CN113999894B - Activated fluorescent coding ferritin nano probe, preparation and application detection method - Google Patents
Activated fluorescent coding ferritin nano probe, preparation and application detection method Download PDFInfo
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Abstract
The invention discloses an activated fluorescence coding ferritin nano probe, a preparation and application detection method, which comprises the following steps: self-assembly and repolymerization into a cage-shaped structure are realized, and a fluorescent coding ferritin nanoparticle solution is obtained; obtaining a nucleic acid assembly S/A/R, and obtaining an activated fluorescence-encoded ferritin nano-probe, wherein the nano-probe is divided into two functional areas, and fluorescence-encoded ferritin nano-particles are used for qualitative analysis; the nucleic acid assembly anchored on the surface of the fluorescent encoding ferritin nanoparticle generates a signal through an 'activated' signal mode for quantitative detection of a target. The invention realizes high-sensitivity and high-specificity detection of corresponding target mRNA, is applied to synchronous in-situ detection and imaging analysis of lung cancer related mRNA in living single cells 7, is convenient for preparing activated fluorescence-encoded ferritin nano probes, avoids complex functional modification, and provides a simple preparation method for constructing functional nano probes.
Description
Technical Field
The invention relates to the field of activated fluorescence-encoded ferritin nano probes, belongs to the field of biology, and particularly relates to an activated fluorescence-encoded ferritin nano probe, a preparation method and an application detection method.
Background
The mRNA related to lung cancer is closely related to the occurrence and development processes of lung cancer, so that the combined detection of multiple lung cancer-related mRNAs in tissues or cells simultaneously has important significance for early auxiliary diagnosis, treatment evaluation and prognosis judgment of lung cancer. At present, no specific tumor marker is shown to accurately and independently diagnose lung cancer, so that multiple markers are usually required to be jointly detected to improve the accuracy of diagnosis.
Currently, methods for achieving multiple mRNA detection mainly include reverse transcription PCR, northern blotting, and microarray chips. Where amplification-free methods are used for analysis, the amount of sample often required is relatively large; the amplification-based method is easy to cause pollution due to excessive steps, so that the accuracy of the detection result is affected. Furthermore, these methods often require destruction of the tissue or cells, and it is difficult to monitor mRNA in situ at the single cell level, both temporally and spatially. Also considering the complexity of gene expression in cells, detecting the average level of mRNA expression in a cell population may result in the loss of some important information associated with mRNA expression and cell function, and thus methods that can detect multiple mrnas in situ at the single cell level are highly desirable.
The fluorescent in situ hybridization technology is a detection technology using fluorescent substance labeled probes, and can realize in-situ detection of single mRNA or multiple mRNAs at single cell level, however, the technology needs pretreatment processes such as fixing and treating cells, can not realize detection of target objects in living cells, and has the defects of high imaging background and the like. Therefore, it is highly desirable to establish a technique that is easy to operate, has high imaging contrast, and can be applied to living cells, so as to realize synchronous in-situ detection and imaging of multiple mRNAs at the level of single cells.
In view of this, the present study constructed novel "active" fluorescence-encoded ferritin nanoprobes based on the principle of fluorescence encoding. On the basis of respectively detecting 7 lung cancer related mRNAs by using the 'activated' fluorescent coded ferritin nano probes in the system research solution, the constructed nano probe system is further used for in-situ detection and imaging analysis of 7 mRNAs corresponding to living single cells, and provides new methods and technical support for early auxiliary diagnosis, prognosis judgment and the like of lung cancer. The activated fluorescent coded ferritin nano-probe is divided into two functional areas, wherein fluorescent coded ferritin nano-particles (coated with fluorescein and rhodamine in different proportions) are used for qualitative analysis; the nucleic acid assembly anchored on the surface of the fluorescent encoded ferritin nanoparticle generates a signal in the presence of the target through an 'activated' signal mode for quantitative detection of the target.
Disclosure of Invention
The invention mainly solves the technical problem of providing the activated fluorescent coding ferritin nano probe which realizes high-sensitivity and high-specificity detection of corresponding target mRNA in solution, is further successfully applied to synchronous in-situ detection and imaging analysis of lung cancer related mRNA in living single cell 7, simultaneously has simple preparation of the activated fluorescent coding ferritin nano probe, avoids complex functional modification process and provides a simple preparation method for construction of the functional nano probe, and a preparation and application detection method.
In order to solve the technical problems, the invention adopts a technical scheme that: a preparation method of an activated fluorescence-encoded ferritin nano probe comprises the following steps:
s1, adjusting the pH value of APO solution of APO ferritin, adding sodium fluorescein solution and rhodamine B solution at the same time, and adjusting the pH value of APO solution of APO ferritin again, so that self-assembly and repolymerization of APO solution of APO ferritin into a cage-shaped structure are realized under the condition, and meanwhile, dye is packed into an internal cavity of APO, and fluorescent coded ferritin nanoparticle solution is obtained;
s2, taking a signal probe S, a recognition probe R and an anchor-breaking probe A which have equal volumes and final concentrations of 0.5-1.5 mu mol/L, and hybridizing for 25-35min at the hybridization temperature of 18-25 ℃ to obtain a nucleic acid assembly S/A/R;
s3, adding a fluorescent coding ferritin nanoparticle solution which has the same volume as the signal probe S and has the final concentration of 150-250nmol/L into the nucleic acid assembly S/A/R obtained in the step S2, simultaneously placing the solution into a constant-temperature oscillator with the temperature of 23-27 ℃ for reacting for 1.5-2.5 hours, removing unbound nucleic acid through an ultrafiltration centrifuge tube, and collecting the solution reserved in the tube core to obtain the activated fluorescent coding ferritin nano probe.
In a preferred embodiment, the step S1 further includes the following steps:
s101, wetting a filter membrane by using MilliQ water, filtering APO through the filter membrane by using a syringe, collecting filtrate, repeating the steps, and transferring the filtrate to an ultrafiltration tube for centrifugation to obtain APO solution with a certain concentration;
s102, adding a plurality of MilliQ water into the APO solution obtained in the step S101, adding an HCl solution to adjust the pH value, and oscillating at the same time to fully depolymerize the APO.
In a preferred embodiment, in the step S1, the feeding ratio of the sodium fluorescein solution to the rhodamine B solution is set to seven, and correspondingly, seven activated fluorescence-encoded ferritin nanoprobes are obtained in the step S3.
In a preferred embodiment, in step S2, the anchoring probe a is marked with adamantane for observing self-assembled binding of the anchoring chain to ferritin by Zeta potential characterization.
An activated fluorescent coded ferritin nano-probe is prepared by the method.
An activated fluorescence coding ferritin nano-probe application detection method is used for detecting lung cancer related mRNA, and specifically comprises the following steps:
SS1, optimizing detection experimental conditions, including optimizing the loading concentration ratio of fluorescent coding ferritin nano-particles to a nucleic acid assembly, the dosage of an activated fluorescent coding ferritin nano-probe and the fluorescence recovery time of hybridization of the activated fluorescent coding ferritin nano-probe and a target;
SS2, establishing a detection standard curve, namely respectively diluting seven targets into different concentration gradients, setting 3 parallel concentrations, measuring fluorescence response signals of the targets, linearly fitting obtained experimental results, and respectively establishing 7 standard curves for lung cancer related mRNA detection;
SS3, determining detection limit and detection specificity;
SS4, synchronous in situ detection and imaging.
In a preferred embodiment, the cytotoxicity of the activated fluorescent encoded ferritin nanoprobe is examined before step SS4, which comprises the following steps:
s201, inoculating cells;
s202, adding an object to be detected;
s203, adding a CCK-8 reagent;
s204, measuring an OD value;
s205, calculating the cell survival rate.
In a preferred embodiment, in step SS4, the method specifically comprises the following steps:
s301, inoculating A549 cells into eight glass bottom laser confocal dishes respectively, and placing the dishes in a cell incubator for incubation, wherein the incubation time is set to be 12 hours;
s302, changing liquid of the A549 cells, respectively adding 7 activated fluorescence-encoded ferritin nano probes (FENPS probes) into seven laser confocal dishes, simultaneously adding an activated fluorescence-encoded ferritin nano probe (FENPS probe) system into the rest of the laser confocal dishes, and continuously incubating for 4 hours;
s303, absorbing and discarding the supernatant, and washing the supernatant for a plurality of times by using a buffer solution;
s304, adding buffer solution, and respectively carrying out imaging and data acquisition on the eight samples by using a laser confocal microscope.
In a preferred embodiment, in step S302, the activated fluorescence-encoded ferritin nanoprobe system is a mixture of 7 activated fluorescence-encoded ferritin nanoprobes.
In a preferred embodiment, in step S304, dye Flu uses 488nm laser as excitation light source, and fluorescence collection band is 495-545nm; the dye RB uses a 552nm laser as an excitation light source, and the fluorescence collection wave band is 560-620nm; dye Cy5.5 uses 638nm laser as excitation light source, fluorescence collection wave band is 680-730nm, and uses 63x oil mirror to image.
The beneficial effects of the invention are as follows: the method realizes high-sensitivity and high-specificity detection of corresponding target mRNA in the solution, is further successfully applied to synchronous in-situ detection and imaging analysis of lung cancer related mRNA in the living single cell 7, simultaneously facilitates preparation of the activated fluorescent coded ferritin nano probe, avoids a complex functional modification process, and provides a simple preparation method for construction of the functional nano probe.
Drawings
For a clearer description of the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly introduced below, it being obvious that the drawings in the description below are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art, wherein:
FIG. 1 is a schematic diagram of the construction of an activated fluorescence-encoded ferritin nanoprobe and its use in mRNA detection according to an embodiment of the method for preparing and applying the activated fluorescence-encoded ferritin nanoprobe of the present invention;
FIG. 2 is a schematic diagram showing the principle of synchronous in-situ detection of 7 mRNA in a cell by using an activated fluorescence-encoded ferritin nanoprobe system according to an embodiment of the method for detecting activated fluorescence-encoded ferritin nanoprobe, preparation and application of the invention;
FIG. 3 is an ultraviolet-visible absorption spectrum of APO@flu/RB (Flu/RB=3:3) in an embodiment of an activated fluorescence-encoded ferritin nanoprobe, a preparation and application detection method of the present invention;
FIG. 4 is a graph showing fluorescence spectra of fluorescence-encoded ferritin nanoparticles in an embodiment of the activated fluorescence-encoded ferritin nanoprobe, method of preparing and applying the detection method of the present invention;
FIG. 5 shows the recognition performance of 7 activated fluorescence-encoded ferritin nanoprobes on a target in an embodiment of the method for detecting the preparation and application of activated fluorescence-encoded ferritin nanoprobes according to the present invention;
FIG. 6 shows the specific results of an activated fluorescence-encoded ferritin nanoprobe for detection of a corresponding target in an embodiment of the method for preparing and applying an activated fluorescence-encoded ferritin nanoprobe according to the present invention;
FIG. 7 shows the cytotoxicity results of the activated fluorescence-encoded ferritin nanoprobe according to an embodiment of the present invention, a method for preparing and using the same.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-7, in one embodiment of the present invention, an activated fluorescence-encoded ferritin nanoprobe, a method for preparing the same, and a method for detecting the same are provided, which specifically include the following embodiments:
example 1
A preparation method of an activated fluorescence-encoded ferritin nano probe comprises the following steps:
s1, regulating the pH value of APO solution of APO of apoferritin to 0.5-3.5, adding sodium fluorescein solution Flu and rhodamine B solution RB, the pH value of the APO solution of the APO is regulated to 7.5-8.5, self-assembling and repolymerizing the material into a cage-like structure under the condition; loading dye into the APO inner cavity, and obtaining fluorescent coding ferritin nanoparticle solution;
s2, taking a signal probe S, a recognition probe R and an anchor-breaking probe A which have equal volumes and final concentrations of 0.5-1.5 mu mol/L, and hybridizing for 25-35min at the hybridization temperature of 18-25 ℃ to obtain a nucleic acid assembly S/A/R;
s3, adding a fluorescent coding ferritin nanoparticle solution which has the same volume as the signal probe S and has the final concentration of 150-250nmol/L into the nucleic acid assembly S/A/R obtained in the step S2, simultaneously placing the solution into a constant-temperature oscillator with the temperature of 23-27 ℃ for reacting for 1.5-2.5 hours, removing unbound nucleic acid through an ultrafiltration centrifuge tube, and collecting the solution reserved in the tube core to obtain the activated fluorescent coding ferritin nano probe.
The step S1 is preceded by the following steps:
s101, wetting a filter membrane by using MilliQ water, filtering APO through the filter membrane by using a syringe, collecting filtrate, repeating the steps, and transferring the filtrate to an ultrafiltration tube for centrifugation to obtain APO solution with a certain concentration;
wetting with MilliQ water at 0.22 μm, filtering APO with syringe, collecting filtrate, washing the filter twice with MilliQ water, transferring filtrate to ultrafiltration tube, and centrifuging for 3-5min to obtain filtrate with concentration of 1×10 - 4 APO solution of mol/L;
s102, adding a plurality of MilliQ water into the APO solution obtained in the step S101, adding an HCl solution to adjust the pH value, and oscillating at the same time to fully depolymerize the APO.
Taking a plurality of APO solutions obtained in the step S101, adding a plurality of MilliQ water, slowly adding a proper amount of HCl solution to adjust the pH to 2, and placing the APO solution in a horizontal shaking table to vibrate for 10min to fully decompose the APO.
In the step S1, the feeding ratio of the sodium fluorescein solution to the rhodamine B solution is set to be seven, and correspondingly, seven activated fluorescence-encoding ferritin nano probes obtained in the step S3 are provided.
In the step S1, the feeding ratio of the sodium fluorescein solution Flu and the rhodamine B solution RB is seven, namely Flu: rb=6: 0. flu: rb=5:1, flu: rb=4:2, flu: rb=3:3, flu: rb=2:4, flu: rb=1:5 and Flu: rb= 0:6, and correspondingly, there are seven active fluorescent-encoded ferritin nanoprobes obtained in step S3.
In step S2, adamantane for observing self-assembled binding between the anchor chain and ferritin through Zeta potential characterization is marked on the anchor breaking probe a.
In a specific implementation process, the pH of a Apoferritin (APO) solution is adjusted to be 2, at the moment, a fluorescein sodium solution (Flu) and a rhodamine B solution (RB) with different proportions are added, wherein the feeding ratios (Flu/RB) of Flu and RB are 6:0, 5:1, 4:2, 3:3, 2:4, 1:5 and 0:6 respectively, then the pH is adjusted to be 8, the Apoferritin (APO) solution is self-assembled and is re-polymerized into a cage-shaped structure under the condition, and dyes are packed into an APO internal cavity, so that 7 fluorescent encoding ferritin nano-particles are prepared.
The information corresponding to 7 fluorescently encoded nanoparticles is as follows: apo@flu/RB (Flu/rb=6:0); apo@flu/RB (Flu/rb=5:1); apo@flu/RB (Flu/rb=4:2); apo@flu/RB (Flu/rb=3:3); apo@flu/RB (Flu/rb=2:4); apo@flu/RB (Flu/rb=1:5); apo@flu/RB (Flu/rb= 0:6).
The preparation process comprises the following steps:
(1) Firstly, the APO is pretreated (the preservation solution is replaced by deionized water), and the specific operation is as follows: firstly, a filter membrane with the diameter of 0.22 mu m is wetted by MilliQ water, then APO is filtered by the filter membrane by a 1ml syringe, filtrate is collected, then the filter membrane is washed twice by MilliQ water, the filtrate is transferred to an ultrafiltration tube for centrifugation for 4min, APO in the tube core of the inner tube is collected, an ultraviolet absorption standard curve is established by utilizing the characteristic absorption peak of the APO at 280nm, and the standard curve is utilizedThe concentration of APO collected after concentration was 1X 10 -4 mol/L, sealing and preserving at 4 ℃ for standby.
(2) apo@flu/RB (Flu/rb=6:0) was prepared as follows: accurately taking 6 mu l of APO, then adding 439 mu l of MilliQ water, slowly adding a proper amount of 0.1mol/LHCl solution to adjust the pH to 2, and placing the mixture in a horizontal shaking table to oscillate for 10min so as to fully depolymerize the APO; 300 μl of the solution was slowly added dropwise at a concentration of 2×10 -3 Stirring the Flu solution with the mol/L ratio for 20min to fully and uniformly mix the solution; and adding a proper amount of 0.1mol/LNaOH to regulate the pH value of the solution to 8, and oscillating for 2 hours to enable APO to fully self-assemble and re-polymerize. The solution reaction was then used to remove the precipitate produced by the solution reaction using a 0.22 μm filter membrane and the filtrate was collected. And the filtrate was centrifuged for 4min with an ultrafiltration tube (30 kDa,15 ml) to remove free Flu from the solution. Finally, absorbing the fluorescent coding ferritin nanoparticle solution in the inner tube core, sealing and preserving at 4 ℃ for later use.
Other fluorescence-encoded ferritin nanoparticles were prepared in a similar manner to apo@flu/RB (Flu/rb=6:0), with only the Flu and RB feed ratios (Flu/RB) being adjusted to 5:1, 4:2, 3:3, 2:4, 1:5, 0:6 to prepare apo@flu/RB (Flu/rb=5:1), respectively; apo@flu/RB (Flu/rb=4:2); apo@flu/RB (Flu/rb=3:3); apo@flu/RB (Flu/rb=2:4); apo@flu/RB (Flu/rb=1:5); apo@flu/RB (Flu/rb= 0:6). And then carrying out transmission electron microscope, ultraviolet spectrum and fluorescence spectrum characterization on the prepared fluorescence coding nano-particles, and measuring the number of the dye entrapped in each APO in the 7 fluorescence coding ferritin nano-particles.
Example 2
The activated fluorescence-encoded ferritin nano-probe is prepared by the method, specifically, based on the depolymerization/self-assembly performance of APO depending on pH, flu and RB with different feeding ratios are simultaneously loaded into an APO internal cavity by regulating and controlling the pH value of a solution, and 7 fluorescence-encoded ferritin nano-particles are constructed (shown in figure 2); secondly, constructing a nucleic acid assembly formed by the anchoring probe (A), the recognition probe (R) and the signal probe (S) based on a nucleic acid self-assembly technology; finally, the nucleic acid assembly was self-assembled and anchored to the surface of the fluorescent encoded nanoparticle by utilizing the property that adamantane can be effectively inserted into the protein, thereby preparing an activated fluorescent encoded ferritin nanoprobe (FENPS) (fig. 1). The 7 kinds of FENPSs (FENPS 1, FENPS2, FENPS3, FENPS4, FENPS5, FENPS6, FENPS 7) were used to specifically recognize the corresponding 7 kinds of mRNAs (EGFR, CD151, TK1, epCAM, CD91, PD-L1, VEGF) respectively (nucleic acid strand names and their corresponding base sequences, see Table 1 below).
Here, the preparation of the FENPS1 is described as an example: firstly, 100 mu L (the final concentration is 1 mu mol/L) of a signaling probe (S1), a recognition probe (R1) and an anchor probe (A) of an EGFR probe system are hybridized for 30min at room temperature to form a nucleic acid assembly (S1/A/R1), 100 mu L of prepared APO@flu/RB (Flu: RB=6:0) is added, the mixture is placed in a constant temperature oscillator for reaction for 2h at the temperature of 25 ℃, unbound nucleic acid is removed by ultrafiltration centrifugation, and the solution reserved in a tube core is collected, so that the prepared FENPS1 (APO@flu/RB@S1/A/R1, flu/RB=6:0) is stored in a refrigerator at the temperature of 4 ℃ for standby. The preparation methods of the FENPS2, the FENPS3, the FENPS4, the FENPS5, the FENPS6 and the FENPS7 are similar to the FENPS1, and the FENPS2 (APO@flu/RB@S2/A/R2, flu/RB=5:1), the FENPS3 (APO@flu/RB@S3/A/R3, flu/RB=4:2), the FENPS4 (APO@flu/RB@S4/A/R4, flu/RB=3:3), the FENPS5 (APO@flu/RB@S5/A/R5, flu/RB=2:4), the FENPS6 (APO@flu/RB@S6/A/R6, flu/RB=1:5) and the FENPS7 (APO Flu/RB@S7/A/R7/RB= 0:6).
The procedure involved in the preparation of the fluorescence-encoded ferritin nanoprobe was then characterized accordingly: (1) Reacting the anchoring probe A marked with adamantane with a certain amount of APO in a constant temperature oscillator for 2 hours at the temperature of 25 ℃, removing unbound nucleic acid by ultrafiltration and centrifugation, carrying out Zeta potential characterization on the nucleic acid, and observing whether an anchoring chain can be combined with ferritin in a self-assembly way; (2) To verify whether 7 kinds of FENPSs were successfully prepared and to achieve "active" signal reading in the presence of the target, the fluorescence response properties of FENPS1, FENPS2, FENPS3, FENPS4, FENPS5, FENPS6 and FENPS7 to the target were examined, respectively. Firstly, performing fluorescence signal measurement when the prepared FENPS probe (100 nmol/L) is not added with a corresponding target object, then adding respective corresponding target object solutions (150 nmol/L) to perform hybridization reaction for 0.5h, performing fluorescence signal detection, and comparing the fluorescence signal changes before and after adding the target object to examine the FENPS performance.
TABLE 1 names of nucleic acid chains and their corresponding base sequences
Note that: in solution experiments, EGFR, CD151, TK1, epCAM, CD91, PD-L1, VEGF target sequences were replaced with the corresponding DNA, respectively.
Example 3
An activated fluorescence coding ferritin nano-probe application detection method is used for detecting lung cancer related mRNA, and specifically comprises the following steps:
SS1, optimizing detection experimental conditions, including optimizing the loading concentration ratio of fluorescent coding ferritin nano-particles to a nucleic acid assembly, the dosage of an activated fluorescent coding ferritin nano-probe and the fluorescence recovery time of hybridization of the activated fluorescent coding ferritin nano-probe and a target;
SS2, establishing a detection standard curve, namely respectively diluting seven targets into different concentration gradients, setting 3 parallel concentrations, measuring fluorescence response signals of the targets, linearly fitting obtained experimental results, and respectively establishing 7 standard curves for lung cancer related mRNA detection;
SS3, determining detection limit and detection specificity;
SS4, synchronous in situ detection and imaging.
Before step SS4, the cytotoxicity of the activated fluorescence-encoded ferritin nano-probe needs to be examined, and the method specifically comprises the following steps:
s201, inoculating cells;
s202, adding an object to be detected;
s203, adding a CCK-8 reagent;
s204, measuring an OD value;
s205, calculating the cell survival rate.
In a specific embodiment, the toxicity of the constructed fluorescence-encoded ferritin nano probe to cells is detected by adopting a CCK-8 experiment. Because the 7 fluorescence-encoded ferritin nanoprobes of the experiment are consistent in construction materials, FENPS4 (Flu/RB=3:3) is selected for cytotoxicity experiment investigation, and the specific operation steps are as follows:
(1) Inoculating cells: a549 cells in good culture state were digested and were prepared to a concentration of 1X 10 by cell counting 4 Mu.l of the cell suspension was then added to each well of a 96-well plate to spread the cells uniformly in the 96-well plate, and a negative control group and a blank control group were set, respectively.
(2) Adding an object to be detected: after 24h of cell culture, waste liquid was aspirated, 10. Mu.l of fluorescent-encoded ferritin nanoprobe (0.05, 0.1, 0.5, 1.0. Mu. Mol/L, respectively) was then added to the experimental group, and 90. Mu.l of RPMI-1640 medium without fetal bovine serum was added, and 100. Mu.l of RPMI-1640 medium without fetal bovine serum was added to the control group and the blank group, each group was provided with 5 replicates.
(3) Adding CCK-8 reagent: after cell culture for 24 hours, 10 mu.l of CCK-8 reagent is added into each hole of an experiment group, a control group and a blank group, the mixture is fully and uniformly mixed, the mixture is placed into a cell culture box for culture for 1 to 4 hours, OD value measurement can be carried out by continuously observing the light orange color of liquid in the holes, the experiment is finally carried out for 2 hours, and the absorbance at 450nm is optimal at the moment.
(4) OD value measurement: the absorbance at 450nm wavelength in 96-well plate was detected with an enzyme-labeled instrument under light-shielding condition.
(5) Cell viability calculation: cell viability= (OD 450 Experimental well-OD 450 Blank hole)/(OD 450 Control well-OD 450 Blank wells) x 100%.
In step SS4, the method specifically includes the following steps:
s301, inoculating A549 cells into eight glass bottom laser confocal dishes respectively, and placing the dishes in a cell incubator for incubation, wherein the incubation time is set to be 12 hours;
s302, changing liquid of the A549 cells, respectively adding 7 activated fluorescence-encoded ferritin nano probes (FENPS probes) into seven laser confocal dishes, simultaneously adding an activated fluorescence-encoded ferritin nano probe (FENPS probe) system into the rest of the laser confocal dishes, and continuously incubating for 4 hours;
s303, absorbing and discarding the supernatant, and washing the supernatant for a plurality of times by using a buffer solution;
s304, adding buffer solution, and respectively carrying out imaging and data acquisition on the eight samples by using a laser confocal microscope.
In step S302, the activated fluorescence-encoded ferritin nanoprobe system is a mixture of 7 activated fluorescence-encoded ferritin nanoprobes.
In the step S304, dye Flu uses 488nm laser as excitation light source, and fluorescence collection wave band is 495-545nm; the dye RB uses a 552nm laser as an excitation light source, and the fluorescence collection wave band is 560-620nm; dye Cy5.5 uses 638nm laser as excitation light source, fluorescence collection wave band is 680-730nm, and uses 63x oil mirror to image.
Specifically, a549 cells (1×10 5 And (3) inoculating into 8 laser confocal dishes with glass bottoms of 20mm in diameter, placing the dishes into a cell incubator for 12h of incubation, and changing the liquid of the cells. Then, 7 kinds of FENPS probes were respectively added in the No. 1-7 laser confocal dish, the FENPS probe system (mixture of 7 kinds of FENPS probes) was added in the No. 8 laser confocal dish, incubation was continued for 4 hours, then the supernatant was aspirated and washed three times with PBS buffer, finally 500. Mu.l of PBS buffer was added, and the above 8 samples were imaged and data were collected with a laser confocal microscope, respectively.
Laser confocal microscope imaging conditions: the dye Flu uses a 488nm laser as an excitation light source, and the fluorescence collection wave band is 495-545nm; the dye RB uses a 552nm laser as an excitation light source, and the fluorescence collection wave band is 560-620nm; dye Cy5.5 uses 638nm laser as excitation light source, fluorescence collection wave band is 680-730nm, and uses 63x oil mirror to image. Finally, decoding and analyzing the laser confocal imaging result, so that synchronous in-situ detection and imaging analysis of 7 mRNAs in the living single cell are realized.
The principle of the fluorescence-encoded ferritin nanoprobe system for the synchronous in-situ detection and imaging of 7 mRNAs in cells is shown in FIG. 2. Based on the system optimization of FENPS (FENPS 1, FENPS2, FENPS3, FENPS4, FENPS5, FENPS6, FENPS 7) to realize detection of corresponding 7 target mRNAs (EGFR, CD151, TK1, epCAM, CD91, PD-L1, VEGF) in the solution, the method is further used for synchronous in-situ detection and imaging of 7 mRNAs in cells.
When the fluorescent-encoded ferritin nanoprobe recognizes a target (the detection of the target is exemplified by FENPS 1), FENPS1 is divided into two functional regions, wherein fluorescent-encoded ferritin nanoparticles (Flu/rb=6:0) are used for egfr mrna qualitative analysis; the nucleic acid assembly (S1/A/R1) generates a fluorescent (Cy5.5) signal in the presence of the target via an "activated" signal pattern for quantitative detection of EGFR mRNA. The Cy5.5 is adopted as a quantitative fluorescence signal, and the main consideration is that the excitation wavelength and the emission wavelength are both larger than 600nm, so that the influence of the autobackground fluorescence of organisms can be effectively filtered, the background signal can be effectively reduced when cells are imaged, and the imaging contrast and sensitivity are improved. Different mRNA in the cell has the characteristic of different spatial distribution positions, so that the synchronous in-situ detection of 7 mRNA in the cell can be realized by simultaneously incubating 7 FENPS probes with the target cell. Because of the complex intracellular environment, the intracellular code ratio will be different from the code ratio in the solution, and analysis is needed by collecting the confocal laser imaging data of 7 kinds of FENPS after incubation with the cells respectively. And collecting fluorescent signals of bright spots in Flu channels and fluorescent signals of bright spots in corresponding RB channels in a laser confocal imaging diagram, and obtaining corresponding coding ratio ranges of 7 FENPSs in cells so as to perform decoding processing during synchronous in-situ detection and imaging and realize qualitative analysis of 7 mRNA in the cells.
Subsequently, laser confocal imaging images after 7 FENPSs were incubated with cells simultaneously were decoded. The specific decoding process comprises the following steps: firstly, selecting signal points in a Cy5.5 channel, and considering that one signal point is one copy of target mRNA; secondly, by analyzing the fluorescence signal ratio of the signal point in the corresponding Flu channel and RB channel, the fluorescence ratio is matched with the coding ratio range corresponding to the 7 probes obtained in the cells, and which target object the signal point is can be confirmed, so that the qualitative analysis and quantitative detection of target mRNA are realized; finally, the copy numbers corresponding to 7 target mRNAs can be obtained by carrying out the decoding treatment on all luminous point signals in the Cy5.5 channel, so that the synchronous in-situ detection (including qualitative and quantitative) and imaging analysis of 7 mRNAs in a living single cell are realized.
In an embodiment, in the performance characterization of fluorescence-encoded ferritin nanoparticles,
(1) By utilizing the transmission electron microscope characterization, the morphology of APO, APO@flu/RB (Flu/RB=6:0), APO@flu/RB (Flu/RB=3:3) and APO@flu/RB (Flu/RB= 0:6) is subjected to transmission electron microscope characterization, white protein shells and black internal cavities can be observed, and meanwhile, the APO is in a spherical structure, has the particle size of about 10nm and has good dispersion performance. After the dye is entrapped, the sizes of APO@flu/RB (Flu/RB=6:0), APO@flu/RB (Flu/RB=3:3) and APO@flu/RB (Flu/RB= 0:6) are unchanged, and the APO@flu/RB is still in a spherical structure, which indicates that the APO with the pH adjusted can still be successfully self-assembled and then polymerized into the spherical structure, has a stable structure and can be successfully used for the entrapment of dye substances.
(2) Uv-vis absorption spectrum characterization was performed on APO, flu, RB and apo@flu/RB (Flu/rb=3:3) using uv-vis absorption spectrum and fluorescence spectrum characterization, respectively (fig. 3). APO has a characteristic absorption peak at 280 nm; flu has a characteristic absorption peak around 480 nm; RB has a characteristic absorption peak at 555 nm. The spectrum corresponding to APO@flu/RB (Flu/RB=3:3) has an absorption peak at 280nm, which represents that APO is provided, and Flu and RB characteristic absorption peaks at 480nm and 555nm are corresponding, which shows that Flu and RB are loaded into an APO internal cavity at the same time, and shows that APO@flu/RB (Flu/RB=3:3) is successfully prepared.
The fluorescence spectra of 7 kinds of fluorescence-encoded ferritin nanoparticles are shown in fig. 4, the fluorescence intensity of Flu at 515nm is gradually reduced along with the gradual reduction of Flu feed ratio, the fluorescence intensity of RB at 575nm is gradually enhanced along with the gradual increase of RB feed ratio, meanwhile, the fluorescence intensity ratio of Flu and RB is changed along with the change of feed ratio, and the fluorescence intensity of each ratio-encoded is obviously distinguished. The fluorescence spectrum is combined with the transmission electron microscope characterization and the ultraviolet visible absorption spectrum, which shows that 7 fluorescence encoding ferritin nano particles are successfully prepared, thereby laying a foundation for FENPS preparation.
In the investigation of the characteristics of the characterization,
(1) The number of APO-entrapped dyes in 7 fluorescent-encoded ferritin nanoparticles prepared by using the characterization of the number of APO-entrapped dyes in fluorescent-encoded ferritin nanoparticles according to different feed ratios (Flu/RB: 6:0, 5:1, 4:2, 3:3, 2:4, 5:1, 0:6) shows that the number of APO-entrapped dyes in fluorescent-encoded ferritin nanoparticles shows a decreasing trend along with the decrease of the Flu feed ratio, the number of entrapped RBs shows an increasing trend along with the increase of the RB feed ratio, and the number of entrapped RBs shows that 7 fluorescent-encoded ferritin nanoparticles are successfully prepared, and the total number of dyes entrapped in each APO is about 65, as shown in table 2:
table 2: number of each APO-entrapped dye in 7 fluorescence-encoded ferritin nanoparticles
(2) In the performance characterization of the fluorescent coded ferritin nano-probe, the combination of APO and the anchor-breaking probe (A) is characterized by utilizing the Zeta potential in the Zeta potential characterization. A. The Zeta potentials of APO and APO@A were-13.1 mV, -15.6mV and-32.3 mV, respectively. After the self-assembled anchoring probe (adamantane-labeled nucleic acid strand) formed apo@a on the surface of ferritin, its potential was more negative, indicating that the anchoring probe could successfully anchor to ferritin surface by adamantane/protein interactions.
(3) In the performance of identifying the target object by the fluorescent coded ferritin nanoprobe, the identification performance of 7 kinds of FENPSs (FENPS 1, FENPS2, FENPS3, FENPS4, FENPS5, FENPS6 and FENPS 7) on the target object is respectively examined through a fluorescent hybridization experiment. Taking the FENPS1 as an example, the corresponding result is shown in fig. 5A. Buffer is the signal of hybridization Buffer; FENPS1 is the signal of FENPS1 alone (without EGFR nucleic acid sequence); fenps1+egfr the fluorescence signal generated by the addition of FENPS1 to the EGFR nucleic acid sequence, cy5.5 fluorescence was activated. The results of the target object recognition performance researches of fig. 5A, fig. 5B, fig. 5C, fig. 5D, fig. 5E, fig. 5F, and fig. 5G are respectively the results of the target object recognition performance researches of the FENPS1, the FENPS2, the FENPS3, the FENPS4, the FENPS5, the FENPS6, and the FENPS7, which indicate that the 7 FENPS probes can be activated by the respective corresponding target objects, and can be effectively used for recognition of the corresponding target objects.
Example 4
In the case of using the fluorescent-encoded ferritin nanoprobe for the detection of mRNA in a solution, since the construction principle of 7 kinds of FENPSs is the same, optimization experiments were performed on the representatives of FENPS1, FENPS4, FENPS6, and FENPS 7. The trend of fluorescence signals of the FENPS1, the FENPS4, the FENPS6 and the FENPS7 is observed to be consistent, namely, along with the continuous increase of the concentration ratio of the fluorescence-encoded ferritin particles to the nucleic acid assembly, the fluorescence signals are gradually enhanced, when the concentration ratio of the fluorescence-encoded ferritin particles to the nucleic acid assembly is 1:5, the signals are strongest, and when the concentration ratio is 1:10, the signals are reduced, so that the concentration ratio of the fluorescence-encoded ferritin nanoparticles to the nucleic acid assembly is 1:5, and the FENPS is prepared.
The dosage of the FENPS probe is optimized by adopting a single factor experiment, and when the probe concentration is 100nmol/L, the fluorescence signal is strongest, so that when 7 FENPS probes are used for detecting target objects, the experiment is carried out by adopting 100nmol/L (concentration quantification is carried out by using the APO content in the fluorescence-coded ferritin nano probe).
The fluorescence recovery time of hybridization of the FENPS and the target object is optimized by adopting a single factor test, and when the reaction time of the probe and the target object is 30min, the fluorescence signal is strongest, so that in the subsequent test, the hybridization time of the FENPS and the target object is selected to be 30min.
The response of 7 FENPSs to different concentrations of target was studied separately. EGFR is in the concentration range of 5nmol/L to 100nmol/L, and the corresponding linear equation is: y= 8187.0202X-41420.2583 (X is C) EGFR Y is fluorescence intensity F-F0), R 2 = 0.9963; CD151 is in the concentration range of 2nmol/L to 40nmol/L, and the corresponding linear equation is: y=562.8346x+1857.4131 (X is C) CD151 Y is fluorescence intensity F-F0), R 2 =0.9960; TK1 is in the concentration range of 2nmol/L to 75nmol/L, and the corresponding linear equation is: y= 5755.7759X-8774.0345 (X is C) TK1 Y is fluorescence intensity F-F0), R 2 = 0.9753; epCAM is in the concentration range of 5nmol/L to 100nmol/L, and the corresponding linear equation is: y= 7114.0549X-66648.1992 (X is C) EpCAM Y is fluorescence intensity F-F0), R 2 = 0.9913; CD91 is in the concentration range of 1nmol/L to 15nmol/L, and the corresponding linear equation is: y=692.1446x+753.1007x, r 2 = 0.9976; in the concentration range of 20nmol/L to 100nmol/L, the corresponding linear equation is: y= 8724.6672X-157041.5156, r 2 = 0.9883 (X is C) CD91 Y is fluorescence intensity F-F0); PD-L1 is in 5nmol/L-75nmol/L concentration range, and the corresponding linear equation is: y=3440.0212x+30236.0932 (X is C) PD-L1 Y is fluorescence intensity F-F0), R 2 = 0.9901; VEGF is in the concentration range of 5nmol/L to 75nmol/L, and the corresponding linear equation is: y=8629.1338x+107740.3794, r 2 =0.9961 (X is C) VEGF Y is fluorescence intensity F-F0), R 2 ＝0.9901。
The detection limit results of 7 FENPS probes for 7 target mRNAs are obtained in the solution, and the detection limit results are respectively: (1) EGFR mRNA detection limit is 1nmol/L; (2) CD151mRNA detection limit is 1nmol/L; (3) TK1mRNA detection limit is 1nmol/L; (4) EpCAMmRNA detection limit is 2nmol/L; (5) the limit of detection of CD91mRNA is 0.5nmol/L; (6) the detection limit of PD-L1mRNA is 1nmol/L; (7) VEGFmRNA detection limit was 0.5nmol/L.
The identification performance of 7 kinds of FENPS (FENPS 1, FENPS2, FENPS3, FENPS4, FENPS5, FENPS6, FENPS 7) on the target object was examined by fluorescence hybridization experiments. Taking the FENPS1 as an example, the corresponding result is shown in fig. 5A. Buffer is the signal of hybridization Buffer; FENPS1 is the signal of FENPS1 alone (without EGFR nucleic acid sequence); fenps1+egfr the fluorescence signal generated by the addition of FENPS1 to the EGFR nucleic acid sequence, cy5.5 fluorescence was activated. The results of the study on the recognition performance of the target objects by the FENPS1, the FENPS2, the FENPS3, the FENPS4, the FENPS5, the FENPS6 and the FENPS7 in fig. 5A, 5B, 5C, 5D, 5E, 5F and 5G show that the 7 FENPS probes can be "activated" by the respective corresponding target objects, and can be effectively used for recognition of the corresponding target objects, and fig. 5A: FENPS1 is used for EGFR mRNA recognition; fig. 5B: FENPS2 is used for CD151mRNA recognition; fig. 5C: FENPS1 is used for TK1mRNA recognition; fig. 5D: FENPS1 is used for EpCAMmRNA recognition; fig. 5E: FENPS1 is used for CD91mRNA recognition; fig. 5F: FENPS1 is used for PD-L1mRNA recognition; fig. 5G: FENPS1 was used for VEGFmRNA recognition. In FIG. 5, curve a is hybridization buffer, curve b is fluorescence spectrum of FENPS probe alone, and curve c is fluorescence spectrum generated after FENPS probe is added to the corresponding target.
The specific results of detecting 7 lung cancer related mRNAs are shown in FIG. 6, and FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, and FIG. 6G are specific results of detecting EGFR, CD151, TK1, EPCAM, CD91, PD-L1, and VEGF, respectively. Wherein, mis-1 is a single base mismatched sequence, mis-2 is a double base mismatched sequence, mis-3 is a three base mismatched sequence, and cmy-c is a random interfering sequence. From fig. 6, the trend of the detection results of 7 lung cancer related mRNA is consistent, the fluorescence intensity of the mismatched sequences is significantly lower than that of the target object with the same concentration, and single base mismatches can be effectively distinguished, which indicates that the detection method based on the FENPS construction can realize the high-specificity detection of the corresponding target object.
Fluorescence-encoded ferritin nanoprobes (FENPS) are used for specific results corresponding to target detection. Wherein, FIG. 6A FENPS1 detects the specificity result of EGFR; FIG. 6B FENPS2 shows the specificity results of CD 151; FIG. 6C FENPS3 detection TK1 specificity results; FIG. 6D FENPS4 detects the specificity of EPCAM; FIG. 6E FENPS5 shows the specificity results of CD91 detection; FIG. 6F FENPS6 shows the specificity result of detecting PD-L1; FIG. 6G FENPS7 shows the results of detection of VEGF specificity.
In the above examples, fluorescence-encoded ferritin nanoprobes were used for simultaneous in situ detection and imaging of 7 mrnas within a cell, wherein CCK-8 was used in the cytotoxicity of the probes to study the cytotoxicity of FENPS. Since the compositions of 7 kinds of FENPSs were similar, cytotoxicity studies were performed on the behalf of FENPS4 (Flu/rb=3:3), and the results are shown in fig. 7. The viability of the cells without the FENPS probe was defined as 100%, the other groups were corresponding to the cell activities after incubation with the cells for 24 hours at different concentrations, and it was found from the graph that the smaller the probe concentration, the higher the cell activity was 93% when the FENPS4 probe concentration was 0.5. Mu. Mol/L, and the cell activity was still 90% or more when the probe concentration was as high as 1. Mu. Mol/L, and the FENPS concentration selected for intracellular detection in the experiment was 0.4. Mu. Mol/L; according to the number of Flu and RB dyes contained in the APO of the FENPS preparation material, the cytotoxicity of the Flu-RB (3:3) mixture with equivalent weight is selected, and the cell viability is obviously reduced when the concentration is more than 25 mu mol/L. The results show that compared with a single fluorescent dye, the FENPS probe formed by the APO-encapsulated dye has lower cytotoxicity and provides a guarantee for the detection of mRNA in living cells in the later period.
In another implementation process, the synchronous in-situ detection and imaging of 7 lung cancer related mRNAs in the cells are performed, the FENPS probe system (7 FENPS probes) is used for synchronous in-situ detection and imaging of 7 mRNAs in the A549 cells, the cell morphology is good, and fluorescent light-emitting areas in four channels are well overlapped in cytoplasm and are consistent with biological characteristics of mRNA mostly distributed in cytoplasm. Subsequently, laser confocal imaging images after 7 FENPSs were incubated with cells simultaneously were decoded. The specific decoding process comprises the following steps: firstly, selecting signal points in a Cy5.5 channel, and considering that one signal point is one copy of target mRNA; secondly, by analyzing the fluorescence signal ratio of the signal point in the corresponding Flu channel and RB channel, the fluorescence ratio is matched with the coding ratio range corresponding to the 7 probes obtained in the cells, and which target object the signal point is can be confirmed, so that the qualitative analysis and quantitative detection of target mRNA are realized; finally, the copy numbers corresponding to 7 target mRNAs can be obtained by carrying out the decoding processing on all luminous point signals in the Cy5.5 channel.
After decoding the confocal imaging images of more than 100 cells, the expression levels of 7 mRNAs in the A549 cells detected by the method are obtained, and the detection result shows that the expression levels of 7 mRNAs in the A549 cells are different, wherein the expression level of EGFR is highest (the average copy number in a single cell is 34), then VEGF (the average copy number in a single cell is 32), PD-L1 (the average copy number in a single cell is 29), TK1 (the average copy number in a single cell is 28), CD151 (the average copy number in a single cell is 17), CD91 (the average copy number in a single cell is 16), and the lowest expression level is EpCAM (the average copy number in a single cell is 15). The above research results show that the fluorescence-encoded ferritin nano-probe can successfully realize synchronous in-situ detection (including qualitative and quantitative) and imaging analysis of 7 mRNAs in a living single cell.
Therefore, the invention has the following advantages:
1. by utilizing the fluorescence coding principle, a series of novel 'activated' fluorescence coding ferritin nano probes (FENPS) are constructed, wherein the FENPS is divided into two functional areas, and the fluorescence coding ferritin nano particles are used for qualitative analysis of a target object; the nucleic acid assembly generates a signal through an activated signal mode in the presence of a target object for quantitative detection of the target object, and based on the signal, the nucleic acid assembly realizes high-sensitivity and high-specificity detection of corresponding target mRNA in a solution and is further successfully applied to synchronous in-situ detection and imaging analysis of lung cancer related mRNA in a living single cell 7;
2. the simple preparation of the activated fluorescent coding ferritin nano-probe is realized by utilizing a self-assembly mode (including APO self-assembly, nucleic acid self-assembly and adamantane/protein interaction mediated self-assembly), so that a complex functional modification process is avoided, and a simple preparation method is provided for the construction of the functional nano-probe;
3. by the method, the accuracy of lung cancer diagnosis is greatly improved, the patient can be timely treated according to the detection result, and meanwhile, the method is beneficial to the research of lung cancer diseases and has great practical significance.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.
Claims (3)
1. The preparation method of the activated fluorescent coded ferritin nano probe is characterized by comprising the following steps of:
s1, regulating the pH value of APO solution of APO of apoferritin to 0.5-3.5, adding sodium fluorescein solution Flu and rhodamine B solution RB, the pH value of the APO solution of the APO is regulated to 7.5-8.5, self-assembling and re-polymerizing the material into a cage-shaped structure; loading dye into the APO inner cavity, and obtaining fluorescent coding ferritin nanoparticle solution;
s2, taking a signaling probe S, a recognition probe R and an anchoring probe A which have equal volumes and final concentrations of 0.5-1.5 mu mol/L, hybridizing for 25-35min at the hybridization temperature of 18-25 ℃ to obtain a nucleic acid assembly S/A/R, wherein the signaling probe S is an EGFR signaling probe, a CD151 signaling probe, a TK1 signaling probe, an EpCAM signaling probe, a CD91 signaling probe, a PD-L1 signaling probe and a VEGF signaling probe; the recognition probes R are EGFR recognition probes, CD151 recognition probes, TK1 recognition probes, epCAM recognition probes, CD91 recognition probes, PD-L1 recognition probes and VEGF recognition probes,
wherein, the nucleic acid sequence of the EGFR signaling probe is as follows:
Cy5.5-CTCCCTCAGCCACCCTTTTTTTTTTTTTTTTTTTTT，
the nucleic acid sequence of the CD151 signaling probe is as follows:
Cy5.5-CGAGACAGTGAGTGGTTTTTTTTTTTTTTTTTTTTT，
the nucleic acid sequence of the TK1 signal probe is as follows:
Cy5.5-GAGAAGGAGGTCGAGTTTTTTTTTTTTTTTTTTTTT，
the nucleic acid sequence of the EpCAM signal probe is as follows:
Cy5.5-TTGCCGCAGCTCAGGTTTTTTTTTTTTTTTTTTTTT，
the nucleic acid sequence of the CD91 signal probe is as follows:
Cy5.5-GGAGAGAAGTAGCAGTTTTTTTTTTTTTTTTTTTTT，
the nucleic acid sequence of the PD-L1 signaling probe is as follows:
Cy5.5-ACCACCAATTCCAAGTTTTTTTTTTTTTTTTTTTTT，
the nucleic acid sequence of the VEGF signaling probe is:
Cy5.5-CATGCCAAGTGGTCCCAGTTTTTTTTTTTTTTTTTTTTT，
the nucleic acid sequence of the EGFR recognition probe is as follows:
GGTACATATGGGTGGCTGAGGGAG-BHQ3，
the nucleic acid sequence of the CD151 recognition probe is as follows:
TGAGOGGATCCACTCACTGTCTOG-BHQ3，
the nucleic acid sequence of the TK1 recognition probe is as follows:
CCCAATCAOCTCGACCTOCTTCTC-BHQ3，
the nucleic acid sequence of the EpCAM recognition probe is:
CACATTCTTCCTGAGCTGOGGCAA-BHQ3，
the nucleic acid sequence of the CD91 recognition probe is as follows:
CCTCTGGTCCTGCTACTTCTCTCC-BHQ3，
the nucleic acid sequence of the PD-L1 recognition probe is as follows:
CTCCTCTCTCTTGGAATTGGTGGT-BHQ3，
the nucleic acid sequence of the VEGF recognition probe is:
TGCAGCCTGGGACCACTTGGCATG-BHQ3，
the nucleic acid sequence of the anchor breaking probe A is as follows:
Ad-AAAAAAAAAAAAAAAAAAAA；
s3, adding a fluorescent coding ferritin nanoparticle solution which has the same volume as the signal probe S and has the final concentration of 150-250nmol/L into the nucleic acid assembly S/A/R obtained in the step S2, simultaneously placing the solution into a constant-temperature oscillator with the temperature of 23-27 ℃ for reacting for 1.5-2.5 hours, removing unbound nucleic acid through an ultrafiltration centrifuge tube, and collecting the solution reserved in a tube core to obtain an activated fluorescent coding ferritin nano probe;
in the step S1, the feed ratio of the sodium fluorescein solution Flu and the rhodamine B solution RB was set to seven, flu: rb=6: 0. flu: rb=5:1, flu: rb=4:2, flu: rb=3:3, flu: rb=2:4, flu: rb=1:5 and Flu: rb= 0:6, correspondingly, seven active fluorescent-encoded ferritin nanoprobes obtained in step S3;
in step S2, adamantane for observing self-assembled binding between the anchor chain and ferritin through Zeta potential characterization is marked on the anchor breaking probe a.
2. The method for preparing the activated fluorescent-encoded ferritin nanoprobe according to claim 1, wherein the step S1 is preceded by the steps of:
s101, wetting a filter membrane by milliQ water, filtering APO through the filter membrane by a syringe, collecting filtrate,
repeating the steps, and transferring the filtrate to an ultrafiltration tube for centrifugation to obtain APO solution with a certain concentration;
s102, adding a plurality of MilliQ water into the APO solution obtained in the step S101, adding an HCl solution to adjust the pH value, and oscillating at the same time to fully depolymerize the APO.
3. An activated fluorescent-encoded ferritin nanoprobe, prepared by the method of any one of claims 1 to 2.
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