CN114755460A - MiRNA accurate addressing and ultra-sensitive detection equipment and method - Google Patents
MiRNA accurate addressing and ultra-sensitive detection equipment and method Download PDFInfo
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Abstract
The invention relates to a miRNA precise addressing and ultra-sensitive detection device and method, and the method comprises the following steps: preparing an AFM probe, wherein the AFM probe comprises an AFM tip and an AFM cantilever, and the AFM tip is obtained by depositing a two-dimensional film on the surface of a diamond containing an NV color center through an atomic layer; and positioning and detecting the target miRNA coupled magnetic ball by using the AFM probe in combination with a magnetic field module and a light path system, wherein the surface of the target miRNA coupled magnetic ball is modified with a DNA probe with a modifier and the target miRNA. According to the method, the ODMR spectrum is obtained according to the change of the magnetic field intensity through the magnetic effect between the AFM probe of the diamond NV color center and the magnetic ball, magnetic imaging is realized, the fluorescence intensity change spectrogram is obtained through fluorescence resonance energy transfer and fluorescence enhancement of the diamond NV color center and a modifier, and therefore accurate addressing and ultra-sensitive detection of in-vitro miRNA are realized through the cooperation of AFM and a light path system.
Description
Technical Field
The invention relates to the field of biological detection, in particular to miRNA (micro ribonucleic acid) accurate addressing and ultrasensitive detection equipment and a method.
Background
The clinical methods of labeling, diagnosis and monitoring of characteristic biomolecules (markers) have gradually become a new trend for early diagnosis of malignant tumors (cancers). The expression levels of miRNA in serum, saliva and urine have obvious corresponding relation with the occurrence and evolution process of cancer, the traditional miRNA detection methods such as Northern blot hybridization have the defects of time and labor consumption, low sensitivity, poor specificity, large sample demand, poor repeatability and the like, and at present, the innovation of high-level ultra-sensitive measurement of low-abundance miRNA and a corresponding detection strategy are still urgently needed for early diagnosis of cancer.
The NV color center of the diamond is the most common luminescent defect in the diamond and consists of a nitrogen atom substituted for carbon and a hole, and the energy level structure of the NV color center of the diamond shows that the ground state and the excited state are both spin tristates. At room temperature, the zero phonon line of the NV color center of the diamond is positioned near 637nm, the fluorescence is strongest at 700nm, and the absorption is strongest and is positioned in a green light wave band. The diamond NV color center is suitable for biological cell imaging and intracellular electromagnetic field and temperature measurement due to high stability and low cytotoxicity.
In the prior art, the patent with the application number of 202010224841.6 named as a scanning detection system based on a diamond NV color center improves the signal collection efficiency, but cannot realize real-time and dynamic detection of in-vitro miRNA information.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides miRNA accurate addressing and ultrasensitive detection equipment and a method, aiming at solving the problem of real-time and dynamic detection of in-vitro miRNA information.
The technical scheme adopted by the invention is as follows:
in one aspect, the present invention provides a miRNA precisely addressing and ultrasensitive detection apparatus, comprising: the device comprises an AFM system, a two-dimensional film generation system, an optical path system, a magnetic field module and a microwave system;
The AFM probe of the AFM system comprises an AFM tip and an AFM cantilever, wherein the AFM tip is obtained by depositing a two-dimensional film on the surface of a diamond containing an NV color center through an atomic layer; the AFM system is used for addressing a target miRNA coupling magnetic ball, namely a sample to be detected;
the two-dimensional film generation system is used for generating a two-dimensional film on the surface of the diamond containing the NV color center through atomic layer deposition;
the magnetic field module is used for applying a magnetic field to the sample detection area;
the optical path system and the AFM system are used in an integrated manner, the optical path system emits exciting light and collects and scans the NV color center of the diamond of the AFM tip of the sample to be detected and fluorescence signals of dye molecules and metal nano particles in the sample to be detected so as to realize signal collection;
the microwave system is used for modulating the signal so as to improve the detection precision;
based on the principle of Fluorescence Resonance Energy Transfer (FRET) and fluorescence Metal Enhancement (MEF), all systems are used in a linkage manner, AFM detection is realized simultaneously in the magnetic imaging and fluorescence detection processes of a sample to be detected, and dynamic information acquisition of the sample to be detected is realized, so that the aim of realizing accurate addressing and ultrasensitive detection of one or more miRNAs in vitro by using quantum characteristic regulation and control of diamond NV color centers is fulfilled.
The further technical scheme is as follows:
each system configuration includes:
the optical path system includes: a laser, an avalanche photodiode, a photon counter, an Olympus inverted microscope, a spectrometer, a pinhole and a dichroic mirror;
the objective lens of the Olympus inverted microscope is inverted, a transparent sample stage is arranged above the inverted microscope, exciting light emitted by a laser is emitted to the surface of a sample to be detected through the objective lens, and an optical path system acquires an emission spectrum, a fluorescence life and magnetic resonance parameters of the sample to be detected through the objective lens;
the AFM system comprises an atomic force microscope which is a cantilever beam type;
the integrated use process of the optical system and the AFM system comprises the following steps: the transparent sample stage is arranged in the atomic force microscope chamber, when the light path system emits laser below a sample to be detected and collects signals, the AFM needle point scans the sample to be detected above the sample at the same time, and dynamic and real-time collection of NV color center fluorescence and magnetic resonance characteristic information of the diamond is realized by using the signals collected by the AFM system and the light path system;
the microwave system comprises a microwave source, a microwave switch, a high-power amplifier and a circulator; after the microwave system is connected and debugged, a microwave source generates a microwave field, the microwave field is capacitively coupled to the omega-type resonator to provide a uniform peak field measurement area, and the quantum characteristics of the NV color center of the diamond are modulated through fixed frequency to realize the initialization of the NV color center qubit and the specific detection and analysis;
The magnetic field module is a permanent magnet or an electromagnet, and the influence on the self-selection characteristic of the NV color center is realized by adjusting the direction and the size of a magnetic field and the distance between the magnetic field module and the NV color center so as to realize the magnetic imaging function;
the two-dimensional film generation system is an atomic layer deposition platform.
On the other hand, the invention provides a miRNA accurate addressing and ultrasensitive detection method, which comprises the following steps:
preparing an AFM probe, wherein the AFM probe comprises an AFM tip and an AFM cantilever, and the AFM tip is obtained by depositing a two-dimensional film on the surface of a diamond containing an NV color center through an atomic layer;
and positioning and detecting a target miRNA coupling magnetic sphere by using the AFM probe in combination with a magnetic field module and a light path system, wherein the surface of the target miRNA coupling magnetic sphere is modified with a DNA probe with a modifier and a target miRNA.
The further technical scheme is as follows:
the preparation of the AFM probe comprises the following steps:
a) etching the blocky diamond containing the NV color center by adopting a reactive ion etching process to obtain the diamond with the conical tip;
b) placing the diamond with the conical tip in an atomic layer deposition reaction cavity, and modifying a two-dimensional film on the surface;
c) processing the main body part at the rear end of the conical tip by adopting a diamond etching process to obtain a complete diamond AFM tip;
d) And assembling the diamond AFM tip and the AFM cantilever into a complete probe.
The thickness of the two-dimensional film is 0.5-100 nm.
The AFM probe is combined with a magnetic field module and a light path system to position and detect the target miRNA coupling magnetic ball, and the method comprises the following steps:
spin-coating the target miRNA coupling magnetic spheres on a to-be-detected sample area of a quartz glass slide;
the magnetic field module applies a magnetic field to the sample area to be tested, and the direction of the magnetic field is parallel to the NV axis;
the laser of the light path system generates exciting light to act on the NV color center of the AFM probe, the AFM probe is used for scanning the sample to be detected to obtain the magnetic field intensity of different position points, and the magnetic ball modified with miRNA is magnetically imaged through the change of the magnetic field intensity to realize the positioning of the miRNA coupling magnetic ball; meanwhile, the light path system collects fluorescence signals in the movement range of the AFM probe, and the fluorescence change in the movement range of the AFM probe is reflected in real time through the spectral scanning imaging function, so that the ultra-sensitive detection of miRNA is realized.
The AFM probe is combined with a magnetic field module and a light path system to position and detect the target miRNA coupling magnetic ball, and the method further comprises the following steps:
the signal is modulated and amplified using a microwave system.
Preparing a target miRNA coupling magnetic ball, comprising:
Preparing a magnetic sphere solution and a probe DNA solution, the surfaces of which are modified with streptavidin;
taking 5-10 mu l of the probe DNA solution, adding the probe DNA solution into 100-150 mu l of the magnetic ball solution, and incubating for 12-15h at room temperature; centrifugally washing the incubated solution by using a buffer solution to remove probe DNA which is not firmly connected to obtain a treated magnetic ball solution;
uniformly mixing 10-20 mu l of target miRNA solution with 100-150 mu 1 of the treated magnetosphere solution, incubating at room temperature, and centrifugally washing with buffer solution to remove redundant miRNA to obtain target miRNA coupled magnetospheres;
the two ends of the probe DNA are respectively modified with biotin and modifiers, and the modifiers comprise dye molecules and metal nanoparticles.
The magnetic ball solution is a solution containing magnetic balls with one or more diameters;
for the solution containing the magnetic spheres with various diameters, the probe DNA solution is a solution containing various DNAs, the target miRNA coupling magnetic spheres are a coupling magnetic sphere solution containing various target miRNAs, and the various DNAs are used for respectively and correspondingly mixing with the magnetic spheres with various diameters and respectively pairing with the various target miRNAs.
The invention has the following beneficial effects:
the invention realizes AFM detection in the process of fluorescence detection of the sample, and can realize dynamic information acquisition of the sample. The target miRNA is coupled with the magnetic ball surface, the DNA probe with the modifier and the target miRNA are modified on the surface of the magnetic ball, the AFM probe with the diamond NV color center obtains an ODMR spectrum according to the change of the magnetic field strength through the magnetic effect between the AFM probe and the magnetic ball, magnetic imaging is achieved, a fluorescence resonance energy transfer and fluorescence enhancement of the diamond NV color center and the modifier are obtained, a fluorescence intensity change spectrogram is obtained, and therefore ultra-sensitive detection and addressing of the miRNA in vitro are achieved through cooperation of the AFM and a light path system. The invention optimizes and controls the quantum characteristics of the NV color center AFM needle point of the diamond through the atomic layer deposition film, and improves the sensitivity of signal detection.
Drawings
Fig. 1 is a schematic structural diagram of a detection apparatus according to embodiment 1 of the present invention.
In the figure: 1. a microwave source; 2. a microwave switch; 3. a high power amplifier; 4. a circulator; 5. an omega-type resonator; 6. a tapered tip; 7. a body portion; 8. an atomic force microscope; 9. a transparent sample stage; 10. an olympus inverted microscope; 11. a laser; 12. a dichroic mirror; 13. a small hole; 14. a spectrometer; 15. an avalanche photodiode; 16. a photon counter; 17. a magnetic field module; 18. and (5) testing the sample to be tested.
Detailed Description
The following describes embodiments of the present invention with reference to the drawings.
The application provides a miRNA accurate addressing and super sensitive check out test set, includes: the device comprises an AFM system, a two-dimensional film generation system, a light path system, a magnetic field module and a microwave system;
the AFM probe of the AFM system comprises an AFM tip and an AFM cantilever, wherein the AFM tip is obtained by depositing a two-dimensional film on the surface of a diamond containing an NV color center through an atomic layer;
the two-dimensional film generation system is used for generating a two-dimensional film on the surface of the diamond containing the NV color center through atomic layer deposition;
the magnetic field module is used for applying a magnetic field to the sample detection area;
The optical path system is used for generating exciting light to act on an NV color center of an AFM needle point of a scanning sample, and acquiring signals to obtain an optical detection magnetic resonance spectrum;
the microwave system is used to modulate and amplify the signal.
The technical scheme of the miRNA accurate addressing and ultrasensitive detection device of the present application is further described in specific example 1 below.
Example 1
As shown in fig. 1, the detection apparatus includes: the device comprises an AFM system, an optical path system, a microwave system, a magnetic field module 17 and a two-dimensional film generation system. All systems are fused and integrated for use, and dynamic real-time signal acquisition of the sample is realized.
Wherein, the AFM system comprises an atomic force microscope 8 which is a cantilever type;
wherein, the optical path system includes: a laser 11, an avalanche photodiode 15, a photon counter 16, an olympus inverted microscope 10, a spectrometer 14, a pinhole 13, a dichroic mirror 12 and the like; the parts of the optical path system are sequentially connected and assembled, wherein an objective lens of an Olympus inverted microscope 10 is inverted, a transparent sample stage 9 is arranged above the objective lens, exciting light emitted by a laser 11 is emitted to the surface of a sample through the objective lens, and the optical path system acquires an emission spectrum, a fluorescence life and magnetic resonance parameters of the sample through the objective lens;
The microwave system consists of a microwave source 1, a microwave switch 2, a high-power amplifier 3 and a circulator 4; after the microwave system is sequentially connected and debugged, a microwave field is generated by a microwave source 1, the microwave field is capacitively coupled to an omega-type resonator 5 to provide a uniform peak field measurement area, and the fluorescence of the NV color center of the diamond is modulated by fixed frequency to realize the initialization of the quantum bit of the NV color center and the specific detection and analysis;
the magnetic field module 17 is a permanent magnet or an electromagnet, and the influence of the magnetic field module on the self-selection characteristic of the NV color center is realized by adjusting the direction and the size of the magnetic field, the distance between the module and the NV color center and the like, so that the magnetic imaging function is realized.
The two-dimensional film generation system is an atomic layer deposition platform;
wherein, the integrated use process of optical system and AFM system: the sample stage 9 is arranged in a cavity of the atomic force microscope 8, a sample area to be detected is arranged on the sample stage 9, a sample 18 to be detected is coated in the sample area to be detected, the light path system emits laser below the sample, the AFM needle point scans the sample above the sample, the light path system collects signals based on Fluorescence Resonance Energy Transfer (FRET) and fluorescence Metal Enhancement (MEF) principles, and the microwave system realizes amplification and modulation of the signals, so that the NV color center fluorescence and magnetic resonance characteristic information of the diamond can be dynamically and real-timely collected through linkage use of the AFM system, the light path system and other systems.
Addressing the miRNA coupling magnetic ball, namely the sample 18 to be detected, by an AFM system; the light path system emits exciting light and collects fluorescence signals of a diamond NV color center of the AFM needle point, dye molecules in a sample and metal nano particles to realize signal collection; the microwave system realizes signal modulation; based on the principle of Fluorescence Resonance Energy Transfer (FRET) and fluorescence Metal Enhancement (MEF), AFM detection is realized simultaneously in the processes of magnetic imaging and fluorescence detection of a sample by using all systems in a linkage manner, and dynamic information acquisition of the sample can be realized, so that the aims of realizing accurate addressing and ultrasensitive detection of one or more miRNAs in vitro by using quantum characteristic regulation and control of diamond NV color centers are fulfilled.
The application also provides a miRNA accurate addressing and ultrasensitive detection method, which comprises the following steps:
preparing an AFM probe, wherein the AFM probe comprises an AFM tip and an AFM cantilever, and the AFM tip is obtained by depositing a two-dimensional film on the surface of a diamond containing an NV color center through an atomic layer;
and positioning and detecting a target miRNA coupled magnetic ball by using the AFM probe in combination with a magnetic field module and a light path system, wherein the surface of the target miRNA coupled magnetic ball is modified with a DNA probe with a modifier and a target miRNA.
Wherein, the preparation of the AFM probe comprises the following steps:
a) etching a blocky diamond containing NV color centers by adopting a reactive ion etching process to obtain a diamond with a conical tip;
b) placing the diamond with the conical tip in an atomic layer deposition reaction cavity, and modifying a two-dimensional film on the surface;
c) processing the main body part at the rear end of the conical tip by adopting a diamond etching process to obtain a complete diamond AFM tip;
d) and assembling the diamond AFM tip and the AFM cantilever into a complete probe.
Wherein the thickness of the two-dimensional film is 0.5-100 nm.
According to the scheme, the diamond tip containing the NV color center is subjected to two-dimensional thin film modification, so that the quantum characteristics of the diamond tip are further regulated and controlled. By adjusting the thickness of the two-dimensional film, the fluorescence signal of the NV color center of the diamond can be adjusted in three aspects of the depth, the energy transfer and the thickness of the protective layer of the NV color center, so that the transverse relaxation time and the longitudinal relaxation time of the NV color center are improved, the fluorescence intensity and the service life of the NV color center are improved, the isolation degree between the NV color center and the external environment is improved, and the quantum coherence time and the fluorescence detection efficiency of the NV color center of the diamond are improved.
The AFM probe is combined with a magnetic field module and an optical path system to position and detect the target miRNA coupling magnetic ball, and the method comprises the following steps:
Spin-coating the target miRNA coupling magnetic spheres on a to-be-detected sample area of a quartz glass slide;
the magnetic field module applies a magnetic field to a sample area to be detected;
a laser of the light path system generates exciting light to act on an NV color center of an AFM probe, the AFM probe is used for scanning the sample to be detected to obtain the magnetic field intensity of different position points, and the magnetic ball modified with miRNA is magnetically imaged through the change of the magnetic field intensity to realize the positioning of the miRNA coupling magnetic ball; meanwhile, the fluorescence property excited by the NV color center is changed due to FRET and MEF between a modifier in a sample to be detected and the NV color center, a light path system collects a fluorescence signal in the movement range of the AFM probe, and the fluorescence change in the movement range of the AFM probe is reflected in real time through a spectrum scanning imaging function, so that the ultra-sensitive detection of miRNA is realized.
Optical detection magnetic resonance spectra can be obtained by the optical path system collecting the sample signals to indicate the additional zeeman splitting due to the local magnetic field of the magnetic nanoparticles.
Further comprising:
the signal is modulated and amplified using a microwave system.
The scheme realizes the precise addressing and the ultra-sensitive detection of the miRNA in vitro through the cooperation of magnetic imaging and fluorescent signal detection. And the AFM detection of the sample is ensured to be realized simultaneously in the fluorescence detection process, and the dynamic information acquisition of the sample can be realized.
The preparation method of the target miRNA coupled magnetic ball comprises the following steps:
preparing a magnetic sphere solution and a probe DNA solution, the surfaces of which are modified with streptavidin;
taking 5-10 mu l of the probe DNA solution, adding the probe DNA solution into 100-150 mu l of the magnetic ball solution, and incubating for 12-15h at room temperature; centrifugally washing the incubated solution by using a buffer solution to remove probe DNA which is not firmly connected to obtain a treated magnetic ball solution;
uniformly mixing 10-20 mu l of target miRNA solution with 100-150 mu 1 of the treated magnetosphere solution, incubating at room temperature, and centrifugally washing with buffer solution to remove redundant miRNA to obtain target miRNA coupled magnetospheres;
the two ends of the probe DNA are respectively modified with biotin and modifiers, and the modifiers comprise dye molecules and metal nanoparticles.
Wherein the magnetic sphere solution is a solution containing magnetic spheres with one or more diameters;
for the solution containing the magnetic spheres with various diameters, the probe DNA solution is a solution containing various DNAs, the target miRNA coupling magnetic spheres are a coupling magnetic sphere solution containing various target miRNAs, and the various DNAs are used for respectively and correspondingly mixing with the magnetic spheres with various diameters and respectively pairing with the various target miRNAs.
The target miRNA coupling magnetic ball prepared by the scheme has one or more miRNAs, and simultaneous identification and detection of the miRNAs are realized.
The technical scheme of the miRNA accurate addressing and ultrasensitive detection method of the present application is further described in specific example 2 and specific example 3 below.
Example 2
Firstly, preparing an AFM probe, which comprises the following specific steps:
1) etching a massive diamond containing NV color centers by utilizing a reactive ion etching process to obtain a conical tip 6, which specifically comprises the following steps:
depositing a SiNx layer on a diamond substrate by Plasma Enhanced Chemical Vapor Deposition (PECVD); then spin-coating a negative electron resist (HSQ) layer on the SiNx, wherein the HSQ layer is used for electron beam exposure; patterning using an Electron Beam Lithography (EBL) system, and then HSQ was dissolved away in 25% tetramethylammonium hydroxide (TMAH); etching the SiNx layer using a Reactive Ion Etching (RIE) system for pattern transfer; etching by Inductively Coupled Plasma (ICP) to obtain a corresponding nano-diamond structure, and finally etching by using an HF wet method to remove residues of HSQ or SiNx;
2) placing the blocky diamond with the conical tip 6 in an atomic layer deposition reaction cavity, and modifying a two-dimensional film on the surface of the blocky diamond;
3) obtaining a complete diamond AFM needle point (comprising a conical structure 6 and a main body part 7 extending from the rear end of the conical structure 6) by a diamond etching process;
4) And assembling the diamond NV color center tip modified by the two-dimensional film and the cantilever of the AFM probe into a complete probe by an AFM probe assembling process.
After the assembled AFM probe is installed, the AFM system is adjusted to the tapping mode for use.
Preparing a sample to be detected, namely a miRNA-155 coupling magnetic ball, and specifically comprising the following steps:
1) preparing a magnetic ball (the diameter is 200nm) the surface of which is modified with streptavidin, and probe DNA the two ends of which are respectively modified with biotin and gold nanoparticles, wherein the probe DNA is a straight chain;
2) adding 5 mul of probe DNA solution into 100 mul of magnetic ball solution, and incubating for 12h at room temperature;
3) centrifugation was carried out three times using TTL buffer (100mM Tris, 1M LiCl, 0.1% Tween-20, pH 8.0) at 4 ℃ to remove the weakly ligated probe DNA;
4) mixing 10 mu l of miRNA-155 solution to be detected with 100 mu l of magnetic sphere solution, incubating for 2h at room temperature, and centrifuging and washing for three times at 4 ℃ by using 0.01M PBS (pH 7.4) buffer solution to remove excessive miRNA-155, thereby obtaining the miRNA-155 coupled magnetic sphere.
Thirdly, detecting the sample, which comprises the following steps:
1) dripping 5 mul of miRNA-155 coupling magnetic ball solution on a quartz glass slide, and spin-coating at the rotating speed of 600rpm for 5 min;
2) placing the glass slide on a heating table at 30 ℃ and drying for 10 min;
3) Placing the glass slide on an object stage, firstly, modulating an objective lens by about 1mm from a sample, and focusing a light path system;
4) AFM adjusts the needle point to a proper position;
5) adjusting parameters of a magnetic field module to ensure that the direction of the magnetic field is parallel to an NV (non-volatile memory) axis and the magnetic field intensity is 55G;
6) opening a laser of the optical path system, selecting 510nm picosecond laser, and simultaneously starting AFM scanning;
7) in the process of scanning a sample to be detected by an AFM (atomic force microscope) needle point, a light path system collects corresponding fluorescence signals of a diamond NV (nitrogen-doped N-oxide) color center, dye molecules and metal nanoparticles to realize signal collection, and records key information of magnetic resonance parameters, fluorescence intensity, fluorescence life and the like of the sample, so that accurate addressing and ultra-sensitive detection of miRNA-155 are realized.
Specifically, a tapping mode of AFM is adopted to scan a sample area to be detected so as to obtain magnetic field strengths of different position points, and magnetic imaging is carried out on the magnetic ball modified with miRNA through the change of the magnetic field strengths; and further realizing the positioning of the miRNA coupling magnetic spheres.
Specifically, FRET and MEF between the decoration and the NV color center in the sample can cause the fluorescence property excited by the NV color center to change, the AFM needle point can change the fluorescence intensity due to the influence of the decoration at different positions, and the light path system can reflect the fluorescence change in the movement range of the AFM needle point in real time through the spectral scanning imaging function, so that the accurate positioning and the ultra-sensitive detection of miRNA can be realized.
Example 3
First, an AFM probe was prepared in the same manner as in example 2.
Secondly, preparing a sample to be detected, namely miRNA-155 and miRNA-182 coupling magnetic spheres, and specifically comprising the following steps:
1) preparing a probe DNA (linear chain and hairpin) with streptavidin-modified magnetic spheres (the diameters of 200nm and 300nm respectively) and biotin and fluorescent dye molecules modified at two ends respectively;
wherein the linear chain DNA correspondingly realizes base complementary pairing with miRNA-155, and the dye molecule modified at one end of the linear chain DNA is Cy 5;
the hairpin-shaped DNA and miRNA-182 realize base complementary pairing, and a dye molecule modified at one end of the hairpin-shaped DNA is Cy 3;
2) respectively taking 5 mu l of probe DNA solution, adding the probe DNA solution into 100 mu l of magnetic sphere solution, and incubating for 12h at room temperature;
3) using TTL buffer (100mM Tris, 1M LiCl, 0.1% Tween-20, pH 8.0) at 4 ℃ centrifugal washing three times to remove the connection of probe DNA;
4) mixing 10 mu l of miRNA-155 and miRNA-182 solution to be detected with 100 mu l of magnetic sphere solution respectively, incubating for 2h at room temperature, and centrifuging and washing for three times at 4 ℃ by using 0.01M PBS (pH 7.4) buffer solution to remove redundant miRNA-155 and miRNA-182, so as to obtain miRNA-155 and miRNA-182 coupling magnetic spheres;
5) and respectively taking 20 mul of miRNA-155 and miRNA-182 coupling magnetic sphere solution, and mixing to obtain miRNA-155 and miRNA-182 coupling magnetic sphere solution for later use.
Thirdly, detecting the sample, which comprises the following steps:
1) 5 mul of miRNA-155 and miRNA-182 coupling magnetic ball solution are dripped on a quartz glass slide, and spin-coating is carried out for 5min at the rotating speed of 600 rpm;
2) placing the glass slide on a heating table at 30 ℃ and drying for 10 min;
3) placing the glass slide on an object stage, firstly, modulating an objective lens by about 1mm from a sample, and focusing a light path system;
4) AFM adjusts the needle point to a proper position;
5) adjusting parameters of a magnetic field module to ensure that the direction of the magnetic field is parallel to an NV axis and the magnetic field intensity is 55G;
6) turning on a laser, selecting 510nm picosecond laser, and simultaneously starting AFM scanning;
7) in the AFM needle point scanning process, a light path system collects corresponding fluorescence signals of a diamond NV color center, dye molecules and metal nanoparticles to realize signal collection, key information such as magnetic resonance parameters, fluorescence intensity and fluorescence service life of a sample is recorded, different map information can be caused by magnetic spheres with different particle sizes, DNAs with different shapes and different dye molecules, and accurate addressing and ultra-sensitive detection of miRNA-155 and miRNA-182 are synchronously realized by analyzing the map information.
According to the method, AFM detection is realized in the sample fluorescence detection process, and dynamic information of the sample can be acquired. Modifying the surface of the magnetic ball with a DNA probe with a modifier and miRNA, obtaining an ODMR spectrum according to the change of the magnetic field intensity by the diamond NV color center probe of AFM and the magnetic ball through the magnetic effect, and realizing magnetic imaging; the diamond NV color center obtains a fluorescence intensity change spectrogram through fluorescence resonance energy transfer and fluorescence enhancement with a modifier, and the ultra-sensitive detection and addressing of the miRNA in vitro are realized through the synergistic effect of the AFM and the optical path system.
Those of ordinary skill in the art will understand that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. The utility model provides a miRNA accurate addressing and super sensitive check out test set which characterized in that includes: the device comprises an AFM system, a two-dimensional film generation system, an optical path system, a magnetic field module (17) and a microwave system;
the AFM probe of the AFM system comprises an AFM tip and an AFM cantilever, wherein the AFM tip is obtained by depositing a two-dimensional film on the surface of a diamond containing an NV color center through an atomic layer: the AFM system is used for addressing a target miRNA coupling magnetic ball, namely a sample to be detected;
the two-dimensional film generation system is used for generating a two-dimensional film on the surface of the diamond containing the NV color center through atomic layer deposition;
the magnetic field module is used for applying a magnetic field to the sample detection area;
The optical path system and the AFM system are used in an integrated mode, the optical path system emits exciting light and collects and scans diamond NV color centers of AFM tips of samples to be detected and fluorescence signals of dye molecules and metal nanoparticles in the samples to be detected to achieve signal collection;
the microwave system is used for modulating and amplifying the signal;
the systems are used in a linkage manner, AFM detection is realized simultaneously in the magnetic imaging and fluorescence detection processes of the sample to be detected, and dynamic information acquisition of the sample to be detected is realized, so that the aims of realizing precise addressing and ultra-sensitive detection of one or more miRNA in vitro by using quantum characteristic regulation and control of the diamond NV color center are fulfilled.
2. The miRNA precise addressing and ultrasensitive detection device according to claim 1, wherein each system has a structure of:
the optical path system includes: the device comprises a laser (11), an avalanche photodiode (15), a photon counter (16), an Olympus inverted microscope (10), a spectrometer (14), a pinhole (13) and a dichroic mirror (12);
the objective lens of the Olympus inverted microscope (10) is inverted, a transparent sample stage (9) is arranged above the inverted objective lens, exciting light emitted by a laser (11) is emitted to the surface of a sample to be detected through the objective lens, and an optical path system acquires an emission spectrum, a fluorescence life and magnetic resonance parameters of the sample to be detected through the objective lens;
The AFM system comprises an atomic force microscope (8), wherein the atomic force microscope (8) is of a cantilever type;
the integrated use process of the optical system and the AFM system comprises the following steps: the transparent sample stage (9) is arranged in a cavity of the atomic force microscope (8), when the light path system emits laser below a sample to be detected and collects signals, the AFM needle point scans the sample to be detected above the sample at the same time, and dynamic and real-time acquisition of diamond NV color center fluorescence and magnetic resonance characteristic information is realized by using the signals collected by the AFM system and the light path system;
the microwave system comprises a microwave source (1), a microwave switch (2), a high-power amplifier (3) and a circulator (4); after the microwave system is connected and debugged, a microwave source (1) generates a microwave field, the microwave field is capacitively coupled to an omega-type resonator (5), a uniform peak field measurement area is provided, and quantum characteristics of an NV color center of the diamond are modulated through fixed frequency so as to realize NV color center qubit initialization and specific detection and analysis;
the magnetic field module (17) is a permanent magnet or an electromagnet, and the influence on the NV color center self-selection characteristic is realized by adjusting the direction and the size of a magnetic field and the distance between the magnetic field module (17) and the NV color center so as to realize the magnetic imaging function;
the two-dimensional film generation system is an atomic layer deposition platform.
3. A miRNA precise addressing and ultrasensitive detection method is characterized by comprising the following steps:
preparing an AFM probe, wherein the AFM probe comprises an AFM tip and an AFM cantilever, and the AFM tip is obtained by depositing a two-dimensional film on the surface of a diamond containing an NV color center through an atomic layer;
and positioning and detecting a target miRNA coupling magnetic sphere by using the AFM probe in combination with a magnetic field module and a light path system, wherein the surface of the target miRNA coupling magnetic sphere is modified with a DNA probe with a modifier and a target miRNA.
4. The method for miRNA precise addressing and ultrasensitive detection according to claim 3, wherein the preparing the AFM probe comprises:
a) etching the blocky diamond containing the NV color center by adopting a reactive ion etching process to obtain the diamond with the conical tip;
b) placing the diamond with the conical tip in an atomic layer deposition reaction cavity, and modifying a two-dimensional film on the surface;
c) processing the main body part at the rear end of the conical tip by adopting a diamond etching process to obtain a complete diamond AFM tip;
d) and assembling the diamond AFM tip and the AFM cantilever into a complete probe.
5. The method for miRNA precise addressing and ultrasensitive detection according to claim 3, wherein the thickness of the two-dimensional film is 0.5-100 nm.
6. The miRNA precise addressing and ultrasensitive detection method according to claim 3, wherein the positioning and detection of the target miRNA coupled magnetic sphere by using the AFM probe in combination with a magnetic field module and an optical path system comprises:
spin-coating the target miRNA coupling magnetic spheres on a to-be-detected sample area of a quartz glass slide;
the magnetic field module applies a magnetic field to the sample area to be tested, and the direction of the magnetic field is parallel to the NV axis;
the laser of the light path system generates exciting light to act on the NV color center of the AFM probe, the AFM probe is used for scanning the sample to be detected to obtain the magnetic field intensity of different position points, and the magnetic ball modified with miRNA is magnetically imaged through the change of the magnetic field intensity to realize the positioning of the miRNA coupling magnetic ball; meanwhile, the light path system collects fluorescence signals in the movement range of the AFM probe, and the fluorescence change in the movement range of the AFM probe is reflected in real time through the spectral scanning imaging function, so that the ultra-sensitive detection of miRNA is realized.
7. The method for accurate addressing and ultrasensitive detection of miRNA according to claim 6, wherein the AFM probe is used in combination with a magnetic field module and an optical path system to position and detect a target miRNA coupled magnetic sphere, further comprising:
The signal is modulated and amplified by a microwave system.
8. The method for accurately addressing and ultrasensitive detecting the miRNA according to claim 3, wherein preparing the target miRNA coupled magnetic spheres comprises:
preparing a magnetic sphere solution and a probe DNA solution, the surfaces of which are modified with streptavidin;
taking 5-10 mu l of the probe DNA solution, adding the probe DNA solution into 100-150 mu l of the magnetic ball solution, and incubating for 12-15h at room temperature; centrifugally washing the incubated solution by using a buffer solution to remove probe DNA which is not firmly connected to obtain a treated magnetic ball solution;
uniformly mixing 10-20 mu l of target miRNA solution with 100-150 mu l of the treated magnetosphere solution, incubating at room temperature, and centrifugally washing with buffer solution to remove redundant miRNA to obtain target miRNA coupled magnetospheres;
the two ends of the probe DNA are respectively modified with biotin and modifiers, and the modifiers comprise dye molecules and metal nanoparticles.
9. The method for the accurate addressing and the ultrasensitive detection of miRNA according to claim 8, wherein the magnetic sphere solution is a solution comprising magnetic spheres of one or more diameters;
for the solution containing the magnetic spheres with various diameters, the probe DNA solution is a solution containing various DNAs, the target miRNA coupling magnetic spheres are a coupling magnetic sphere solution containing various target miRNAs, and the various DNAs are used for respectively and correspondingly mixing with the magnetic spheres with various diameters and respectively pairing with the various target miRNAs.
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