kit including the above-mentioned bismuthene nanosheet fluorescence quenching biosensor.
  • the present invention The application of the above-mentioned bismuthene nanosheet fluorescence quenching biosensor in miRNA detection is also provided.
  • the present invention realizes high-resolution detection of specific miRNAs, including qualitative detection and quantitative detection, and is applied to in vitro clinical diagnosis, such as clinical diagnosis of Alzheimer’s disease (AD), Clinical diagnosis of tumors, etc., have huge application prospects and market value.
  • the present invention provides a biosensor based on the fluorescence quenching of bismuthene nanosheets to solve the cumbersome operation of the existing miRNA detection methods, the large demand for RNA samples, and the inability to distinguish between miRNAs with small differences and detection. Problems such as low resolution.
  • a biosensor based on fluorescence quenching of bismuthene nanosheets comprising fluorescently labeled single-stranded nucleic acid probes and bismuthene nanosheets.
  • the single-stranded nucleic acid probes are complementary to specific miRNAs, and the single-stranded nucleic acid probes are adsorbed on Surface of bismuthene nanosheets;
  • the lateral dimension of the bismuthene nanosheets is greater than 30 nm, and the thickness is 0.4-20 nm.
  • the XRD diffraction peaks of the bismuth ene nanosheets are located at 22.4°, 27.1°, 37.9°, 39.6°, 45.6°, 48.7°, 56° and 59.3°, respectively corresponding to (003), (012 ), (104), (110), (006), (202), (024), (107) crystal planes (JCPDS No. 44-1246).
  • the Raman spectrum of the bismuthene nanosheets respectively exhibit vibration peaks at 65.6 cm -1 and 91 cm -1.
  • the fluorescently labeled single-stranded nucleic acid probe is a fluorescein imide (FAM) labeled single-stranded nucleic acid probe.
  • FAM fluorescein imide
  • the fluorescently labeled single-stranded nucleic acid probe can also be fluorescein isothiocyanate (FITC), tetrachlorofluorescein (TET), hexachlorofluorescein (HEX), carboxyl tetrafluorescein Single-stranded nucleic acid probes labeled with fluorescein molecules commonly used to label nucleotide probes, such as methylrhodamine (TAMRA).
  • FITC fluorescein isothiocyanate
  • TET tetrachlorofluorescein
  • HEX hexachlorofluorescein
  • TAMRA methylrhodamine
  • the single-stranded nucleic acid probe is a single-stranded DNA probe.
  • the single-stranded nucleic acid probe may also be a single-stranded RNA probe.
  • sequence of the single-stranded DNA probe is 5'-TCAACATCAGTCTGATAAGCTA-3', and the single-stranded DNA probe is complementary to miRNA-21.
  • the sequence of the single-stranded DNA probe can also be complementary to other miRNA markers, such as Alzheimer's disease (AD) marker miRNA, tumor marker miRNA, and intervertebral discs that have been reported in the literature.
  • AD Alzheimer's disease
  • tumor marker miRNA tumor marker miRNA
  • intervertebral discs intervertebral discs that have been reported in the literature.
  • Degeneration (IDD) marker miRNA it can also be plant miRNA, for example, rice miRNA528.
  • the length of the single-stranded DNA probe is 15 to 36 nt; more preferably, the length of the single-stranded DNA probe is 19 to 23 nt.
  • a biosensor based on the fluorescence quenching of bismuthene nanosheets has a special composite structure: fluorescein-single-stranded nucleic acid probe Needle-bismuthene nanosheets, that is, fluorescently labeled single-stranded nucleic acid probes are adsorbed on the surface of the bismuthene nanosheets through intermolecular forces (van der Waals forces), thereby combining to form the special composite structure.
  • the detection principle is: with the help of the unique fluorescence quenching effect of the bismuthene nanosheets, the fluorescence of the fluorescein-single-stranded nucleic acid probe-bismuthene nanosheet complex is quenched.
  • the specific miRNA or serum (or other sample liquid containing miRNA) is complementary to the nucleic acid probe, the miRNA and the bismuthene nanosheets compete for binding to the single-stranded nucleic acid probe, and the miRNA and the single-stranded nucleic acid probe have a greater affinity.
  • the significant quenching effect of bismuth attributed the weak fluorescence charge transfer between bismuth and dye molecules to the ground state of bismuth.
  • dsDNA double-stranded DNA
  • the formation of dsDNA weakens the adsorption force of bismuthene nanosheets and single-stranded nucleic acid probes.
  • Single-stranded nucleic acid probes (at this time miRNA and single-stranded nucleic acid probes) The needles combine to form dsDNA) to get rid of the constraints of bismuthene nanosheets.
  • the fluorescence quenching effect of the bismuthene nanosheets is difficult to continue to play a role, and the composite structure system shows a certain degree of fluorescence recovery.
  • the biosensor based on the fluorescence quenching of bismuthene nanosheets of the present invention has extremely high detection limit for detecting specific miRNA, and can detect miRNA with a concentration as low as 60pM; it also has extremely high detection specificity and can distinguish single base errors.
  • the biosensor based on the fluorescence quenching of bismuthene nanosheets also has the advantages of simple operation, fast detection speed, and less sample demand.
  • the present invention also provides a miRNA detection kit to solve the cumbersome detection operation of the existing miRNA detection kit, the large amount of RNA samples, the low detection limit, the inability to distinguish between miRNAs with small differences, and the detection Problems such as low resolution.
  • a miRNA detection kit includes a fluorescently-labeled single-stranded nucleic acid probe and a fluorescence quencher, the single-stranded nucleic acid probe is complementary to a specific miRNA, and the fluorescence quencher includes bismuthene nanosheets;
  • it also includes a standard concentration of specific miRNA
  • the specific miRNA of the standard concentration binds to the single-stranded nucleic acid probe through competition, and is used to measure the fluorescence recovery intensity and make a standard line.
  • the specific miRNA at the standard concentration includes the specific miRNA at the following concentrations: 0 nM, 0.5 nM, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM and 10 nM.
  • the fluorescence quencher contains 1-500 ⁇ g/ml bismuthene nanosheets. More preferably, the fluorescence quencher contains 20-80 ⁇ g/ml bismuthene nanosheets. More preferably, the fluorescence quencher contains 50 ⁇ g/ml bismuthene nanosheets.
  • the miRNA detection kit includes a fluorescently labeled single-stranded nucleic acid probe and a fluorescence quencher.
  • Fluorescence-labeled single-stranded nucleic acid probes have a fluorescence effect, that is, they emit corresponding fluorescence when irradiated by a certain excitation light.
  • the fluorescently labeled single-stranded nucleic acid probe is incubated with the fluorescence quencher, the fluorescently labeled single-stranded nucleic acid probe is adsorbed on the surface of the bismuthene nanosheet by means of van der Waals force, forming fluorescein-single-stranded nucleic acid probe-bismuth Ene nanosheet composite.
  • the fluorescence-labeled single-stranded nucleic acid probes produce fluorescence quenching.
  • miRNA or serum (or other sample solution containing miRNA) complementary to single-stranded nucleic acid probes is added to the complex structure system, miRNA and bismuthene nanosheets compete to bind to single-stranded nucleic acid probes, and miRNA and single-stranded nucleic acid The probes combine to form a dsDNA structure.
  • dsDNA weakens the adsorption force of the bismuthene nanosheets and the single-stranded nucleic acid probes, and the single-stranded nucleic acid probes (at this time miRNA and single-stranded nucleic acid probes combine to form dsDNA) get rid of the bismuthene nanosheets A certain degree of fluorescence recovery appears. By detecting the degree of fluorescence recovery, by detecting the concentration of specific miRNA or specific miRNA in serum, the function of clinical diagnosis can be realized.
  • the miRNA detection kit of the present invention has extremely high detection limits, capable of detecting 60pM miRNA; also has extremely high detection specificity, capable of distinguishing single-base mismatch; at the same time, the miRNA detection kit also has simple operation, Advantages such as fast detection speed and low sample demand.
  • the present invention also provides the application of a biosensor based on the fluorescence quenching of bismuthene nanosheets in the detection of miRNA to solve the cumbersome detection operation, large demand for RNA samples, and detection of existing miRNA detection technologies. Problems such as low limit, inability to distinguish miRNAs with small differences, and low detection resolution.
  • the present invention uses the biosensor based on the fluorescence quenching of bismuthene nanosheets to detect miRNA.
  • the fluorescence quenching miRNA detection kit is prepared through the biosensor based on the fluorescence quenching of the bismuthene nanosheets, and finally applied to miRNA In qualitative or quantitative detection, it has a very high detection limit, capable of detecting 60pM miRNA; it also has a high detection specificity, capable of distinguishing single-base mismatches.
  • the application of the biosensor based on the fluorescence quenching of bismuth ene nanosheets to detect miRNA also has simple operation, low technical requirements for the operator, and fast detection speed.
  • the fluorescence recovery measurement can be performed and automatically calculated by the computer. Output miRNA levels.
  • the biosensor based on the fluorescence quenching of bismuthene nanosheets requires less sample when detecting miRNA, and can achieve absolute quantification by forming a double-stranded structure between nucleic acid molecules without amplification.
  • Figure 1 is a diagram of the preparation and characterization of the bismuthene nanosheets of the present invention.
  • AFM morphology shows that there are few layers of bismuthene nanosheets on the mica.
  • Figure 2 is a spectrum test diagram of the bismuthene nanosheets of the present invention.
  • a) Linear normalized absorption spectra of FAM-ssDNA solutions with a bismuth concentration of 0 ⁇ g/ml (far right), 50 ⁇ g/ml (middle) and 100 ⁇ g/ml (far left).
  • Fig. 3 is a fluorescence quenching test effect diagram of the biosensor based on the fluorescence quenching of bismuthene nanosheets of the present invention.
  • d Fluorescence image of FAM-ssDNA and bismuthene nanosheet solution incubated with miRNA-21.
  • FAM-ssDNA probe top
  • FAM-ssDNA/miRNA-21 the second line from top to bottom
  • FAM-ssDNA/miRNA-21@bismuth nanosheet the third line from top to bottom
  • Fluorescence spectra of the FAM-ssDNA probe solution when there are different concentrations of miRNA-21 in the bismuthene nanosheet solution the concentration of miRNA-21 corresponding to the thirteen lines from top to bottom decreases.
  • bismuthene nanosheets we use bismuthene nanosheets to solve this problem, and use photoluminescence and optical pump detection spectroscopy to clarify the quenching mechanism.
  • the significant quenching effect of the bismuthene nanosheets is attributed to the weak fluorescent charge transfer between the bismuthene nanosheets and the dye molecules in the ground state.
  • bismuthene nanosheets are used as a fluorescence quencher to detect lung cancer-related miRNA-21 biomarkers, thereby providing a rapid, sensitive and selective detection platform for miRNA sensing.
  • a biosensor based on the fluorescence quenching of bismuthene nanosheets including fluorescently labeled single-stranded nucleic acid probes and bismuthene nanosheets.
  • the single-stranded nucleic acid probes are adsorbed on the surface of the bismuthene nanosheets by means of van der Waals force to form unique fluorescence.
  • the detection principle is: with the unique fluorescence quenching effect of the bismuthene nanosheets, the fluorescence of the fluorescein-single-stranded nucleic acid probe-bismuthene nanosheet complex is quenched.
  • the significant quenching effect of bismuth attributed the weak fluorescence charge transfer between bismuth and dye molecules to the ground state of bismuth. Using this mechanism, bismuth nanomaterials were used as a fluorescence quencher.
  • a specific miRNA or serum (or other sample solution containing miRNA) complementary to a single-stranded nucleic acid probe is added to the complex structure system, the miRNA and the bismuthene nanosheets compete to bind to the single-stranded nucleic acid probe, and the miRNA and the single-stranded nucleic acid probe The affinity of stranded nucleic acid probes is greater, and single-stranded nucleic acid probes tend to form dsDNA structures. miRNA and single-stranded nucleic acid probes combine to form a double-stranded DNA (dsDNA) structure. The formation of dsDNA weakens the adsorption force of bismuthene nanosheets and single-stranded nucleic acid probes.
  • dsDNA double-stranded DNA
  • Single-stranded nucleic acid probes (at this time miRNA and single-stranded nucleic acid probes)
  • the needles combine to form dsDNA) to get rid of the constraints of bismuthene nanosheets.
  • the fluorescence quenching effect of the bismuthene nanosheets is difficult to continue to play a role, and the composite structure system shows a certain degree of fluorescence recovery.
  • the degree of fluorescence recovery detecting the concentration of specific miRNA or specific miRNA in serum, the function of clinical diagnosis is realized.
  • the biosensor based on the fluorescence quenching of bismuthene nanosheets of the present invention has extremely high detection limit for detecting specific miRNA, and can detect miRNA with a concentration as low as 60pM; it also has extremely high detection specificity and can distinguish single base errors.
  • the biosensor based on the fluorescence quenching of bismuthene nanosheets also has the advantages of simple operation, fast detection speed, and less sample demand.
  • the lateral dimension of the bismuthene nanosheet is greater than 30 nm, and the thickness is 0.4-20 nm.
  • the lateral size and thickness of the bismuthene nanosheets can ensure that the single-stranded nucleic acid probes are adsorbed on the surface of the bismuthene nanosheets by means of van der Waals force, so that the single-stranded nucleic acid probes will not be separated from the bismuthene nanosheets due to insufficient adsorption force, resulting in detection The result is not accurate.
  • the XRD diffraction peaks of the bismuthene nanosheets are located at 22.4°, 27.1°, 37.9°, 39.6°, 45.6°, 48.7°, 56° and 59.3°, corresponding to (003) of the bismuth nanocrystals, respectively. (012), (104), (110), (006), (202), (024), (107) crystal planes (JCPDS No. 44-1246).
  • JCPDS No. 44-1246 crystal planes
  • the Raman spectrum of the bismuthene nanosheets respectively exhibit typical peaks at 65.6 cm -1 and 91 cm -1 , corresponding to the Eg and Alg vibration modes of bismuth, respectively.
  • the present invention prepares bismuthene nanosheets, and subsequently uses the bismuthene nanosheets to act as a fluorescence quencher.
  • the fluorescently labeled single-stranded nucleic acid probe is a fluorescein imide (FAM) labeled single-stranded nucleic acid probe.
  • FAM fluorescein imide
  • FAM is a commonly used nucleic acid probe and is widely used in fluorescent label detection. It has the advantages of rapid reaction with amino groups, stable binding products and easy control.
  • the fluorescently labeled single-stranded nucleic acid probe can also be fluorescein isothiocyanate (FITC), tetrachlorofluorescein (TET), hexachlorofluorescein (HEX), carboxytetramethylrhodamine ( TAMRA) and other common single-stranded nucleic acid probes labeled with fluorescein molecules, which are commonly used to label nucleotide probes, have similar fluorescent labeling and fluorescent detection effects.
  • FITC fluorescein isothiocyanate
  • TET tetrachlorofluorescein
  • HEX hexachlorofluorescein
  • TAMRA carboxytetramethylrhodamine
  • the single-stranded nucleic acid probe is a single-stranded DNA (ssDNA) probe.
  • the ssDNA probe has the advantages of stable molecular structure and easy combination with miRNA to form a stable double-stranded structure, which is convenient for control and fluorescent detection.
  • the sequence of the single-stranded DNA probe is 5'-TCAACATCAGTCTGATAAGCTA-3', and the single-stranded DNA probe is complementary to miRNA-21, wherein the sequence of miRNA-21 is 5'-UAGCUUAUCAGACUGAUGUUGA-3' .
  • miRNA-21 is a recognized carcinogenic miRNA. According to literature reports, abnormally elevated miRNA-21 expression levels have been detected in a variety of tumor specimens and cell lines, including breast cancer, cervical cancer, lung cancer, pancreatic cancer, prostate cancer, colorectal cancer, glioma, and cholangiocarcinoma.
  • the sequence of the single-stranded DNA probe can also be complementary to other miRNA markers, such as Alzheimer's disease (AD) marker miRNA, tumor marker miRNA, and intervertebral disc degeneration (IDD) that have been reported in the literature.
  • Marker miRNA can also be plant miRNA, such as rice miRNA528. Relying on the same detection principle, that is, miRNA competes with the single-stranded DNA probe in a biosensor based on the fluorescence quenching of bismuthene nanosheets (fluorescein-single-stranded nucleic acid probe-bismuthene nanosheet complex), and the fluorescence is restored, Realize the qualitative or quantitative detection of miRNA.
  • the single-stranded nucleic acid probe may also be a single-stranded RNA probe.
  • optimizing the fluorescence detection system for example, by removing the RNase in the fluorescence detection system or adding a reagent that inhibits RNA degradation to the fluorescence detection system, it can also ensure that the single-stranded RNA probe and miRNA are combined into a stable double-stranded structure to ensure fluorescent labeling
  • the single-stranded nucleic acid probes get rid of the constraints of bismuthene nanosheets and restore fluorescence intensity.
  • the length of the single-stranded DNA probe is 15 to 36 nt; more preferably, the length of the single-stranded DNA probe is 19 to 23 nt.
  • the single-stranded DNA probe binds to the miRNA to form a double-stranded structure. As the length of the single-stranded DNA probe increases, the pairing specificity of DNA-miRNA binding is stronger, that is, the detection accuracy is higher; however, the single-stranded length is too long DNA probes will prolong the DNA-miRNA binding time, which is not conducive to rapid detection.
  • the present invention has discovered through extensive explorations that a single-stranded DNA probe with a length of 15 to 36 nt can not only ensure rapid DNA-miRNA binding, but also ensure the specificity of DNA-miRNA binding. Sex. More preferably, when the length of the single-stranded DNA probe is 19 to 23 nt, it has a faster detection rate, while ensuring the high specificity of DNA-miRNA binding, which can well meet the clinical requirements for miRNA detection and be used in clinical practice. diagnosis.
  • a miRNA detection kit includes two parts, a fluorescently labeled single-stranded nucleic acid probe and a fluorescence quencher, wherein the single-stranded nucleic acid probe is complementary to a specific miRNA, and the fluorescence quencher contains bismuthene nanosheets.
  • the miRNA detection kit may include two parts of reagents, the first part of the reagent includes a certain concentration of fluorescently labeled single-stranded nucleic acid probe, and the second part of the reagent includes a certain concentration of fluorescence quencher.
  • the fluorescently labeled single-stranded nucleic acid probe can be incubated with the fluorescence quencher to form a fluorescein-single-stranded nucleic acid probe-bismuthene nanosheet complex, which is based on the fluorescence of the bismuthene nanosheet In the quenched biosensor, at this time, the fluorescence of the fluorescein-single-stranded nucleic acid probe-bismuthene nanosheet complex is quenched by the unique fluorescence quenching effect of the bismuthene nanosheet.
  • a specific miRNA or serum (or other sample solution containing miRNA) complementary to a single-stranded nucleic acid probe is added to the complex structure system, the miRNA and the bismuthene nanosheets compete to bind to the single-stranded nucleic acid probe, and the miRNA and the single-stranded nucleic acid probe The affinity of stranded nucleic acid probes is greater. miRNA and single-stranded nucleic acid probes combine to form a double-stranded DNA (dsDNA) structure. The formation of dsDNA weakens the adsorption force of bismuthene nanosheets and single-stranded nucleic acid probes.
  • dsDNA double-stranded DNA
  • Single-stranded nucleic acid probes (at this time miRNA and single-stranded nucleic acid probes)
  • the needles combine to form dsDNA) to get rid of the constraints of bismuthene nanosheets.
  • the fluorescence quenching effect of the bismuthene nanosheets is difficult to continue to play a role, and the composite structure system shows a certain degree of fluorescence recovery.
  • the degree of fluorescence recovery By detecting the degree of fluorescence recovery and detecting the concentration of specific miRNA or specific miRNA in serum, the function of clinical miRNA diagnosis can be realized.
  • the miRNA detection kit further includes a specific miRNA at a standard concentration.
  • the standard concentration of specific miRNA usually includes several concentration gradients of specific miRNA standard solutions.
  • the miRNA standard solution competes with single-stranded nucleic acid probes to detect the fluorescence quenching intensity generated by the competitive binding of the miRNA standard solution, and is produced based on the above-mentioned correlation detection results.
  • the standard line is used to calculate the concentration of specific miRNA in the sample in the later stage.
  • the specific miRNA at the standard concentration includes the specific miRNA at the following concentrations: 0 nM, 0.5 nM, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, and 10 nM.
  • concentrations 0 nM, 0.5 nM, 1 nM, 2 nM, 4 nM, 6 nM, 8 nM, and 10 nM.
  • the fluorescence recovery intensity also increases, that is, there is a positive correlation trend between miRNA concentration and fluorescence recovery intensity.
  • the increase in the concentration of a specific miRNA at the standard concentration is linearly positively correlated with the fluorescence recovery intensity.
  • the standard curve within this concentration range can be used for quantitative detection and calculation of sample miRNA.
  • the fluorescence quencher contains 1-500 ⁇ g/ml bismuthene nanosheets. More preferably, the fluorescence quencher contains 20-80 ⁇ g/ml bismuthene nanosheets. More preferably, the fluorescence quencher contains 50 ⁇ g/ml bismuthene nanosheets.
  • the fluorescence quencher at the above concentration can ensure that the fluorescently-labeled single-stranded nucleic acid probe is fully quenched, and the subsequent dsDNA structure is detached from the surface of the bismuthene nanosheet to achieve fluorescence recovery.
  • the fluorescence quencher needs to ensure that the fluorescently labeled single-stranded nucleic acid probe is fully quenched, so the concentration of the fluorescence quencher also determines the concentration of the single-stranded nucleic acid probe.
  • concentration of the fluorescence quencher also determines the concentration of the single-stranded nucleic acid probe.
  • different types of miRNA detection kits can be selected based on the approximate concentration range of the specific miRNA in the sample to be tested.
  • the commercially available bulk bismuth was treated by a sonochemical method, and a few-layer bismuthene nanosheets were prepared by a top-down liquid phase peeling method, and the morphology and chemical composition of the prepared bismuthene nanosheets were characterized.
  • Liquid-phase ultrasonic treatment is an effective method to break the weak van der Waals force to prepare multilayer bismuth.
  • the ultra-thin bismuth nanosheets with good dispersion were successfully prepared using the ultrasonic liquid phase peeling method of the probe. Specifically, 30 mg of bismuth powder was ground and added to the solvent (300 ml). The bismuth powder solution was ultrasonically treated in an ice bath with a power of 420W for 6h. Then, the solution was treated by ultrasound with a 1080W probe for 24h. Subsequently, the solution was centrifuged at 7000 rpm for 30 min. Finally, collect the bismuthene nanosheets. The obtained bismuthene nanosheets are dispersed in the above-mentioned solvent and stored in a refrigerator at 4° C. for later use.
  • the solvent can be selected as a combination of N-methyl-2-pyrrolidone (NMP) solution, ethanol, and isopropanol, in different proportions. In this embodiment, NMP is selected.
  • the bismuthene nanosheets prepared above were characterized by atomic force microscopy (AFM), high-resolution transmission electron microscopy (HRTEM), XRD spectroscopy and Raman spectroscopy.
  • the atomic force microscope (AFM) morphology of a typical bismuthene nanosheet is shown in Figure 1 b.
  • the overall lateral size of the bismuthene nanosheets is greater than 800 nm and the thickness is about 1 nm, indicating that the thickness of the bismuthene nanosheets is two layers.
  • FIG 1 c the structure of the bismuthene nanosheets was confirmed by high-resolution transmission electron microscopy (HRTEM).
  • the well-resolved crystal lattices show their single crystal properties.
  • the lattice spacing of 0.328nm can match the (012) crystal plane of bismuth, which is consistent with the XRD spectrum.
  • the XRD spectrum was measured as shown in Figure 1 d.
  • the XRD diffraction peaks of the few-layer bismuthene nanosheets are located at 22.4°, 27.1°, 37.9°, 39.6°, 45.6°, 48.7°, 56° and 59.3°, which correspond to the (003), (012), and (012) of the bismuthene nanosheets, respectively.
  • fluorescein imide is used as the popular fluorescein label of the molecular beacon to label the probe ssDNA.
  • FAM fluorescein imide
  • Figure 2 a shows the normalized absorption spectra of FAM-ssDNA added with different concentrations of bismuth (0, 50 ⁇ g/ml and 100 ⁇ g/ml). All spectra are corrected for the background absorption of bismuth. Compared with FAM-ssDNA, after adding bismuth solution (50 ⁇ g/ml), peaks of 2.72eV and 2.57eV were observed, and a new band appeared at 2.49eV. When the concentration of bismuthene nanosheets was further increased to 100 ⁇ g/ml, the position of the blue peak changed from 2.49eV to 2.41eV, and the two substances in the mixed solution had the same absorption rate.
  • FIG. 2 b shows the normalized fluorescence spectrum of the sample used for absorption measurement in Figure 2 a.
  • the sample FAM-ssDNA showed an emission peak at 2.40eV.
  • a significant shift (0.09 eV) was observed in the emission spectrum, which clearly indicates the weak fluorescent nature of the charge transfer complex.
  • This embodiment uses the femtosecond pump detection technology to obtain the femtosecond transient absorption spectrum of the bismuthene nanosheets, revealing the fluorescence quenching mechanism of the bismuthene nanosheets, that is, the fluorescence charge transfer process with a weak ground state.
  • Fig. 3 e shows a two-dimensional graph of the transient absorption change of FAM–ssDNA in the absence and presence of bismuth nanosheets.
  • the graph is a function of wavelength and probe delay up to a 4ns time window.
  • the change and span of the color in the vertical direction represent the transient absorption signal intensity and dynamic response of the sample, respectively.
  • the lower ⁇ A value of the FAM-ssDNA-bismuth complex may be due to (i) the linear absorption of the bismuthene nanosheets in the mixed solution and (ii) the quenching of the excited state in the laser pulse.
  • the dynamic response of the complex in all wavelength ranges is much longer than that of FAM-ssDNA.
  • the large dynamic response of FAM-ssDNA is an inherent characteristic of the charge transfer complex. Therefore, even in the presence of bismuth atoms, the energy transfer process that shortens the decay dynamic response of FAM-ssDNA is not obvious.
  • the weak ground state fluorescent charge transfer complex is the main contribution to the strong suppression (ie fluorescence quenching) of FAM-ssdDNA fluorescence.
  • the dye-labeled ssDNA probe can be adsorbed on the surface of the bismuth nanosheet through the van der Waals force between the nucleobase and the base surface of the bismuth nanosheet.
  • the present invention anticipates that because the bismuthene nanosheets are used as high-efficiency quenchers, the dye-labeled ssDNA can be completely quenched on the bismuthene nanosheets.
  • the sequence of ssDNA and the sequence of the target miRNA (miRNA-21) are shown in Table 1. In view of the presence of the target miRNA, the formation of dsDNA weakens the interaction between the dye-labeled DNA probes on the bismuth surface, resulting in the recovery of fluorescence.
  • FIG. 3 b depicts a fluorescence microscope image of a 10 -6 M FAM-ssDNA probe solution.
  • the picture in c in Figure 3 shows that once the bismuthene nanosheets are added, the fluorescence of the FAM-ssDNA probe solution will be quenched sharply, which proves that the bismuth nanosheets have excellent quenching ability.
  • fluorescence recovery was found under the added target miRNA-21, which was mainly attributed to the complementary binding between FAM-ssDNA and target miRNA-21, which was then desorbed from the bismuth nanosheet.
  • FAM-labeled ssDNA shows strong emission at 525nm wavelength. After adding bismuth nanosheets, the fluorescence of the probe is quenched to almost insignificant. Due to the weak interaction between dsDNA and bismuth nanosheets, the fluorescence intensity is greatly increased to 400 (red curve).
  • f shows the changes in the fluorescence spectrum of FAM-ssDNA after adding miRNA-21 at different concentrations from 0 to 500 nM. As the concentration of miRNA-21 increases, the fluorescence intensity gradually increases.
  • g in Figure 3 shows a comparison of the fluorescence recovery response of miRNA-21 with a base match and a base mismatch. As the concentration of miRNA-21 increased, it was significantly enhanced, while the response of miRNA-21 mismatched with one base did not change significantly.
  • the enlarged view of fluorescence recovery in g in Figure 3 shows that as the concentration of miRNA-21 increases linearly in the range of 0 to 10 nM (inset, g in Figure 3), the standard curve within this concentration range can be well applied to sample miRNA -21 quantitative detection. The limit detection is 60pM (3 times the signal-to-noise ratio).
  • the biosensor based on the fluorescence quenching of bismuthene nanosheets can be well applied to the detection of sample miRNAs, with extremely high detection limits, capable of detecting miRNAs with a concentration as low as 60pM; With high detection specificity, it can distinguish single-base mismatches; at the same time, the biosensor based on bismuthene nanosheet fluorescence quenching also has the advantages of simple operation, fast detection speed, and less sample demand.

Abstract

本发明提供一种基于铋烯纳米片荧光淬灭的生物传感器，包括荧光标记的单链核酸探针及铋烯纳米片，所述单链核酸探针与特定miRNA互补，所述单链核酸探针吸附于铋烯纳米片表面；所述单链核酸探针吸附于铋烯纳米片表面时，荧光淬灭；所述特定miRNA竞争结合单链核酸探针时，荧光恢复。

Description

一种基于铋烯纳米片荧光淬灭的生物传感器、miRNA检测试剂盒及应用
本发明要求2019年12月13日递交的发明名称为“一种基于铋烯纳米片荧光淬灭的生物传感器、miRNA检测试剂盒及应用”的申请号201911283041.5的在先申请优先权，上述在先申请的内容以引入的方式并入本文本中。
技术领域
本发明涉及生物医学和分析化学技术领域，具体涉及一种用于临床诊断的试剂，更具体涉及一种基于铋烯纳米片荧光淬灭的生物传感器，以及包括该基于铋烯纳米片荧光淬灭的生物传感器的miRNA检测试剂盒，本发明还涉及该基于铋烯纳米片荧光淬灭的生物传感器在检测miRNA上的应用。
背景技术
MicroRNA(miRNA)是一种机体内源性表达的单链小分子RNA，位于基因组非编码区，本身不具有开放阅读框(open reading frame，ORF)，具有高度保守性，时序性和组织特异性。miRNA广泛存在于各种真核细胞中，不编码任何蛋白质，长度仅为20～24nt。成熟的miRNA5’端有一个磷酸基团，3’端为羟基，由具有发夹状结构的约70～90nt的单链RNA前体经过Dicer酶加工后形成。成熟的miRNA形成RNA诱导的基因沉默复合体(RNA-induced silencing complex，RISC)作用于靶点mRNA，通过对mRNA剪切或抑制其翻译过程而调控基因的表达。目前人们还不明确miRNA及其靶mRNA之间的作用机制。
最近有研究指出，miRNA在细胞生长、发育、分化、死亡等生物过程中起着重要的作用，同时，miRNA参与了血细胞生成、胰岛素分泌、神经系统构成和人类癌细胞生长等不同的过程，因此，非常有必要开发出有效的检测工具来测定miRNA。目前检测miRNA的方法主要有Northern印迹分析，微点阵(microarray)分析和实时定量PCR(quantitative Real-Time PCR)。Northern印迹分析的不足之处在于：Northern分析的过程涉及大量人工操作，对检验人员的技术要求较高，并且每次仅有一条miRNA探针与一个RNA印迹杂交，因此，它不适用于大规模的临床筛选检测。微点阵分析的不足之处在于：需要足够的RNA样本，无法区分差异很小的miRNA，检测分辨率低，难以达到临床检测要求。实时定量PCR需要进行RNA反转录及后续扩增，往往只能进行相对定量或者定性分析，难以达到临床绝对定量检测要求。
基于现有的miRNA检测的局限性，开发出一种新型的miRNA检测方法、检测试剂以及配套的检测设备，成为当下临床miRNA检测研究的一大热点。荧光光谱法是一种快速、实时的样品方法，其具有其它方法无法比拟的精准定量优势。不幸的是，众所周知，找到理想的荧光淬灭剂非常困难，而且需要进一步通过超快光学和分子电子学研究确定荧光淬灭剂的淬灭机理，研究工作的难度不言而喻。
目前，二维材料(2D Nanosheet)已在生物医学、电子、环境和能源应用中引起了广泛 关注。近年来，对生物传感器应用的2D材料的研究最多的是石墨烯、黑磷(BP)、二维过度金属硫化物(TMDs)、三族硫族化物等。VA族元素被称为“光子原”，为单元素烯应用打开了一个新的未开发区域。尽管VA族中的几种2D材料(例如BP，砷和锑)已应用于生物医学和分析化学领域，但应用于生物传感领域仍是挑战。作为具有强自旋轨道耦合的VA族中最重的原子量，铋具有出色的金属和拓扑性质，例如快速的载流子迁移，出色的生物相容性，强的光-材料相互作用，化学和热稳定性能，这在生物传感器中表现出巨大的应用前景。用荧光探针检测miRNA的关键在于荧光淬灭机制，包括
Figure PCTCN2020124037-appb-000001
共振能量转移(FRET)和电荷转移(CT)。迫切需要找到一种能够淬灭染料分子的荧光以检测miRNA，并最终实现miRNA定性和定量检测的2D纳米材料。
发明内容
有鉴于此，本发明提供了一种基于铋烯纳米片荧光淬灭的生物传感器，本发明还提供了一种包括上述基于铋烯纳米片荧光淬灭的生物传感器的miRNA检测试剂盒，本发明还提供了上述基于铋烯纳米片荧光淬灭的生物传感器在miRNA检测方面的应用。本发明基于铋烯纳米片的荧光淬灭效果，实现对特定miRNA的高分辨率检测，包括定性检测及定量检测，应用于体外临床诊断上，例如阿兹海默症(AD)的临床诊断、肿瘤的临床诊断等，具有巨大的应用前景及市场价值。
第一方面，本发明提供了一种基于铋烯纳米片荧光淬灭的生物传感器，以解决现有的miRNA检测方法存在的操作繁琐、RNA样本需求量大、无法区分差异很小的miRNA、检测分辨率低等问题。
一种基于铋烯纳米片荧光淬灭的生物传感器，包括荧光标记的单链核酸探针及铋烯纳米片，所述单链核酸探针与特定miRNA互补，所述单链核酸探针吸附于铋烯纳米片表面；
所述单链核酸探针吸附于铋烯纳米片表面时，荧光淬灭；
所述特定miRNA竞争结合单链核酸探针时，荧光恢复。
本发明一具体实施方式中，所述铋烯纳米片的横向尺寸大于30nm，厚度为0.4～20nm。
优选地，所述铋烯纳米片的XRD衍射峰位于22.4°，27.1°，37.9°，39.6°，45.6°，48.7°，56°和59.3°，分别对应铋纳米晶的(003)，(012)，(104)，(110)，(006)，(202)，(024)，(107)晶面(JCPDS No.44-1246)。
优选地，所述铋烯纳米片的Raman光谱在65.6cm -1和91cm -1处分别表现出振动峰。
优选地，所述荧光标记的单链核酸探针为荧光素亚酰胺(FAM)标记的单链核酸探针。
本发明另一具体实施方式中，所述荧光标记的单链核酸探针还可以为异硫氰酸荧光素(FITC)、四氯荧光素(TET)、六氯荧光素(HEX)、羧基四甲基罗丹明(TAMRA)等常见用于标记核苷酸探针的荧光素分子标记的单链核酸探针。
优选地，所述单链核酸探针为单链DNA探针。
在其他可选的实施方式中，所述单链核酸探针还可以为单链RNA探针。
优选地，所述单链DNA探针的序列为5’-TCAACATCAGTCTGATAAGCTA-3’，该单链DNA探针与miRNA-21互补。
在其他可选的实施方式中，所述单链DNA探针的序列还可以与其它miRNA标志物互补，例如文献已经报道的阿兹海默症(AD)标志物miRNA、肿瘤标志物miRNA、椎间盘退变(IDD)标志物miRNA；还可以是植物miRNA，例如可以是水稻miRNA528。
在其他可选的实施方式中，所述单链DNA探针的长度为15～36nt；更优选地，所述单链DNA探针的长度为19～23nt。
本发明第一方面所述的一种基于铋烯纳米片荧光淬灭的生物传感器，该基于铋烯纳米片荧光淬灭的生物传感器为一种特殊的复合物结构：荧光素-单链核酸探针-铋烯纳米片，即荧光标记的单链核酸探针通过分子间作用力(范德华力)吸附于铋烯纳米片表面，由此结合成该特殊的复合物结构。检测原理为：借助于铋烯纳米片独特的荧光淬灭效果，使得该荧光素-单链核酸探针-铋烯纳米片复合物荧光淬灭，当向该复合物结构体系中添加与单链核酸探针互补的特定miRNA或者血清(或者是其它包含miRNA样本液)时，miRNA与铋烯纳米片竞争结合单链核酸探针，且miRNA与单链核酸探针的亲和力更大。铋的显著淬灭作用将铋与染料分子之间的基态弱荧光电荷转移归因于铋，利用该机制，铋纳米材料被用于显示荧光淬灭剂。miRNA与单链核酸探针结合形成双链DNA(dsDNA)结构，dsDNA的形成削弱了铋烯纳米片与单链核酸探针的吸附力，单链核酸探针(此时miRNA与单链核酸探针结合形成dsDNA)摆脱铋烯纳米片的束缚。脱离铋烯纳米片后，铋烯纳米片的荧光淬灭效应难以继续发挥作用，该复合物结构体系出现一定程度的荧光恢复。通过检测荧光恢复程度，检测特定miRNA或者血清中特定miRNA的浓度，实现临床诊断的功能。本发明基于铋烯纳米片荧光淬灭的生物传感器用于检测特定miRNA具有检出限极高，能够检出浓度低至60pM的miRNA；还具有极高的检测特异性，能够区分单碱基错配的情况；同时该基于铋烯纳米片荧光淬灭的生物传感器还具有操作简单、检测速度快、样本需求量少等优势。
第二方面，本发明还提供了一种miRNA检测试剂盒，以解决现有miRNA检测试剂盒存在的检测操作繁琐、RNA样本需求量大、检出限低、无法区分差异很小的miRNA、检测分辨率低等问题。
一种miRNA检测试剂盒，包括荧光标记的单链核酸探针及荧光淬灭剂，所述单链核酸探针与特定miRNA互补，所述荧光淬灭剂包含铋烯纳米片；
所述荧光淬灭剂结合单链核酸探针时，荧光淬灭；
所述特定miRNA竞争结合单链核酸探针时，荧光恢复。
优选地，还包括标准浓度的特定miRNA；
所述标准浓度的特定miRNA通过竞争结合单链核酸探针，用于测定荧光恢复强度及制作标准线。
优选地，所述标准浓度的特定miRNA包括以下浓度的特定miRNA：0nM、0.5nM、1nM、2nM、4nM、6nM、8nM和10nM。
优选地，所述荧光淬灭剂包含1～500μg/ml铋烯纳米片。更优选地，所述荧光淬灭剂包含20～80μg/ml铋烯纳米片。更优选地，所述荧光淬灭剂包含50μg/ml铋烯纳米片。
本发明第二方面所述的一种miRNA检测试剂盒，其包括荧光标记的单链核酸探针及荧光淬灭剂。荧光标记的单链核酸探针具有荧光效应，即接受一定激发光照射时，发出相对应的荧光。当将荧光标记的单链核酸探针与荧光淬灭剂一起孵育时候，荧光标记的单链核酸探针借助于范德华力吸附于铋烯纳米片表面，形成荧光素-单链核酸探针-铋烯纳米片复合物。借助于铋烯纳米片的淬灭效应，荧光标记的单链核酸探针产生荧光淬灭。当向该复合物结构体系中添加与单链核酸探针互补的miRNA或者血清(或者是其它包含miRNA样本液)时，miRNA与铋烯纳米片竞争结合单链核酸探针，miRNA与单链核酸探针结合形成dsDNA结构，dsDNA的形成削弱了铋烯纳米片与单链核酸探针的吸附力，单链核酸探针(此时miRNA与单链核酸探针结合形成dsDNA)摆脱铋烯纳米片的束缚，出现一定程度的荧光恢复。通过检测荧光恢复程度，经检测特定miRNA或者血清中特定miRNA的浓度，实现临床诊断的功能。本发明miRNA检测试剂盒具有检出限极高，能够检出60pM的miRNA；还具有极高的检测特异性，能够区分单碱基错配的情况；同时该miRNA检测试剂盒还具有操作简单、检测速度快、样本需求量少等优势。
第三方面，本发明还提供了一种基于铋烯纳米片荧光淬灭的生物传感器在检测miRNA上的应用，以解决现有miRNA检测技术存在的检测操作繁琐、RNA样本需求量大、检出限低、无法区分差异很小的miRNA、检测分辨率低等问题。
一种基于铋烯纳米片荧光淬灭的生物传感器在检测miRNA上的应用。
本发明基于铋烯纳米片荧光淬灭的生物传感器在检测miRNA上的应用，通过该基于铋烯纳米片荧光淬灭的生物传感器制备出荧光淬灭型miRNA检测试剂盒，并最终应用于miRNA的定性或者定量检测上，具有检出限极高，能够检出60pM的miRNA；还具有极高的检测特异性，能够区分单碱基错配的情况。同时，应用基于铋烯纳米片荧光淬灭的生物传感器检测miRNA还具有操作简单，对操作人员的技术要求不高，检测速度快，添加检测样本后即可进行荧光恢复测量并通过计算机自动计算、输出miRNA水平。另外，该基于铋烯纳米片荧光淬灭的生物传感器检测miRNA时样本需求量少，无需扩增即可通过核酸分子间形成双链结构实现绝对定量。
本发明的优点将会在下面的说明书中部分阐明，一部分根据说明书是显而易见的，或者可以通过本发明实施例的实施而获知。
附图说明
为更清楚地阐述本发明的内容，下面结合附图与具体实施例来对其进行详细说明。
图1为本发明铋烯纳米片的制备和表征图。a)铋纳米片的制备过程示意图；I，通过探头超声处理铋溶液；II，将溶液离心，并收集含有铋的上清液。b)AFM形貌显示云母上有少层铋烯纳米片。c)少层铋烯纳米片的FFT屏蔽HRTEM图像的傅里叶转换(FFT)图。d)铋烯纳米片的XRD谱(上方线条为铋烯纳米片)。e)少层铋烯纳米片的拉曼光谱，E g(双重简并的面内振动模式)和A 1g(双重简并的面外振动模式)这两个峰代表两种不同的振动模式。
图2为本发明铋烯纳米片的光谱测试图。a)铋浓度为0μg/ml(最右侧)，50μg/ml (中间)和100μg/ml(最左侧)的FAM-ssDNA溶液的线性归一化吸收光谱。b)FAM-ssDNA的典型归一化荧光光谱，其中铋浓度为0μg/ml(最右侧)，50μg/ml(中间)和100μg/ml(最左侧)。c)在泵浦探测发射波长为2.29eV的条件下，FAM-ssDNS和铋烯/FAM-ssDNA的光激发载流子动力学曲线。两条衰减曲线均具有双指数衰减函数规律(下方为0μg/ml，上方为100μg/ml)。d)FAM–ssDNA和铋烯/FAM–ssDNA在各种探针上的瞬态吸收光谱(上部图片的最右侧，从上往下分别对应线条：-0.24ps、500ps、100ps、20ps、0.8ps；下部图片的最右侧，从上往下分别对应线条：-0.7ps、0.8ps、20ps、100ps、500ps)。e)FAM-ssDNA和铋烯/FAM-ssDNA的瞬态吸收光谱的二维(mapping)图。f)铋烯/FAM-ssDNA复合物的示意图。
图3为本发明基于铋烯纳米片荧光淬灭的生物传感器的荧光淬灭试验效果图。a)基于铋烯纳米片的miRNA检测的示意图。b)在混合铋烯纳米片之前，FAM-ssDNA探针溶液(10-6M)的荧光图像。c)FAM-ssDNA探针溶液与铋烯纳米片混合的荧光图像。d)FAM-ssDNA与铋烯纳米片溶液与miRNA-21一起孵育的荧光图像。e)FAM-ssDNA探针(最上方)，FAM-ssDNA/miRNA-21(由上往下第二条线)，FAM-ssDNA/miRNA-21@铋烯纳米片(由上往下第三条线)，FAM-ssDNA@铋烯(由上往下第四条线)的荧光光谱。f)在铋烯纳米片溶液中存在不同浓度的miRNA-21时，FAM-ssDNA探针溶液的荧光光谱(由上往下十三条线对应的miRNA-21浓度递减)。g)荧光强度与miRNA-21/miRNA浓度不匹配之间的关系。每个点对应于具有指示的miRNA浓度的荧光强度。所有误差条是来自五个数据点的荧光强度的标准误差。
具体实施方式
以下所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。
在本专利中，我们使用铋烯纳米片解决了这一问题，并使用光致发光和光学泵浦探测光谱学阐明了淬灭机理。铋烯纳米片的显著淬灭作用归因于铋烯纳米片与染料分子之间的基态弱荧光电荷转移。利用该机制，铋烯纳米片被用于荧光淬灭剂，以检测肺癌相关的miRNA-21生物标记物，从而为miRNA传感提供快速灵敏且选择性的检测平台。
实施例1：基于铋烯纳米片荧光淬灭的生物传感器
一种基于铋烯纳米片荧光淬灭的生物传感器，包括荧光标记的单链核酸探针及铋烯纳米片，单链核酸探针借助于范德华力吸附于铋烯纳米片表面，形成独特的荧光素-单链核酸探针-铋烯纳米片复合物结构，其中，单链核酸探针与特定miRNA互补，用于与特定miRNA结合形成双链结构。
检测原理为：借助于铋烯纳米片独特的荧光淬灭效果，使得该荧光素-单链核酸探针-铋烯纳米片复合物荧光淬灭。铋的显著淬灭作用将铋与染料分子之间的基态弱荧光电荷转移归因于铋，利用该机制，铋纳米材料被用于显示荧光淬灭剂。当向该复合物结构体系中 添加与单链核酸探针互补的特定miRNA或者血清(或者是其它包含miRNA样本液)时，miRNA与铋烯纳米片竞争结合单链核酸探针，且miRNA与单链核酸探针的亲和力更大，单链核酸探针倾向于形成dsDNA结构。miRNA与单链核酸探针结合形成双链DNA(dsDNA)结构，dsDNA的形成削弱了铋烯纳米片与单链核酸探针的吸附力，单链核酸探针(此时miRNA与单链核酸探针结合形成dsDNA)摆脱铋烯纳米片的束缚。脱离铋烯纳米片后，铋烯纳米片的荧光淬灭效应难以继续发挥作用，该复合物结构体系出现一定程度的荧光恢复。通过检测荧光恢复程度，检测特定miRNA或者血清中特定miRNA的浓度，实现临床诊断的功能。本发明基于铋烯纳米片荧光淬灭的生物传感器用于检测特定miRNA具有检出限极高，能够检出浓度低至60pM的miRNA；还具有极高的检测特异性，能够区分单碱基错配的情况；同时该基于铋烯纳米片荧光淬灭的生物传感器还具有操作简单、检测速度快、样本需求量少等优势。
在具体的实施方式中，铋烯纳米片的横向尺寸大于30nm，厚度为0.4～20nm。该横向尺寸及厚度的铋烯纳米片能够保证单链核酸探针借助于范德华力吸附于铋烯纳米片表面，不至于因吸附力不够而使得单链核酸探针脱离铋烯纳米片，造成检测结果不准确。
在具体的实施方式中，铋烯纳米片的XRD衍射峰位于22.4°，27.1°，37.9°，39.6°，45.6°，48.7°，56°和59.3°，分别对应铋纳米晶的(003)，(012)，(104)，(110)，(006)，(202)，(024)，(107)晶面(JCPDS No.44-1246)。通过XRD表征方法，证实本发明制备出了铋烯纳米片，且后续通过铋烯纳米片起到荧光淬灭剂的作用。
在具体的实施方式中，铋烯纳米片的Raman光谱在65.6cm -1和91cm -1处分别表现出典型的峰，分别对应铋的Eg和Alg振动模式。借助于拉曼光谱表征，本发明制备出了铋烯纳米片，且后续通过铋烯纳米片起到荧光淬灭剂的作用。
在具体的实施方式中，荧光标记的单链核酸探针为荧光素亚酰胺(FAM)标记的单链核酸探针。FAM为常用的核酸探针，广泛应用于荧光标记检测，具有与氨基反应迅速、结合产物稳定且易于控制等优点。
在其他实施方式中，荧光标记的单链核酸探针还可以为异硫氰酸荧光素(FITC)、四氯荧光素(TET)、六氯荧光素(HEX)、羧基四甲基罗丹明(TAMRA)等常见用于标记核苷酸探针的荧光素分子标记的单链核酸探针，具有相似的荧光标记及荧光检测效果。
在具体的实施方式中，单链核酸探针为单链DNA(ssDNA)探针，ssDNA探针具有分子结构稳定，易于与miRNA结合形成稳定双链结构等优点，方便控制以及进行荧光检测。
在具体的实施方式中，单链DNA探针的序列为5’-TCAACATCAGTCTGATAAGCTA-3’，该单链DNA探针与miRNA-21互补，其中，miRNA-21的序列为5’-UAGCUUAUCAGACUGAUGUUGA-3’。miRNA-21是一个公认的致癌性miRNA。根据文献报道，多种肿瘤标本以及细胞系都检测出miRNA-21表达水平异常升高，包括乳腺癌、子宫颈癌、肺癌、胰腺癌、前列腺癌、大肠癌、胶质癌和胆管癌等。
在其他实施方式中，单链DNA探针的序列还可以与其它miRNA标志物互补，例如文献已经报道的阿兹海默症(AD)标志物miRNA、肿瘤标志物miRNA、椎间盘退变(IDD) 标志物miRNA；还可以是植物miRNA，例如可以是水稻miRNA528。借助于相同的检测原理，即miRNA竞争结合基于铋烯纳米片荧光淬灭的生物传感器(荧光素-单链核酸探针-铋烯纳米片复合物)中的单链DNA探针，荧光恢复，实现miRNA的定性或者定量检测。
在其他实施方式中，单链核酸探针还可以为单链RNA探针。通过优化荧光检测体系，例如通过去除荧光检测体系中的RNA酶或者向荧光检测体系中添加抑制RNA降解的试剂，同样能够保证单链RNA探针与miRNA结合成稳定的双链结构，确保荧光标记的单链核酸探针摆脱铋烯纳米片的束缚，恢复荧光强度。
在具体的实施方式中，单链DNA探针的长度为15～36nt；更优选地，单链DNA探针的长度为19～23nt。单链DNA探针与miRNA结合形成双链结构，随着单链DNA探针的长度增加，DNA-miRNA结合的配对特异性越强，即检测精准度越高；但是，长度过大的单链DNA探针会导致DNA-miRNA结合时间延长，不利于快速检测。鉴于miRNA的长度普遍分布范围为19～23nt，本发明通过大量的探索，发现长度为15～36nt的单链DNA探针既能保证DNA-miRNA快速结合，同时也能保证DNA-miRNA结合的特异性。更优选的，单链DNA探针的长度为19～23nt时，具有更快的检测速率，同时保证DNA-miRNA结合的高特异性，能够很好地满足临床上miRNA的检测要求，并用于临床诊断。
实施例2：miRNA检测试剂盒
一种miRNA检测试剂盒，包括荧光标记的单链核酸探针及荧光淬灭剂两部分，其中，单链核酸探针与特定miRNA互补，荧光淬灭剂包含铋烯纳米片。例如，miRNA检测试剂盒可以包括两部分试剂，第一部分试剂包括一定浓度的荧光标记的单链核酸探针，第二部分试剂包括一定浓度的荧光淬灭剂。
使用该miRNA检测试剂盒时，可以先将荧光标记的单链核酸探针与荧光淬灭剂一起孵育形成荧光素-单链核酸探针-铋烯纳米片复合物，即基于铋烯纳米片荧光淬灭的生物传感器，此时，借助于铋烯纳米片独特的荧光淬灭效果，使得该荧光素-单链核酸探针-铋烯纳米片复合物荧光淬灭。当向该复合物结构体系中添加与单链核酸探针互补的特定miRNA或者血清(或者是其它包含miRNA样本液)时，miRNA与铋烯纳米片竞争结合单链核酸探针，且miRNA与单链核酸探针的亲和力更大。miRNA与单链核酸探针结合形成双链DNA(dsDNA)结构，dsDNA的形成削弱了铋烯纳米片与单链核酸探针的吸附力，单链核酸探针(此时miRNA与单链核酸探针结合形成dsDNA)摆脱铋烯纳米片的束缚。脱离铋烯纳米片后，铋烯纳米片的荧光淬灭效应难以继续发挥作用，该复合物结构体系出现一定程度的荧光恢复。通过检测荧光恢复程度，检测特定miRNA或者血清中特定miRNA的浓度，实现临床miRNA诊断的功能。
在具体的实施方式中，该miRNA检测试剂盒还包括标准浓度的特定miRNA。标准浓度的特定miRNA通常包括若干浓度梯度的特定miRNA标准溶液，通过miRNA标准溶液竞争结合单链核酸探针，检测miRNA标准溶液竞争结合所产生的荧光淬灭强度，并基于上述关联检测结果制作制作标准线，用于后期计算样本中特定miRNA的浓度。
在具体的实施方式中，标准浓度的特定miRNA包括以下浓度的特定miRNA：0nM、 0.5nM、1nM、2nM、4nM、6nM、8nM和10nM。在本发明中，通过大量的标准浓度的特定miRNA检测试验发现：随着标准浓度的特定miRNA浓度增加，荧光恢复强度同样增加，即出现miRNA浓度与荧光恢复强度的正相关趋势，特别是，在0到10nM范围内，标准浓度的特定miRNA浓度增加与荧光恢复强度呈现线性正相关，该浓度范围内的标准曲线可以用来进行样本miRNA的定量检测及计算。
在具体的实施方式中，荧光淬灭剂包含1～500μg/ml铋烯纳米片。更优选地，荧光淬灭剂包含20～80μg/ml铋烯纳米片。更优选地，荧光淬灭剂包含50μg/ml铋烯纳米片。上述浓度下的荧光淬灭剂能够确保荧光标记的单链核酸探针充分淬灭，保证后续dsDNA结构均从铋烯纳米片表面脱离，实现荧光恢复。
在具体的实施方式中，荧光淬灭剂需要确保荧光标记的单链核酸探针充分淬灭，由此荧光淬灭剂的浓度同样决定了单链核酸探针的浓度。为保证待检测样本中miRNA充分竞争结合单链核酸探针，临床上可以基于待检测样本中特定miRNA的大概浓度范围选择不同型号(其对应的铋烯纳米片浓度不同)的miRNA检测试剂盒。
实施例3：铋烯纳米片的制备与表征
本实施例使用超声化学方法处理市售的块体铋，通过自上而下的液相剥离法制备少层铋烯纳米片，并对制备的铋烯纳米片进行了形态和化学成分的表征。
(1)铋烯纳米片的制备：液相超声处理是打破弱范德华力制备多层铋的有效方法。
如图1中a所示，使用探针超声液相剥离法成功制备了分散良好且超薄的铋纳米片。具体而言，将30mg铋粉末研磨后加入到溶剂(300ml)中。在冰浴中以420W的功率对铋粉末溶液进行超声处理6h。然后，通过1080W的探头超声对溶液处理24h。随后，将溶液在7000rpm下离心30min。最后，收集铋烯纳米片。得到的铋烯纳米片分散在上述溶剂中，存于4℃冰箱待用。在具体制备过程中，溶剂可以选为N-甲基-2-吡咯烷酮(NMP)溶液、乙醇、异丙醇中的一种或几种、不同比例的组合，本实施例中选用NMP。
(2)铋烯纳米片的表征
将上述制备的铋烯纳米片通过原子力显微镜(AFM)、高分辨率透射电子显微镜(HRTEM)、XRD光谱及拉曼光谱表征。
典型的铋烯纳米片的原子力显微镜(AFM)形貌如图1中b所示。铋烯纳米片的整体横向尺寸大于800nm，厚度约为1nm，表明了铋烯纳米片厚度为2层。如图1中c所示，通过高分辨率透射电子显微镜(HRTEM)证实了铋烯纳米片的结构。良好分辨的晶格显示出它们的单晶性质，晶格间距为0.328nm可以与铋的(012)晶面相匹配，与XRD光谱一致。为了进一步研究铋烯纳米片的晶体结构，如图1中d所示测量了XRD光谱。少层铋烯纳米片的XRD衍射峰位于22.4°，27.1°，37.9°，39.6°，45.6°，48.7°，56°和59.3°，分别对应铋烯纳米片的(003),(012),(104),(110),(006),(202),(024),(107)晶面(JCPDS No.44-1246)；图1中e中的拉曼光谱表明，铋烯纳米片在65.6cm -1和91cm -1处分别表现出典型的峰，分别对应铋的Eg和Alg振动模式。
(3)荧光素与铋烯纳米片之间的淬灭机制
为了更好地了解染料分子与铋纳米片之间的强相互作用，使用了荧光素亚酰胺(FAM)作为分子信标的流行荧光素标记，以标记探针ssDNA。我们进行了稳态荧光光谱和时间分辨荧光光谱的测量。
图2中a显示了添加了不同浓度的铋(0、50μg/ml和100μg/ml)的FAM-ssDNA的归一化吸收光谱。所有光谱都根据铋的背景吸收进行了校正。与FAM-ssDNA相比，在添加铋溶液(50μg/ml)后观察到2.72eV和2.57eV的峰，在2.49eV处出现新的谱带。铋烯纳米片浓度进一步提高到100μg/ml时，蓝色峰的位置从2.49eV变为2.41eV，混合溶液中的两种物质具有相同的吸收率。新吸收带的起源以及等渗点的出现表明由于基态电荷转移复合物，系统中形成了新物种(FAM-ssDNA-铋复合物，即本发明中的基于铋烯纳米片荧光淬灭的生物传感器)。图2中b显示了图2中a中用于吸收测量的样品的归一化荧光光谱。样品FAM-ssDNA在2.40eV处显示出发射峰。除了荧光强度的急剧抑制外，在发射光谱中观察到显著的位移(0.09eV)，这清楚地表明电荷转移复合物的弱荧光性质。
(4)少层铋烯纳米片的飞秒瞬态吸收光谱
本实施例利用飞秒泵浦探测技术，获取铋烯纳米片的飞秒瞬态吸收光谱，揭示了铋烯纳米片的荧光淬灭机理即基态弱的荧光电荷转移过程。
为了直接揭示FAM-ssDNA和铋烯纳米片的超快速动力学和电子转移，本发明使用泵浦探测光谱法测量了FAM-ssDNA和铋-FAM-ssDNA复合溶液的飞秒瞬态吸收光谱(图2中c)。与FAM-ssDNA相比(τ1＝3.7ps和τ2＝0.86ns)，铋-FAM-ssDNA复合物表现出更长的衰减动力学响应(τ1＝30ps和τ2＝5.20ns)。本发明比较了在不存在和存在铋的情况下，各种探针延迟下FAM-ssDNA的瞬态吸收光谱(图2中d)。本发明在FAM-ssDNA-铋复合物的瞬态吸收光谱中没有观察到任何明显的新谱带，表明没有激发态相互作用的证据。图3中e展示了在不存在和存在铋纳米片的情况下，FAM–ssDNA的瞬态吸收变化的二维图，该图是波长和长达4ns时间窗口的探针延迟的函数。颜色在垂直方向上的变化和跨度分别表示样品的瞬态吸收信号强度和动态响应。FAM-ssDNA-铋复合物的ΔA值较低可能是由于(i)混合溶液中铋烯纳米片的线性吸收大和(ii)激光脉冲中激发态的淬灭。但是，从图2中d和e中可以清楚地看出，该复合物在所有波长范围内的动态响应都比FAM-ssDNA长得多。铋烯纳米片存在下，FAM-ssDNA的大动态响应是电荷转移复合物的固有特性。因此，甚至在存在铋原子的情况下，使FAM-ssDNA的衰变动力响应变短的能量转移过程也不明显。最后，我们可以推断出，基态弱的荧光电荷转移复合物是对FAM-ssdDNA荧光的强烈抑制(即荧光淬灭)的主要贡献。
效果实施例
基于铋烯纳米片荧光淬灭生物传感器实现对miRNA的超灵敏定量和选择性检测的性能测试
如图3中a所示，染料标记的ssDNA探针可以通过核碱基与铋纳米片基面之间的范德华力吸附在铋烯纳米片的表面上。本发明预期由于铋烯纳米片作为高效淬灭剂，可以在铋烯纳米片上进行染料标记的ssDNA的完全荧光淬灭。ssDNA的序列以及目标miRNA (miRNA-21)的的序列如表1所示。鉴于目标miRNA的存在，dsDNA的形成削弱了铋表面上染料标记的DNA探针之间的相互作用，从而导致了荧光的恢复。
表1.实验中的寡核苷酸序列信息
Figure PCTCN2020124037-appb-000002
首先，为了验证上述传感策略的可行性，我们使用荧光显微镜在高浓度(10-6M)下进行了直接观察荧光图像。图3中b描绘了10 -6M FAM-ssDNA探针溶液的荧光显微镜图像。图3中c中的图片显示，一旦添加了铋烯纳米片，FAM-ssDNA探针溶液的荧光就会被急剧淬灭，这证明了铋纳米片具有出色的淬灭能力。从图3中d，在添加的目标miRNA-21下发现了荧光恢复，这主要归因于FAM-ssDNA与目标miRNA-21之间的互补结合，然后从铋纳米片解吸。
为了进一步验证观察结果，我们进行了图3中e所示的荧光光谱测量。FAM标记的ssDNA(探针)在525nm波长处显示强发射。添加铋纳米片后，探针的荧光淬灭至几乎不明显。由于dsDNA与铋纳米片之间的弱相互作用，荧光强度大大提高到400(红色曲线)。
为了确认该平台的敏感性，将不同浓度的miRNA-21与FAM-ssDNA探针杂交，然后与铋纳米片混合。图3中f显示了添加0至500nM不同浓度的miRNA-21后，FAM-ssDNA荧光光谱的变化。随着miRNA-21浓度的增加，荧光强度逐渐增加。
为了研究该平台的选择，图3中g显示了碱基匹配和一个碱基不匹配的miRNA-21的荧光恢复响应的比较。随着miRNA-21浓度的增加，它显着增强，而与一碱基错配的miRNA-21的响应没有明显变化。图3中g中荧光恢复的放大图显示，随着miRNA-21浓度在0到10nM范围内线性增加(插图，图3中g)，该浓度范围内的标准曲线可以很好地适用于样本miRNA-21的定量检测。极限检测为60pM(信噪比的3倍)。
上述效果实施例均直接证明了基于铋烯纳米片荧光淬灭的生物传感器能够很好地应用于样本miRNA的检测，具有检出限极高，能够检出浓度低至60pM的miRNA；还具有极高的检测特异性，能够区分单碱基错配的情况；同时该基于铋烯纳米片荧光淬灭的生物传感器还具有操作简单、检测速度快、样本需求量少等优势。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (12)

  1. 一种基于铋烯纳米片荧光淬灭的生物传感器，其特征在于，包括荧光标记的单链核酸探针及铋烯纳米片，所述单链核酸探针与特定miRNA互补，所述单链核酸探针吸附于铋烯纳米片表面；
    所述单链核酸探针吸附于铋烯纳米片表面时，荧光淬灭；
    所述特定miRNA竞争结合单链核酸探针时，荧光恢复。
  2. 如权利要求1所述的基于铋烯纳米片荧光淬灭的生物传感器，其特征在于，所述铋烯纳米片的横向尺寸大于30nm，厚度为0.4～20nm。
  3. 如权利要求1所述的基于铋烯纳米片荧光淬灭的生物传感器，其特征在于，所述铋烯纳米片的XRD衍射峰位于22.4°，27.1°，37.9°，39.6°，45.6°，48.7°，56°和59.3°。
  4. 如权利要求1所述的基于铋烯纳米片荧光淬灭的生物传感器，其特征在于，所述铋烯纳米片的Raman光谱在65.6cm -1和91cm -1处分别表现出振动峰。
  5. 如权利要求1所述的基于铋烯纳米片荧光淬灭的生物传感器，其特征在于，所述荧光标记的单链核酸探针为荧光素亚酰胺标记的单链核酸探针。
  6. 如权利要求1所述的基于铋烯纳米片荧光淬灭的生物传感器，其特征在于，所述单链核酸探针为单链DNA探针。
  7. 如权利要求6所述的基于铋烯纳米片荧光淬灭的生物传感器，其特征在于，所述单链DNA探针的序列为5’-TCA ACATCAGTCTGATAAGCTA-3’。
  8. 一种miRNA检测试剂盒，其特征在于，包括荧光标记的单链核酸探针及荧光淬灭剂，所述单链核酸探针与特定miRNA互补，所述荧光淬灭剂包含铋烯纳米片；
    所述荧光淬灭剂结合单链核酸探针时，荧光淬灭；
    所述特定miRNA竞争结合单链核酸探针时，荧光恢复。
  9. 如权利要求8所述的荧光淬灭型miRNA检测试剂盒，其特征在于，还包括标准浓度的特定miRNA；
    所述标准浓度的特定miRNA通过竞争结合单链核酸探针，用于测定荧光恢复强度及制作标准线。
  10. 如权利要求9所述的荧光淬灭型miRNA检测试剂盒，其特征在于，所述荧光淬灭剂包含1～500μg/ml铋烯纳米片。
  11. 如权利要求9所述的荧光淬灭型miRNA检测试剂盒，其特征在于，所述标准浓度的特定miRNA包括以下浓度的特定miRNA：0nM、0.5nM、1nM、2nM、4nM、6nM、8nM和10nM。
  12. 一种基于铋烯纳米片荧光淬灭的生物传感器在检测miRNA上的应用。
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