CN113640266B - Detection method for storing and releasing iron from ferritin in cells - Google Patents
Detection method for storing and releasing iron from ferritin in cells Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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- General Health & Medical Sciences (AREA)
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a detection method for storing and releasing iron from ferritin in cells, which comprises the following operation steps: s1: a step of examining cytotoxicity; s2: a characterization test step of functional ferritin; s3: a performance investigation step of functional ferritin; s4: detecting the iron ions in and out of the solution; s5: visual in situ detection of intracellular ferritin storage and release of iron. Through the mode, the invention further uses the method for visual in-situ detection of iron stored and released in cells based on detection of iron ions in/out of the solution by simple mixing construction of functional ferritin, thereby providing a new method and technical means for research of iron stored and released in cells and diagnosis and treatment of iron related diseases.
Description
Technical Field
The invention relates to a method for detecting functional ferritin in cells, belongs to the field of biology, and particularly relates to a method for detecting ferritin storage and release iron in cells.
Background
Ferritin is an important protein for storing iron in a body, the behavior of storing or releasing iron is closely related to the increase or decrease of the iron content in the body, and the abnormality of the iron content in the body is closely related to the occurrence, progress or prognosis of cardiovascular diseases, parkinsonism, cancers and the like. The ferritin can regulate and control the dynamic balance of the iron content in the organism or cells in a manner of storing and releasing iron, namely, when the available iron of the external source is insufficient, the ferritin can release the stored iron for the organism to use; when iron is excessive in the body, the iron can be absorbed and stored, and the iron level is reduced, so that the body is prevented from oxidative damage and the like. Therefore, the detection of iron storage and release behavior of ferritin is of great importance for the evaluation of iron content changes and iron related diseases.
Iron detection has been widely focused on by researchers, and various methods have been used for detecting iron. The analytical methods such as spectrophotometry, flame atomic absorption, colorimetry and the like are widely used, but are limited in application due to the problems of complex pretreatment, damage to samples or incapability of realizing in-situ detection. The fluorescence imaging technology can effectively solve the problems, has the advantages of high sensitivity, high space-time resolution, no damage, in-situ real-time detection and the like, and can realize the visual research on the cell microstructure from the subcellular or molecular level. However, at present, research on intracellular iron based on fluorescence imaging technology is often focused on how to realize the measurement of intracellular iron content, but related research on in-situ detection of ferritin stored/released iron in cells is rarely reported, and pure CDs can realize the measurement of intracellular iron content, but cannot realize the detection of intracellular ferritin stored/released iron.
In view of this, the present invention intends to construct functional ferritin (Apoferritin, APO) by simple mixing of fluorescent Carbon Dots (CDs) and Apoferritin (APO) with good biocompatibility by a self-assembly method. Based on the detection of the iron ions in/out of the solution, the method is further used for the visual in-situ detection of the iron stored and released by the ferritin in cells, thereby providing a new method and technical means for the research of the mechanism of the iron stored and released by the ferritin and the diagnosis and treatment of iron related diseases.
Disclosure of Invention
The invention mainly solves the technical problem of how to provide a method for constructing functional ferritin through simple mixing, and further uses the method for visualized in-situ detection of ferritin storage and release iron in cells on the basis of detecting ferritin in/out of solution, thereby providing a new method and a detection method of technical means for researching ferritin storage and release iron and diagnosing and treating iron-related diseases.
In order to solve the technical problems, the invention adopts a technical scheme that: there is provided an assay for ferritin storage and release of iron in a cell, the assay comprising the following steps:
s1: examining cytotoxicity;
s2: characterization test of functional ferritin;
s3: performance investigation of functional ferritin;
s4: detecting the iron ions in and out of the solution;
s5: visual in situ detection of intracellular ferritin storage and release of iron.
In a preferred embodiment, in step S2, carbon Dots (CDs) and Apoferritin (APO) are mixed to construct functional ferritin.
In a preferred embodiment, in step S2, the uv-vis absorption spectrum and the fluorescence spectrum are plotted according to the data of Carbon Dots (CDs), apoferritin (APO) and apo@cds, and it is confirmed whether Carbon Dots (CDs) are included in apo@cds or not in the uv-vis absorption spectrum and the fluorescence spectrum.
In a preferred embodiment, in step S2, a Zeta potential analyzer is used to measure Zeta potential of Carbon Dots (CDs) and apo@cds, and stability is determined according to the magnitude of the negative value of the Zeta potential of the nanoparticle;
in a preferred embodiment, in step S2, it is determined that a stable system is formed when the absolute value of the potential of the nanoparticle solution system is greater than 15 mV.
In a preferred embodiment, in step S4 and step S5, the detection of the iron ion in and out ferritin in the solution is performed by adopting a fluorescence visualization mode, and the following operation process is further included:
SS1: detecting ferritin in the solution;
SS2: detecting ferritin ion in the solution;
SS3: detection of ferritin storage and release of iron in cells.
In a preferred embodiment, wherein, in step SS1, when iron ions enter the ferritin cavity and react with Fe in the cavity 3+ Carbon dot reaction with specific response to realize Fe 3+ Detection of ferritin.
In a preferred embodiment, wherein, when Fe 3+ In the concentration range of 5 to 120mol/L, the fluorescence intensity of APO@CDs and Fe 3+ Exhibits a linear relationship with respect to the concentration of (a).
In a preferred embodiment, wherein, in step SS2, APO@CDs and Fe are tested 3+ And determining Fe based on the fluorescence ratio 3+ Whether or not to enter ferritin:
when the fluorescence ratio decreases, then Fe is determined 3+ Entering ferritin;
when the fluorescence ratio increases, it is determined that Fe entering ferritin is sequestered 3+ To make Fe 3+ Ferritin is produced.
In a preferred embodiment, wherein, in step SS3, APO@CDs and Fe are obtained by incubating APO@CDs with cells 3+ Co-incubated cells and APO@CDs, fe 3+ Cells incubated with deferoxamine mesylate (DFO) were imaged under a laser confocal microscope to detect intracellular ferritin storage and release of iron.
The beneficial effects of the invention are as follows: successfully constructs self-assembled functional ferritin, realizes the effect of Fe in solution 3+ Detection of in/out ferritin and further successfully used for visual in situ detection of intracellular ferritin storage and release of iron.
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 showing the construction of functional ferritin (APO@CDs), detection of iron ions in solution into and out of ferritin (B), and detection of ferritin stored and released iron in cells (C) according to an embodiment of the method for detecting ferritin stored and released iron in cells of the present invention;
FIG. 2 is a graph showing the UV-visible absorption spectra of CDs, APO, APO@CDs in an embodiment of the method for detecting ferritin storage and release of iron in cells according to the present invention;
FIG. 3 is a fluorescence spectrum of APO@CDs and CDs in an embodiment of the method for detecting ferritin storage and release in cells of the present invention;
FIG. 4 is a graph showing the response of APO@CDs, CDs to different metal ions in an embodiment of the method for detecting ferritin storage and release of iron in a cell according to the present invention;
FIG. 5 shows the APO@CDs vs. Fe in an embodiment of the method for detecting ferritin storage and release in cells according to the present invention 3+ Is a response to the condition;
FIG. 6 shows APO@CDs and Fe according to an embodiment of the method for detecting ferritin storage and release in cells of the present invention 3+ Response to DFO;
FIG. 7 is a cytotoxicity assay of CDs, APO@CDs according to an embodiment of the method for detecting ferritin storage and release of iron in cells of the present invention;
FIG. 8 is a laser confocal imaging of A549 cells under different processing conditions in an embodiment of the detection method of the invention for ferritin storage and release of iron in cells.
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 FIGS. 1-8, in one embodiment of the present invention, a method for detecting ferritin storage and release of iron in a cell is provided, comprising the following steps:
specifically, referring to fig. 1, based on the pH-dependent depolymerization/recombination characteristics of APO, after APO is depolymerized into a subunit state under acidic conditions, a CDs solution is added, and simple one-step mixing is performed, and the pH of the mixed solution is adjusted to be neutral to enable ferritin subunits to be repolymerized into a spherical structure, and fluorescent CDs are coated in the cavities of the mixed solution, so that functional ferritin (apo@cds) with iron ion response characteristics can be successfully constructed.
By means of Fe in solution 3+ Can quench the fluorescence of APO@CDs and realize the reaction of Fe 3+ Detection of ferritin, wherein Fe 3+ Fluorescence quenching APO@CDs is derived from Fe 3+ The APO can enter an APO cavity through a hydrophilic channel of the APO to react with CDs; thereafter, DFO is used to compete with CDs for Fe binding 3+ So that Fe is 3+ The fluorescent light of APO@CDs is recovered after falling off from the surface of CDs, and Fe can be realized 3+ Ferritin assay (fig. 1B). Successful realization of Fe in solution 3+ Based on the detection of the in/out ferritin, according to the same principle as that in the solution, using a laser confocal microscope to respectively incubate cells of APO@CDs, APO@CDs and Fe 3+ Co-incubated cells and APO@CDs, fe 3+ Imaging cells incubated by DFO, and detecting ferritin storage and release iron in the cells by comparing the cell fluorescence imaging gray values under different incubation conditions while visualizing APO@CDs fluorescence quenching and recovery in the cells.
In an embodiment, a method for detecting ferritin storage and release of iron in a cell comprises the following steps:
s1: a step of examining cytotoxicity;
s2: a characterization test step of functional ferritin;
s3: a performance investigation step of functional ferritin;
s4: detecting the iron ions in and out of the solution;
s5: visual in situ detection of intracellular ferritin storage and release of iron.
In step S1, toxicity investigation is performed on cells, and growth is performedWell conditioned A549 cells were seeded into 96-well plates, 5X 10 cells per well 3 Individual cells, medical cotton balls soaked in 75% medical alcohol are wiped on a 96-well plate, the temperature is 37 ℃, and CO is 5% 2 Culturing for 24 hours under the condition; sucking out cell culture solution, adding 90L 1640 culture medium and 10L APO@CDs and CDs solutions with different concentrations (the final concentrations are 100 g/mL, 250 g/mL, 500g/mL and 1000 g/mL) into each hole, setting a control group and a blank group, and continuously culturing for 12h; sucking out the cell culture solution, adding 90L 1640 culture medium and 10L CCK-8 into each hole, and continuously culturing for 1-4 h; the wavelength is set to be 450nm, the absorbance value is measured by a multifunctional microplate reader, and the cell survival rate is calculated according to the measurement result, wherein the formula is as follows:
apo@cds have significantly reduced cytotoxicity compared to CDs.
Wherein, in the preparation of the functional ferritin, the method comprises the following steps:
(1) APO depolymerization: placing 5 mu L of APO into a 1.5mL centrifuge tube, adding 195 mu L of Milli-Q water subjected to autoclaving, slowly dripping 0.1mol/L HCl solution into the APO solution to adjust the solution to be acidic, depolymerizing the APO, and vibrating for 2 hours at room temperature;
(2) Adding CDs solution: dropwise adding the subunit solution of APO into 100 mu L of CDs solution, and oscillating for 40min;
(3) APO recombination: slowly dripping 0.1mol/L NaOH into the mixed solution to adjust the solution to be neutral after the reaction is finished, so that dissociated subunits are polymerized again into APO with a cage-shaped structure, and meanwhile, CDs are coated in a cavity of the APO, and vibrating for 2 hours at room temperature;
(4) And (3) filtering a membrane: filtering the reaction solution through a 0.22 μm filter membrane (removing sediment possibly generated in the reaction), washing the filter membrane with a proper amount of Milli-Q water after autoclaving, and collecting filtrate;
(5) Centrifugal ultrafiltration: centrifuging the collected filtrate with ultrafiltration tube (30 kDa,15 mL) at 8000r/min at 4deg.C for 4min, removing CDs not covered in APO cavity in the reaction solution, collecting filtrate, adding appropriate amount of Milli-Q water sterilized under high pressure into the tube core, mixing, centrifuging again (centrifuging at 8000r/min at 4deg.C for 4 min), repeating the operation for three times, and collecting filtrate after centrifuging each time;
(6) Collecting a sample: after centrifugation, APO@CDs solution in the tube core is collected and stored in a refrigerator at the temperature of 4 ℃ for later use.
In the characterization of functional ferritin, the apo@cds are necessary, so that the prepared apo@cds need to be characterized to ensure that the apo@cds are prepared successfully, specifically comprising the following test methods:
(1) Characterization of ultraviolet-visible absorption spectra of CDs, APO and APO@CDs;
and measuring ultraviolet-visible absorption spectrums of CDs, APO and APO@CDs by using an ultraviolet spectrophotometer, wherein the scanning range is 230-500nm, and the scanning speed is 400nm/min. Meanwhile, establishing a standard curve by utilizing characteristic absorption peaks of CDs to calculate the entrapment rate of the CDs, wherein the entrapment rate is calculated according to the following formula:
(2) Fluorescence spectrum characterization of CDs and APO@CDs;
and (3) measuring by adopting a fluorescence spectrometer, wherein the excitation wavelength is 366nm, and scanning the fluorescence emission spectra of CDs and APO@CDs.
(3) Zeta potential characterization of CDs and APO@CDs;
the electric potential of CDs and APO@CDs is measured by adopting a nano-particle size and Zeta electric potential analyzer, the volume of the solution is 1mL, and the solvent is Milli-Q water.
In step S2, carbon Dots (CDs) and Apoferritin (APO) are mixed by a self-assembly method to construct functional ferritin. And drawing an ultraviolet visible absorption spectrum and a fluorescence spectrum according to the data of the CDs, the APO and the APO@CDs, and confirming whether carbon dots are contained in the APO@CDs or not in the ultraviolet visible absorption spectrum and the fluorescence spectrum.
Specifically, referring to FIG. 2, the ultraviolet-visible absorption spectra of CDs, APO, APO@CDs are shown in FIG. 2, and the individual CDs solution has a distinct absorption peak at 311 nm; the APO solution alone has a distinct absorption peak at 280 nm; the ultraviolet-visible absorption spectrum of APO@CDs shows an obvious absorption peak at 280nm, which indicates that the APO exists in the APO@CDs, and no obvious CDs absorption peak exists, so that the fluorescence spectrum is further used for characterizing whether the APO@CDs are packed with CDs or not.
In FIG. 3, the position of the emission peak of APO@CDs is 450nm and the position of the emission peak of CDs is 461nm at an excitation wavelength of 366nm, in contrast to the blue shift of the position of the emission peak of APO@CDs. The reason for this may be that the APO cavity encapsulates a plurality of CDs, so that dipole-dipole interactions between the CDs occur, stokes shift occurs, and the fluorescence spectrum of apo@cds changes relative to the CDs. And combining ultraviolet visible absorption spectrum and fluorescence spectrum characterization results to show that APO@CDs are successfully prepared.
In the step S2, a Zeta potential analyzer is adopted to measure the Zeta potential of the carbon dots and APO@CDs, and the stability is judged according to the negative value of the Zeta potential of the nano particles; when the absolute value of the potential of the nanoparticle solution system was greater than 15mV, it was judged that a stable system was formed.
In the step S4 and the step S5, the detection of the ferritin in and out of the solution is carried out by adopting a fluorescence display mode, and the method further comprises the following operation processes:
SS1: detecting ferritin in the solution;
SS2: detecting ferritin ion in the solution;
SS3: detection of ferritin storage and release of iron in cells.
In step SS1, when iron ions enter the ferritin cavity and react with Fe in the cavity 3+ Carbon dot reaction with specific response to realize Fe 3+ Detection of ferritin.
When Fe is 3+ In the concentration range of 5 to 120mol/L, the fluorescence intensity of APO@CDs and Fe 3+ Exhibits a linear relationship with respect to the concentration of (a).
In step SS2, APO@CDs and Fe are tested 3+ And determining Fe based on the fluorescence ratio 3+ Whether or not to enter ferritin:
when fluorescence ratioWhen the Fe content decreases, it is determined that Fe 3+ Entering ferritin;
when the fluorescence ratio increases, it is determined that Fe entering ferritin is sequestered 3+ To make Fe 3+ Ferritin is produced.
In the performance investigation of functional ferritin, in fig. 4: and (3) injection: group 1: blank; group 2: al (Al) 3+ The method comprises the steps of carrying out a first treatment on the surface of the Group 3: ca (Ca) 2+ The method comprises the steps of carrying out a first treatment on the surface of the Group 4: cu (Cu) 2+ The method comprises the steps of carrying out a first treatment on the surface of the Group 5: fe (Fe) 2+ The method comprises the steps of carrying out a first treatment on the surface of the Group 6: fe (Fe) 3+ The method comprises the steps of carrying out a first treatment on the surface of the Group 7: k (K) + The method comprises the steps of carrying out a first treatment on the surface of the Group 8: mg of 2+ The method comprises the steps of carrying out a first treatment on the surface of the Group 9: na (Na) + The method comprises the steps of carrying out a first treatment on the surface of the Group 10: zn (zinc) 2+ 。
Referring to fig. 4, apo@cds are surrounded by a protein shell, and ions or other molecules need to enter through the channels of the coated protein shell to react with the CDs in the apo@cds, and the channels of the protein shell only allow certain ions or small molecules to pass through, thus possibly weakening the response to apo@cds by preventing the entry of certain metal ions. Thus, this study compares the selectivity of apo@cds with CDs. That is, under the condition that the concentrations of APO@CDs and CDs are equal, the APO@CDs and the CDs react with different metal ions with the same concentration respectively, wherein the concentration of the APO@CDs is calculated according to the amount of the entrapped CDs, and the final concentration of each metal ion is 50mol/L. As shown in FIG. 4, APO@CDs and CDs vs. Fe 3+ 、Fe 2+ 、Cu 2+ The response is achieved, but compared with CDs, the quenching effect of the iron ions on APO@CDs is obviously enhanced, so that the sensitivity of the APO@CDs is better than that of the CDs, the iron ions are more specifically responded, and the protein shell can effectively enhance the response of the APO@CDs to the iron ions.
Meanwhile, experiments show that APO@CDs have stable fluorescence luminescence properties at different temperatures. But at different temperatures APO@CDs vs. Fe 3+ There is a certain difference in response of APO@CDs to Fe with increasing temperature (25 ℃,37 ℃, 50 ℃) 3+ The response of the composition is enhanced and then weakened, and the response effect is optimal at 37 ℃.
In the implementation process, a Zeta potential analyzer is adopted to measure Zeta potential of CDs and APO@CDs. In general, the more negative the Zeta potential of the nanoparticle, the more stable it is, and an absolute value of the potential of the nanoparticle solution system of greater than 15mV indicates that it forms a stable system. The potential of the CDs was measured to be-2.45 mV and the potential of the APO@CDs was measured to be-21.1 mV, indicating that the APO@CDs solution was sufficiently stable and superior to the CDs solution alone. And meanwhile, the successful preparation of APO@CDs can be further confirmed.
In step SS3, APO@CDs and Fe are obtained by incubating APO@CDs with cells 3+ Co-incubated cells and APO@CDs, fe 3+ Cells incubated with deferoxamine mesylate (DFO) were imaged under a laser confocal microscope to detect intracellular ferritin storage and release of iron.
In the above process, iron ions can enter the lumen from the protein shell through the triple axis of ferritin. Referring specifically to FIG. 5, the present invention utilizes ferric ions to enter the ferritin cavity and react with Fe in the cavity 3+ CDs reaction with specific response to realize Fe 3+ Detection of ferritin. As can be seen from the figure, APO@CDs+Fe 3+ The fluorescence ratio of (2) is significantly lower than APO@CDs due to Fe 3+ Reacts with APO@CDs, fe 3+ Quenching the fluorescence of APO@CDs, indicating that iron ions entered ferritin, in FIG. 5 (A) APO@CDs vs. Fe 3+ The corresponding fluorescence spectrum is shown in (B) that the corresponding relative fluorescence intensity of (A) is shown in (A), the final concentration of APO@CDs is 172g/mL, fe 3+ Final concentration of 50mol/L, F 0 Is the average value of the fluorescence intensity of APO@CDs solution.
Preferably, APO@CDs and Fe 3+ The results of the DFO reaction are shown in fig. 6, where in the graph, group 1: apo@cds; group 2: APO@CDs+Fe 3+ The method comprises the steps of carrying out a first treatment on the surface of the Group 3: APO@CDs+DFO (100 mol/L); group 4: APO@CDs+Fe 3+ +DFO (100 mol/L); group 5: DFO (100 mol/L) +Fe 3+ +APO@CDs; group 6: APO@CDs+Fe 3+ +DFO (50 mol/L); group 7: APO@CDs+Fe 3+ +DFO (75 mol/L); group 8: APO@CDs+Fe 3+ +DFO (150 mol/L) in which the final concentration of APO@CDs is 172g/mL, fe 3+ Final concentration of 50mol/L, F 0 The average of the fluorescence intensities of group 1 (APO@CDs solution) was determined.
Referring to FIG. 6, in particular, the fluorescence ratio was reduced for group 1 compared to group 2, illustrating Fe 3+ Entering ferritin; fluorescence ratio of group 3 compared to group 1A slight decrease, indicating that DFO may have a slight effect on the fluorescence of apo@cds; the increased fluorescence ratio of group 4 compared to group 2 indicates that DFO is able to sequester Fe into ferritin 3+ To make Fe 3+ Ferritin; the fluorescence ratios of group 3 and group 5 were almost equal compared to groups 1, 3 and 5, indicating Fe 3+ Almost all reacted with DFO without entering ferritin, whereas fluorescence ratios less than group 1 may be due to DFO; group 2, group 6, group 7, group 4 and group 8 showed a gradual increase in fluorescence ratio, indicating that the fluorescence of APO@CDs gradually recovered with increasing DFO concentration, fe 3+ The amount of apoferritin gradually increases. Taken together, apo@cds were shown to be able to effect detection of iron ion in and out ferritin.
Referring to FIG. 7, in the above examples, the cytotoxicity of CDs and APO@CDs was detected using the CCK-8 kit. As can be seen from the graph, the cell viability was 60.0% at a CDs concentration of 500g/mL, but the cell viability was reduced to 27.0% at an increase in CDs concentration of 1000 g/mL; however, when the concentration of APO@CDs is 500g/mL (the concentration is calculated by the amount of entrapped CDs), the survival rate of the cells is more than 90%, and when the concentration reaches 1000g/mL, the survival rate of the cells is still higher than 78.8%, which shows that the toxicity of the APO@CDs to the cells is obviously reduced compared with that of the CDs.
Wherein, APO@CDs mentioned in the invention are functional ferritin, DFO is deferoxamine, CDs are carbon dots, fluorescent carbon dots, and Apoferritin and APO are used as Apoferritin.
Referring to FIG. 8, (a-c) are fluorescent images of laser confocal microscopy under different processing conditions for A549 cells; (d-f) is the bright field corresponding to (a-c); (a) A549 cells incubated at 500g/mL APO@CDs, (b) APO@CDs at 500g/mL, 300mol/L Fe 3+ Co-incubated A549 cells, (c) 500g/mL APO@CDs, 300mol/L Fe 3+ A549 cells incubated with 300mol/L DFO.
Successful realization of Fe in solution 3+ APO@CDs are further used for intracellular ferritin storage and iron release detection based on the in/out ferritin detection. APO@CDs and Fe by incubating APO@CDs with cells 3+ Co-incubated cells and APO@CDs, fe 3+ And methanesulfonic acid removalCells incubated with iron amine (DFO) were imaged under a laser confocal microscope to detect iron storage and release from intracellular ferritin, and it can be seen from the figure that apo@cds incubated cells had bright fluorescence, indicating that apo@cds could enter cells well, a phenomenon associated with good biocompatibility of biogenic apoferritin and transferrin receptor overexpressed on the cancer cell surface was beneficial for apo@cds to enter cells.
APO@CDs and Fe 3+ Fluorescence intensity in co-incubated cells was significantly reduced compared to cells incubated with APO@CDs alone, indicating Fe 3+ The reaction with APO@CDs quenched the fluorescence of APO@CDs, indicating that ferritin stores Fe 3+ 。APO@CDs、Fe 3+ Fluorescence intensity in cells incubated with DFO compared to APO@CDs and Fe 3+ The co-incubated cells had a significant increase, but the fluorescence intensity was reduced compared to cells incubated with APO@CDs alone, indicating that DFO treatment resulted in Fe 3+ From APO@CDs, fluorescence of APO@CDs is recovered, which indicates that ferritin releases Fe 3+ . Taken together, apo@cds allow intracellular detection of ferritin storage and release of iron.
The detection method for storing and releasing iron from ferritin in cells successfully constructs self-assembled functional ferritin (APO@CDs) and realizes the effect of Fe in solution 3+ Detection of in/out ferritin and further successfully used for visual in situ detection of intracellular ferritin storage and release of iron. The successful design and application of the functional ferritin are expected to be used for visualizing the dynamic process of ferritin storage and release of iron, and provide a new method and technical means for researching the mechanism of ferritin storage and release of iron and diagnosing and treating iron-related diseases. (1) APO@CDs vs. Fe in solution 3+ The detection limit of (2) is 2.5mol/L; after treatment by Deferoxamine (DFO), fluorescence of APO@CDs is recovered, which shows that APO@CDs in solution can realize detection of iron ion in/out ferritin; (2) The laser confocal imaging result shows that APO@CDs can realize the visual in-situ detection of ferritin storage and release iron in cells.
Therefore, the invention has the following advantages:
1. the self-assembly mode is utilized to successfully construct functional ferritin (APO@CDs) by simply mixing fluorescent Carbon Dots (CDs) and Apoferritin (APO) with good biocompatibility, and the functional ferritin (APO@CDs) can be used for detecting ferritin in/out of solution and can be further used for visual in-situ detection of ferritin storage and release iron in cells;
2. the sensitivity and the specificity of APO@CDs to the response of iron ions are better than those of the single CDs; APO@CDs have significantly reduced cytotoxicity compared to CDs;
3. provides a new method and technical means for researching iron storage and release of ferritin and diagnosing and treating iron related diseases.
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 (10)
1. A method for detecting storage and release of iron from ferritin in a cell, the method comprising the steps of:
s1: examining cytotoxicity;
s2: characterization test of functional ferritin;
s3: performance investigation of functional ferritin;
s4: detecting the iron ions in and out of the solution;
s5: visual in situ detection of intracellular ferritin storage and release of iron;
in step S1, the investigation of cytotoxicity involves the preparation of functional ferritin, comprising the steps of:
(1) APO depolymerization: placing 5 mu LAPO into a 1.5mL centrifuge tube, adding 195 mu L of Milli-Q water subjected to autoclaving, slowly dropwise adding 0.1mol/LHCl solution into the APO solution to adjust the solution to be acidic, depolymerizing APO, and vibrating for 2h at room temperature;
(2) Adding CDs solution: dropwise adding the subunit solution of APO into the 100 mu LCDs solution, and oscillating for 40min;
(3) APO recombination: after the reaction is finished, slowly dripping 0.1mol/LNaOH into the mixed solution to adjust the solution to be neutral, so that dissociated subunits are polymerized again into APO with a cage-shaped structure, and meanwhile, CDs are coated in a cavity of the APO, and vibrating for 2 hours at room temperature;
(4) And (3) filtering a membrane: filtering the reaction solution through a 0.22 mu m filter membrane, removing sediment possibly generated in the reaction, flushing the filter membrane with a proper amount of Milli-Q water subjected to high-pressure sterilization, and collecting filtrate;
(5) Centrifugal ultrafiltration: centrifuging the collected filtrate with a ultrafilter tube at a temperature of 8000r/min for 4min at a temperature of 4deg.C, removing CDs not coated in the APO cavity in the reaction solution, collecting filtrate, adding appropriate amount of Milli-Q water sterilized under high pressure into the tube core, blowing and mixing with a pipetting gun, centrifuging again, centrifuging at a temperature of 4deg.C for 4min at a temperature of 8000r/min, repeating the operation for three times, and collecting filtrate after each centrifuging is completed;
(6) Collecting a sample: after centrifugation, APO@CDs solution in the tube core is collected and stored in a refrigerator at the temperature of 4 ℃ for later use.
2. The method for detecting ferritin storage and release of iron in cells according to claim 1, wherein in step S2, carbon dots and apoferritin are mixed by self-assembly to construct functional ferritin.
3. The method according to claim 2, wherein in step S2, the uv-vis absorption spectrum and the fluorescence spectrum are plotted according to the data of carbon dots, apo@cds, and it is confirmed whether carbon dots are included in apo@cds in the uv-vis absorption spectrum and the fluorescence spectrum.
4. The method for detecting ferritin storage and release of iron in cells according to claim 3, wherein in step S2, zeta potential measurement is performed on carbon dots, apo@cds using a Zeta potential analyzer, and stability is determined based on the magnitude of the negative value of the Zeta potential of the nanoparticles;
5. the method according to claim 4, wherein in step S2, it is determined that a stable system is formed when the absolute value of the potential of the nanoparticle solution system is greater than 15 mV.
6. The method for detecting ferritin storage and release of iron in cells according to claim 1, wherein in step S4 and step S5, detection of ferritin in and out of solution is performed by fluorescence visualization, further comprising the following steps:
SS1: detecting ferritin in the solution;
SS2: detecting ferritin ion in the solution;
SS3: detection of ferritin storage and release of iron in cells.
7. The method according to claim 6, wherein, in step SS1, when iron ions enter the ferritin cavity and react with Fe in the cavity 3+ Carbon dot reaction with specific response to realize Fe 3+ Detection of ferritin.
8. The method for detecting iron storage and release in cells according to claim 7, wherein when Fe 3+ In the concentration range of 5 to 120mol/L, the fluorescence intensity of APO@CDs and Fe 3+ Exhibits a linear relationship with respect to the concentration of (a).
9. The method for detecting iron storage and release in cells according to claim 6, wherein apo@cds and Fe are tested in step SS2 3+ And determining Fe based on the fluorescence ratio 3+ Whether or not to enterFerritin:
when the fluorescence ratio decreases, then Fe is determined 3+ Entering ferritin;
when the fluorescence ratio increases, it is determined that Fe entering ferritin is sequestered 3+ To make Fe 3+ Ferritin is produced.
10. The method for detecting iron storage and release in cells according to claim 6, wherein, in step SS3, apo@cds and Fe are detected by incubating apo@cds in cells 3+ Co-incubated cells and APO@CDs, fe 3+ Cells incubated with deferoxamine mesylate were imaged under a laser confocal microscope to detect intracellular ferritin storage and release of iron.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5552268A (en) * | 1994-08-01 | 1996-09-03 | Quixote Associates, Inc. | Method for measuring total body tissue iron stores |
| WO2003078467A1 (en) * | 2002-03-14 | 2003-09-25 | Children's Hospital & Research Center At Oakland | Enhancement of iron chelation therapy |
| WO2012137202A1 (en) * | 2011-04-05 | 2012-10-11 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Composition comprising an iron indicator attached to a microparticle and uses of same for quantifying non-transferrin bound iron (ntbi) and cellular labile iron (lci) |
| CN104062287A (en) * | 2014-07-01 | 2014-09-24 | 福建工程学院 | Method for detecting ferritin based on nanogold catalytic chemiluminescence analysis |
| JP2016211889A (en) * | 2015-04-30 | 2016-12-15 | コニカミノルタ株式会社 | Fluorescent probe |
| CN109705841A (en) * | 2018-12-24 | 2019-05-03 | 山西大学 | A kind of transferrins is the gold nano cluster and its preparation method and application of template |
| CN110129032A (en) * | 2019-05-16 | 2019-08-16 | 闽江学院 | A kind of Fluorescence Identification method of ferritin |
| CN110208236A (en) * | 2019-06-28 | 2019-09-06 | 郑州大学 | Ratio-type fluorescent pH nano-probe based on apoferritin, preparation method and applications |
| CN110715915A (en) * | 2019-11-06 | 2020-01-21 | 东北师范大学 | Method for detecting ferric ions based on carbon quantum dots prepared from egg white |
| WO2020239809A1 (en) * | 2019-05-28 | 2020-12-03 | Previpharma Consulting Gmbh | Method for preparing iron-depleted ferritin and use thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080292545A1 (en) * | 2007-04-04 | 2008-11-27 | Yuehe Lin | Functionalized Encoded Apoferritin Nanoparticles and Processes for Making and Using Same |
| JP5382489B2 (en) * | 2008-03-29 | 2014-01-08 | 国立大学法人 奈良先端科学技術大学院大学 | Circularly polarized light-emitting nanoparticles |
| US9291619B2 (en) * | 2011-10-04 | 2016-03-22 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Method for detecting non-transferrin bound iron |
-
2021
- 2021-08-11 CN CN202110918246.7A patent/CN113640266B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5552268A (en) * | 1994-08-01 | 1996-09-03 | Quixote Associates, Inc. | Method for measuring total body tissue iron stores |
| WO2003078467A1 (en) * | 2002-03-14 | 2003-09-25 | Children's Hospital & Research Center At Oakland | Enhancement of iron chelation therapy |
| WO2012137202A1 (en) * | 2011-04-05 | 2012-10-11 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Composition comprising an iron indicator attached to a microparticle and uses of same for quantifying non-transferrin bound iron (ntbi) and cellular labile iron (lci) |
| CN104062287A (en) * | 2014-07-01 | 2014-09-24 | 福建工程学院 | Method for detecting ferritin based on nanogold catalytic chemiluminescence analysis |
| JP2016211889A (en) * | 2015-04-30 | 2016-12-15 | コニカミノルタ株式会社 | Fluorescent probe |
| CN109705841A (en) * | 2018-12-24 | 2019-05-03 | 山西大学 | A kind of transferrins is the gold nano cluster and its preparation method and application of template |
| CN110129032A (en) * | 2019-05-16 | 2019-08-16 | 闽江学院 | A kind of Fluorescence Identification method of ferritin |
| WO2020239809A1 (en) * | 2019-05-28 | 2020-12-03 | Previpharma Consulting Gmbh | Method for preparing iron-depleted ferritin and use thereof |
| CN110208236A (en) * | 2019-06-28 | 2019-09-06 | 郑州大学 | Ratio-type fluorescent pH nano-probe based on apoferritin, preparation method and applications |
| CN110715915A (en) * | 2019-11-06 | 2020-01-21 | 东北师范大学 | Method for detecting ferric ions based on carbon quantum dots prepared from egg white |
Non-Patent Citations (11)
| Title |
|---|
| Apoferritin-based tunable nano-indicator for intracellular pH sensing: Regulating response performances and minimizing effects of system flctuations;Leiliang He et al.;Sensors & Actuators: B. Chemical;第323卷;全文 * |
| Apoferritin-based tunable nano-indicator for intracellular pH sensing: Regulating response performances and minimizing effects of system flctuations;leiliang He et al.;Sensors and Actuators:B.Chemical;第323卷;全文 * |
| Cuiping Han et al..Highly fl uorescent carbon dots as selective and sensitive " on-off-on" probes for iron(III) ion and apoferritin detection and imaging in living cells.Biosensors and Bioelectronics.2016,第83卷全文. * |
| Enzyme Encapsulation by a Ferritin Cage;Stephan Tetter and Donald Hilvert;Angew.Chem.Int.Ed.;第56卷;全文 * |
| Ferritin: A Versatile Building Block for Bionanotechnology;Gunther Jutz et al.;Chem.Rev.;第115卷;全文 * |
| Highly fl uorescent carbon dots as selective and sensitive " on-off-on" probes for iron(III) ion and apoferritin detection and imaging in living cells;Cuiping Han et al.;Biosensors and Bioelectronics;第83卷;全文 * |
| 荧光纳米材料应用于蛋白质分析的研究进展;王琪;王蓓蓓;马美湖;蔡朝霞;;食品科学(第05期);全文 * |
| 铁蛋白反应器储存有毒金属离子的初步研究;林庆梅, 乔玉欢, 黄河清;厦门大学学报(自然科学版)(第06期);全文 * |
| 铁蛋白脱铁机制的建立及脱铁前后光谱变化的研究;刘文营;张建;童军茂;赵广华;;光谱学与光谱分析(第09期);全文 * |
| 铁蛋白表面修饰及其应用;杨彩云等;化学进展(第01期);全文 * |
| 黄豆铁蛋白提取新方法及其与豌豆铁蛋白活性比较;胡菊;廖夏云;邓建军;胡小松;赵广华;;高等学校化学学报(10);全文 * |
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