CN115873596B - Luminescent carbon nanomaterial and application thereof in bone-derived alkaline phosphatase detection - Google Patents
Luminescent carbon nanomaterial and application thereof in bone-derived alkaline phosphatase detection Download PDFInfo
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a luminescent carbon nanomaterial, which is prepared by the following steps: preparing nitrogen-doped carbon dots; and carrying out coordination aggregation on the prepared nitrogen-doped carbon dots and zirconium oxygen clusters to obtain zirconium-coordinated carbon dot dendritic macromolecules (Zr-CD DRs), namely the luminescent carbon nanomaterial. The electrochemiluminescence efficiency of the luminescent carbon nanomaterial is higher than that of the nitrogen-doped carbon dots. The invention also discloses an electrochemiluminescence immunosensor, which comprises an immunosensor containing Zr-CD DRs and a probe containing nickel-phenol coordination nanospheres (Ni-PCS); the Zr-CD DRs are electrochemical luminescence signal labels and electrode modification materials, and the Ni-PCS is a signal quenching material. The electrochemiluminescence immunosensor has excellent sensitivity, specificity, stability and clinical practicability when detecting bone-derived alkaline phosphatase.
Description
Technical Field
The invention relates to a luminescent carbon nanomaterial and application thereof in bone source alkaline phosphatase detection, belonging to the field of electrochemical luminescence sensors.
Background
Bone-derived alkaline phosphatase (BALP) is secreted by osteoblasts, and mainly reflects the activity and function of osteoblasts, and is an important index of bone formation. Currently, the main methods of BALP detection rely on enzyme-linked immunosorbent assay (ELISA) and colorimetric methods. However, the conventional method for detecting BALP by immunological recognition has the problems of poor sensitivity, complex operation, harsh reaction conditions and the like. Therefore, there is a great need to develop simple, enzyme-free and sensitive BALP detection techniques.
Electrochemical luminescence (ECL) immunoassays are widely used because of their high sensitivity, low background, and good specificity. Carbon Dots (CDs) are used as an emerging luminescent carbon nanomaterial, and have the outstanding advantages of low cost, good biocompatibility, easiness in preparation and the like. It was found that the luminescence properties of CDs are largely dependent on their ultra-small size and surface functionality. Therefore, an increase in CDs size significantly reduces the luminous efficiency. In addition, conventional enrichment and immobilization strategies such as electrostatic adsorption and covalent crosslinking may increase the particle size of CDs due to aggregation in practical expansion applications of CDs, resulting in aggregation-induced luminescence quenching effects. In view of this property, it is critical to enrich CDs and maintain a suitable distance to preserve high luminous efficiency of CDs, and thus organic polymer encapsulation can be utilized to enrich CDs. However, organic polymers hinder interactions between CDs and coreactants, thereby reducing ECL performance, which limits their further use in ECL bioassays. Thus, the above-described methods cannot maintain or even increase ECL efficiency while enriching and immobilizing CDs.
Disclosure of Invention
The invention provides a luminescent carbon nanomaterial which can be used for holding or improving ECL efficiency while immobilizing CDs.
In order to achieve the purpose, the technical scheme adopted by the invention is that the luminescent carbon nanomaterial is prepared by the following method: preparing nitrogen-doped carbon dots; and carrying out coordination aggregation on the prepared nitrogen-doped carbon dots and zirconium oxygen clusters to obtain zirconium-coordinated carbon dot dendritic macromolecules (Zr-CD DRs), namely the luminescent carbon nanomaterial.
Further, the nitrogen-doped carbon dots are synthesized by taking citric acid and hydrazine hydrate as raw materials and adopting a solvothermal method.
Further, the solvothermal method for synthesizing the nitrogen-doped carbon dot specifically comprises the following steps: dissolving citric acid in Dimethylformamide (DMF), and then adding hydrazine hydrate dropwise to the DMF solution under stirring to form a mixture; transferring the mixture into a high-pressure reaction kettle with polytetrafluoroethylene lining for solvothermal reaction, cooling to room temperature after the reaction is completed, and centrifuging to obtain an upper solution.
Further, the coordination aggregation of the nitrogen-doped carbon dots and the zirconium oxygen clusters specifically comprises the following steps: zrOCl 2 ·8H 2 O is dissolved in DMF containing nitrogen-doped carbon dots, and is obtained by heating and cooling to room temperature and then separating.
The invention also provides an electrochemiluminescence immunosensor, which comprises an immunosensor containing Zr-CD DRs and a probe containing nickel-phenol coordination nanospheres (Ni-PCS); the Zr-CD DRs are electrochemical luminescence signal labels and electrode modification materials, and the Ni-PCS is a signal quenching material.
Further, the immunosensor containing Zr-CD DRs is a BSA/Ab1/AgNPs/Zr-CD DRs/CS immunosensor, and is prepared by the following steps: s1, dispersing Zr-CD DRs in a chitosan solution, and then dropwise adding the dispersed Zr-CD DRs onto the surface of a pretreated glassy carbon electrode to obtain a Zr-CD DRs/CS modified electrode; s2, silver nano particles (AgNPs) solution is dripped on the surface of the Zr-CD DRs/CS modified electrode, and the AgNPs/Zr-CD DRs/CS modified electrode is obtained; s3, dripping the BALP antibody (Ab 1) to the surface of the AgNPs/Zr-CD DRs/CS modified electrode obtained in the step S2 to obtain an Ab1/AgNPs/Zr-CD DRs/CS modified electrode; s4, dropwise adding a blocking agent BSA solution to the surface of the electrode modified by the Ab1/AgNPs/Zr-CD DRs/CS obtained in the step S3 to obtain the BSA/Ab1/AgNPs/Zr-CD DRs/CS immunosensor.
Further, the probe of the nickel-containing phenol coordination nanosphere (Ni-PCS) is an Ab2-Pt@Ni-PCS quenching probe, and the Ab2-Pt@Ni-PCS quenching probe is prepared by the following steps: synthesizing Ni-PCS by formaldehyde auxiliary metal ligand crosslinking method; dispersing the Ni-PCS solution in deionized water containing polyvinylpyrrolidone (PVP), mixing, adding H 2 PtCl 6 Slightly stirring and then adding a reducing agent NaBH 4 The reaction and separation are carried out to obtain the platinum nano particle functionalized nickelPhenol coordinated nanospheres (Pt@Ni-PCS); and re-dispersing the Pt@Ni-PCS in PBS, and sequentially adding a BALP antibody (Ab 2) and a BSA solution to obtain the Ab2-Pt@Ni-PCS quenching probe.
The invention provides a method for detecting bone-derived alkaline phosphatase, which comprises the following steps: the electrochemiluminescence immunosensor is used as a working electrode to be connected with an electrochemiluminescence detector; taking Phosphate Buffer Solution (PBS) as a detection base solution, and adding a coreactant Triethylamine (TEA) into the PBS for detection; and detecting the intensity of the electrochemiluminescence signal generated by the BALP with different concentrations by an electrochemiluminescence method under the preset condition of the electrochemiluminescence detector.
Further, the electrochemiluminescence detector is an ultra-weak electrochemiluminescence detector.
Further, the preset conditions comprise a photomultiplier with a high voltage of 800V, an electrochemical workstation cyclic voltammetry scanning potential range of 0.2-0.8V and a scanning rate of 0.2V/s.
Compared with the traditional luminescent material, the luminescent carbon nanomaterial prepared by the invention has smaller biotoxicity and excellent luminescent property of Zr-CD DRs; the synthesis method of the zirconium oxygen cluster coordination enrichment CDs ensures that CDs are more stable, ECL quantum efficiency is obviously improved, and ECL signals of the CDs are further improved.
The electrochemical luminescence immunosensor prepared by the invention adopts nickel-phenol coordination nanospheres (Ni-PCS) in the synthesis process, and a large number of active sites are provided for in-situ synthesis of platinum nanoparticles due to the large specific surface area of the Ni-PCS, so that antibodies (Ab 2) are further immobilized; and the Ni-PCS has a remarkable quenching effect on a Zr-CD DRs/TEA system, so that the detection sensitivity of the sensor is improved.
Further, the electrochemical luminescence immunosensor prepared by taking Zr-CD DRs as a luminescent material and Pt@Ni-PCS as a quenching material has excellent sensitivity, specificity, stability and clinical practicability when detecting bone-derived alkaline phosphatase, and the linear range is 1 pg/mL-50 ng/mL, and the detection limit is 24.9fg/mL.
Drawings
FIG. 1 is a schematic diagram of detection of BALP in an embodiment of the present invention, wherein FIG. 1A is the preparation of Zr-CD DRs and FIG. 1B is the preparation of Ab2-Pt@Ni-PCS quenching probe;
FIG. 2 is a topographical image of Zr-CD DRs in example 1 of the present invention, wherein FIG. 2A is a scanning electron microscope image of Zr-CD DRs, and comprises a partially magnified scanning electron microscope image of Zr-CD DRs (scale bar 500 nm); FIG. 2B is a transmission electron microscope image of Zr-CD DRs;
FIG. 3 is a morphology image of Ni-PCS and Pt@Ni-PCS in example 2 of the present invention, wherein FIG. 3A is a transmission electron microscope image of Ni-PCS, FIG. 3B is a transmission electron microscope image of Pt@Ni-PCS, and FIG. 3C is a partially enlarged Pt@Ni-PCS transmission electron microscope image;
FIG. 4 is a graph showing the construction process of the BSA/Ab1/AgNPs/Zr-CD DRs/CS immunosensor of example 2, wherein FIG. 4A is an ECL intensity versus time curve and FIG. 4B is an electrochemical impedance curve;
FIG. 5 shows the viability of MDA-MB-468 cells after 48 hours incubation with CDs and Zr-CD DRs at different concentrations;
FIG. 6 is a calibration curve of ECL strength versus current for the material of example 1, wherein FIG. 6A is a calibration curve of ECL strength versus current for CDs; FIG. 6B is a calibration curve of ECL intensity versus current for Zr-CD DRs;
FIG. 7 is a graph showing the relationship between ECL intensity and BALP concentration of the electrochemical luminescence immunosensor for detecting BALP in example 2. Wherein, FIG. 7A is an electrochemiluminescence intensity versus time curve for detecting different concentrations of BALP using an electrochemiluminescence immunosensor; FIG. 7B is a plot of ECL intensity versus log BALP concentration for an electrochemiluminescence immunosensor, BALP concentration: a=1 pg/mL, b=10 pg/mL, c=100 pg/mL, d=1 ng/mL, e=10 ng/mL, f=50 ng/mL;
FIG. 8 shows ECL intensities for different non-target proteins for the electrochemiluminescence immunosensor for detecting BALP of example 2;
FIG. 9 is a graph of electrochemiluminescence signals of 10 consecutive cycles of the electrochemiluminescence immunosensor for detecting BALP of example 2 when detecting 10pg/mL and 10ng/mL BALP.
Detailed Description
For a better understanding of the nature of the present invention, reference should be made to the following description of the invention taken in conjunction with the accompanying drawings.
The luminescent carbon nanomaterial provided by the invention is prepared by the following method: preparing nitrogen-doped carbon dots; and carrying out coordination aggregation on the prepared nitrogen-doped carbon dots and zirconium oxygen clusters to obtain zirconium-coordinated carbon dot dendritic macromolecules (Zr-CD DRs), namely the luminescent carbon nanomaterial. The morphology structure of the Zr-CD DRs is shown in figure 2.
In the present invention, coordination-induced carbon dot aggregation is achieved by coordination between zirconium ions and carboxyl groups abundant on the surface of the carbon dot. The zirconium-coordinated carbon dot dendritic macromolecules (Zr-CD DRs) are formed by aggregation through coordination action between zirconium oxygen clusters and carboxyl groups of carbon dots, so that efficient and stable enrichment of the carbon dots can be realized. The zirconium oxygen clusters in Zr-CD DRs act as a barrier tape compared to the dispersed carbon dots such that a reasonable distance is maintained between the individual carbon dots to inhibit their aggregation quenching effect, thereby generating a strong and stable ECL signal such that ECL efficiency is significantly improved.
Wherein, the nitrogen-doped carbon point takes citric acid and hydrazine hydrate as raw materials, and is synthesized by adopting a solvothermal method.
The invention provides an electrochemiluminescence immunosensor, which comprises an immunosensor containing Zr-CD DRs and nickel phenol coordination nanospheres (Ni-PCS); the Zr-CD DRs are ECL signal labels and electrode modification materials, and the Ni-PCS is a signal quenching material, so that the high-sensitivity electrochemiluminescence immunosensor is formed, and the high-sensitivity electrochemiluminescence immunosensor is shown in figure 1.
Specifically, the immunosensor is prepared by sequentially assembling Zr-CD DRs and silver nano particles (AgNPs) on the surface of an electrode by taking the Zr-CD DRs as a substrate material, and then sequentially adopting a BALP antibody (Ab 1) and BSA to modify and construct the BSA/Ab1/AgNPs/Zr-CD DRs/CS immunosensor. In some examples of the present invention, chitosan (CS) was used as a film former to immobilize Zr-CD DRs on the electrode surface and adsorb AgNPs with good conductivity, thereby immobilizing antibodies.
As shown in FIG. 3, the invention adopts nickel phenol coordination nanospheres (Ni-PCS) with larger specific surface area, takes the nickel phenol coordination nanospheres as a carrier of platinum nanoparticles, takes the platinum nanoparticles as active sites to immobilize antibodies, and meanwhile, the Ni-PCS has high-efficiency quenching effect on Zr-CD DRs/TEA systems, thereby realizing high-sensitivity detection of an immunosensor.
When the electrochemical luminescence immunosensor is used for detecting BALP, the electrochemical luminescence immunosensor modified by Zr-CD DRs can obtain a stronger ECL signal under the assistance of Triethylamine (TEA) serving as a coreactant. Ab2-Pt@Ni-PCS quenching probe is introduced to the modified electrode interface by using sandwich immune reaction in the presence of BALP. Due to the quenching effect of Pt@Ni-PCS on the Zr-CD DRs/TEA system, a low ECL signal was obtained. With increasing BALP concentration, ECL signal gradually decreases, thereby realizing highly sensitive detection of BALP.
Example 1
1. A luminescent carbon nanomaterial is prepared by the following steps:
(1) And preparing nitrogen-doped carbon dots.
2.1g of citric acid was dissolved in 10mL of N, N-Dimethylformamide (DMF), then 1.0mL of hydrazine hydrate was added dropwise to the above solution under stirring, and the above mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene for solvothermal reaction. After the reaction was heated in an oven at 180℃for 12 hours, it was cooled to room temperature, and then the resulting brownish black solution was centrifuged (12000 rpm,10 minutes) to remove large particle residues. After dialysis of the centrifuged upper solution in deionized water for 48 hours, it was dried at 60℃and the resulting nitrogen-doped Carbon Dots (CDs) powder was stored in a closed brown container and placed at 4℃for further use.
(2) Zirconium coordinated carbon dot dendrimer materials (Zr-CD DRs) were prepared.
ZrOCl 2 ·8H 2 O (30 mg) was dissolved in 10mL of DMF (1 mg/mL) containing the above synthesized nitrogen-doped Carbon Dots (CDs). The mixture was heated in an oil bath at 90 ℃ for 5 hours and then cooled naturally to room temperature. The product was then washed by centrifugation to give Zr-CD DRs. Finally, the Zr-CD DRs precipitate was dried at 60℃and stored at 4℃for further use.
To confirm successful synthesis of Zr-CD DRs, the morphology was characterized by scanning electron microscopy. As shown in FIG. 2A, the morphology of Zr-CD DRs is an irregular multi-layer coral-like structure. In addition, the morphology of the prepared Zr-CD DRs was also characterized by transmission electron microscopy (FIG. 2B). The transmission electron microscope image showed a multi-layered coral structure, similar to the scanning electron microscope image.
2. The Zr-CD DRs prepared above were subjected to a biotoxicity test.
Biocompatibility is an important parameter in evaluating whether a biological material is suitable for use in biomedical applications. In order to evaluate the feasibility of CDs and Zr-CD DRs as good biomedical materials, the present invention adopts CCK-8 experiment to detect cytotoxicity of CDs and Zr-CD DRs prepared in step (2) on human breast cancer cells (MDA-MB-468) cultured in vitro, and the results are shown in FIG. 5. As can be seen from the results of FIG. 5, the cell viability of Zr-CD DRs was significantly higher than that of dispersed CDs in the cell samples of the different concentration groups (10, 50, 100 and 500. Mu.g/mL). In addition, after 48 hours incubation with Zr-CD DRs, the cell viability was about 75%, which was higher than that of the gold nanoparticle group of 1.0. Mu.g/mL, indicating lower cytotoxicity.
3. ECL quantum efficiency of Zr-CD DRs prepared as described above was verified.
The electrochemiluminescence quantum efficiency is an important parameter for quantifying ECL performance, and ECL intensity is proportional to faraday current in an electrochemiluminescence system. The relative ECL quantum efficiency of Zr-CD DRs and CDs is obtained through calculation.
The quantitative relationship between ECL intensity and current of CDs and Zr-CD DRs systems is obtained by the formula (1) and the formula (2):
I CDs ＝k CDs i F ＝2060.12i+3865.78 (1)
wherein I is CDs ECL intensity, k for CDs CDs Slope of linear response curve for CDs, i F Is Faraday current.
I Zr-CD DRs ＝k Zr-CD DRs i F ＝4241.51i+2983.87 (2)
Wherein I is Zr-CD DRs Is ZECL intensity, k of r-CD DRs Zr-CD DRs Is the slope of the linear response curve of Zr-CDDRs.
The relative ECL quantum efficiency ratio (Q) of Zr-CD DRs to CDs can be obtained from equation (3):
wherein,ECL quantum efficiency for Zr-CD DRs, < >>ECL quantum efficiency as CDs.
As can be obtained from the formulas (1) to (3),
the results show that the ECL quantum efficiency of Zr-CD DRs is improved by 2.1 times compared with CDs, which indicates that zirconium oxygen clusters aggregate CDs through coordination interaction to be an effective way for improving the ECL quantum efficiency.
Example 2
1. The preparation of the secondary anti-platinum nano functionalized nickel phenol nanosphere (Ab 2-Pt@Ni-PCS) quenching probe comprises the following preparation steps:
(1) Preparation of Ni-PCS
Synthesizing nickel phenol coordination nanospheres (Ni-PCS) by formaldehyde-assisted metal ligand crosslinking method. The method comprises the following specific steps: (i) 0.1g Pluronic F127 was dissolved in a mixed solution of 4mL ethanol and 23mL deionized water. (ii) 0.25mL of an aqueous ammonia solution (25 wt%) was added dropwise to the mixed solution, and after stirring for 1 hour, 0.1g of tannic acid was added. (iii) After complete dissolution of the tannic acid, 0.19mL of formaldehyde solution (37 wt%) was added and stirring was continued for 24 hours. (iv) NiCl is added 2 Solution (0.05 g NiCl) 2 Dissolved in 1mL of deionized water) was added to the above mixture solution and stirring was continued for 24 hours. (v) Transferring the mixed solution into a high-pressure reaction kettle, and performing hydrothermal treatment in a 100 ℃ oven for 24 hours. (vi) The resulting product (Ni-PCS) was centrifuged and washed (12000 rpm,10 minutes) and then redispersed in deionized water and placed at 4℃for further use. The Ni-PCS had a uniform spherical shape with a diameter of about 500nm and a rough surface, as shown in FIG. 3A.
(2) Preparation of Pt@Ni-PCS
After Ni-PCS was obtained, synthesis of platinum nanoparticle functionalized nickel phenolic coordination spheres (Pt@Ni-PCS) continued. The method comprises the following specific steps: (i) 0.5mL of the prepared Ni-PCS solution was dispersed in 1.5mL of deionized water containing 25mg of polyvinylpyrrolidone (PVP). (ii) After stirring for 30 minutes, 2.4mL H was added 2 PtCl 6 (0.3 mM) and stirring was continued for 10 minutes. (iii) Then 0.125mL NaBH 4 (3 mM) was added dropwise to the above solution, and reacted for 30 minutes. (iv) The mixed solution was centrifuged (12000 rpm,10 minutes) and washed 3 times with ethanol. (v) The resulting final product Pt@Ni-PCS was redispersed in deionized water and stored at 4℃for further use.
As shown in FIGS. 3B and 3C, platinum nanoparticles with a diameter of about 5nm were uniformly distributed on the surface of the sphere, indicating that Pt@Ni-PCS was successfully synthesized.
(3) Preparation of Ab2-Pt@Ni-PCS
100. Mu.L of BALP antibody (Ab 2, 10. Mu.g/mL) was added dropwise to 1mL of the Pt@Ni-PCS solution prepared above, followed by stirring at 4℃for 12 hours, followed by blocking the non-specific adsorption sites with BSA solution (0.5 wt%), and finally the prepared Ab2-Pt@Ni-PCS quenching probe was stored at 4℃for further use.
2. Preparation of BSA/Ab1/AgNPs/Zr-CD DRs/CS immunosensor
(1) 3.4mg AgNO 3 Dissolved in 20mL deionized water cooled to about 10 ℃. Subsequently, 4.53mg NaBH 4 And 24mg sodium citrate were added to the AgNO 3 The solution was stirred in an ice bath for 30 minutes. Finally, the prepared AgNPs solution was stored in a brown closed container and placed in storage at 4 ℃ for further use.
(2) Polishing a Glassy Carbon Electrode (GCE) with the diameter of 4mm to a mirror surface by using alumina powder with the diameter of 0.05 mu m, and then washing the mirror surface by using deionized water; zr-CD DRs were dispersed in a 0.1wt% Chitosan (CS) solution and added dropwise to the pretreated GCE surface after complete dispersion. After naturally airing, obtaining the stable Zr-CD DRs/CS film. Subsequently 10. Mu.L of AgNPs solution was dropped on the Zr-CD DRs/CS modified GCE surface and incubated for 4 hours at room temperature. Then 10. Mu.L of 10. Mu.g/mL BALP antibody (Ab 1) was added dropwise to the modified electrode surface and reacted at 4℃for 8 hours. Finally, 10. Mu.L of BSA (0.25 wt%) solution was dropped onto the modified electrode, and the non-specific adsorption sites were blocked by incubation at 4℃for 40 minutes, to prepare a BSA/Ab1/AgNPs/Zr-CD DRs/CS immunosensor.
(3) Electrochemiluminescence and electrochemical impedance characterization of the immunosensor prepared in step (2).
To verify the electrode modification procedure, ECL characterization was performed on electrodes of varying degrees of modification in PBS containing coreactant TEA, as shown in FIG. 4A, where a is bare GCE, b is Zr-CD DRs/CS/GCE, c is AgNPs/Zr-CD DRs/CS/GCE, d is Ab1/AgNPs/Zr-CD DRs/CS/GCE, e is BSA/Ab1/AgNPs/Zr-CD DRs/CS/GCE, f is BALP/BSA/Ab1/AgNPs/Zr-CD DRs/CS/GCE, g is Ab 2-Pt@Ni-BALP/BSA/1/AgNPs/Zr-CD DRs/CS/GCE.
Bare GCE electrodes exhibit almost negligible ECL signals (curve a). The Zr-CD DRs/CS film is modified on the surface of the electrode, and a stronger ECL signal (curve b) is obtained. After subsequent incubation of the AgNPs on the modified electrodes described above, the ECL signal was further enhanced (curve c) due to the good conductivity of the AgNPs. ECL signal was sequentially attenuated with continued incubation of Ab1, BSA and target BALP at the modified electrode surface (d-f curve). This continuous ECL signal attenuation is due to the fact that the above materials are all non-electroactive materials that hinder electron transfer. After introduction of the Ab2-Pt@Ni-PCS quenching probe by sandwich immune reaction, ECL signal was significantly reduced (curve g) because Ni-PCS was able to effectively quench ECL signal of Zr-CD DRs/TEA system. The ECL signal changes described above indicate successful construction of immunosensors.
To further evaluate whether the electrochemical luminescence immunosensor was successful, the present invention was performed in 5mM [ Fe (CN) with 0.1M KCl 6 ] 3-/4- Electrochemical impedance characterization was performed in the system. As shown in fig. 4B, the diameter of the semicircle represents the electron transfer resistance (Ret). Bare GCE presentation due to free electron transferA smaller semicircular area (curve a) is shown. After modification of Zr-CD DRs/CS on GCE, a larger semicircular region was observed (curve b), indicating that less conductive chitosan impeded electron transfer. Since AgNPs have good conductivity, after adsorption of AgNPs on the chitosan surface, the semicircular area (curve c) is reduced compared to curve b. After sequential modification of the electrode with Ab1, BSA, BALP and Pt@Ni-PCS-Ab2 quenching probes, the resistance increases in turn (curve d-g) due to the inhibitory effect of the protein as an inert electron layer.
In summary, the electrochemical luminescence and the electrochemical impedance characterization show successful preparation of immunosensors.
3. The preparation of the electrochemiluminescence immunosensor for detecting BALP comprises the following steps:
(1) A series of BALP standard solutions (10 mu L) with different concentrations are dripped on the surface of a BSA/Ab1/AgNPs/Zr-CD DRs/CS immunosensor and reacted for 1 hour, and then the electrode interface is washed by PBS (pH 7.4);
(2) And (3) dripping 10 mu L of the prepared Ab2-Pt@Ni-PCS quenching probe onto the surface of an electrode, placing the electrode at the temperature of 4 ℃ for reaction for 1 hour, and then flushing the electrode with PBS (pH 7.4) to obtain the electrochemiluminescence immunosensor for detecting BALP.
4. The detection performance of the electrochemiluminescence immunosensor prepared as described above was analyzed, and the results are shown in fig. 7. Wherein, FIG. 7A shows that the concentration of the target BALP is in the range of 1 pg/mL-50 ng/mL, and the response ECL signal of the electrochemiluminescence immunosensor prepared by the invention gradually decreases along with the increase of the concentration of the target BALP. While the corresponding calibration curve (fig. 7B) shows that ECL signal is logarithmically related to the concentration of BALP, its linear equation is i= -1122.75lg c+7385.0, where I is ECL intensity, c is target BALP concentration, correlation coefficient (R 2 ) 0.9979. Thus, the detection Limit (LOD) of the electrochemiluminescence immunosensor prepared in the invention is 24.9fg/mL through calculation. In conclusion, the electrochemiluminescence immunosensor prepared by the invention has excellent sensitivity in detecting bone-derived alkaline phosphatase.
5. The specificity and stability of the electrochemiluminescence immunosensor prepared as described above were evaluated.
Alpha Fetoprotein (AFP), laminin (LN), prostate Specific Antigen (PSA) and Human Serum Albumin (HSA) were selected as interfering proteins for detection. As a result, as shown in FIG. 8, the concentrations of AFP, LN, PSA and HAS were 10ng/mL.
As can be seen from FIG. 8, the ECL intensity of the interfering protein remained at a high level, with no significant difference from the blank, despite the concentration of the interfering protein as high as 10ng/mL. Whereas at a BALP concentration of 1ng/mL, ECL intensity was significantly reduced. In addition, the ECL strength of the mixture containing 1ng/mL BALP and 10ng/mL interfering protein (AFP, LN, PSA and HSA) was similar to that of 1ng/mL BALP alone. The results show that the electrochemiluminescence immunosensor prepared by the invention has excellent specificity.
FIG. 9 shows that the immunosensor prepared by the invention continuously scans ECL signals for 10 cycles when detecting 10pg/mL and 10ng/mL BALP, wherein ECL intensity is relatively stable, RSD is 2.52% and 0.92% respectively, and the electrochemical luminescence immunosensor prepared by the invention has good stability.
Example 3
The electrochemiluminescence immunosensor prepared in example 2 was used to detect BALP as follows:
(1) Parameter setting: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to 800V, the cyclic voltammetry scanning potential range of an electrochemical workstation is set to 0.2-0.8V, and the scanning rate is set to 0.2V/s;
(2) An Ag/AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode, and the working electrode is connected in a cassette of an electrochemical luminescence detector, so that an electrochemical workstation and the electrochemical luminescence detector are connected together;
(3) Detection was performed in 2mL of 0.1mol/L PBS (pH 8.0) containing 35mmol/L of coreactant TEA, and the intensity of the electrochemiluminescence signal generated by the different concentrations of BALP was detected by electrochemiluminescence;
(4) And drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the BALP concentration logarithm.
Example 4
The electrochemiluminescence immunosensor prepared in example 2 was used to detect bone-derived alkaline phosphatase (BALP) in serum by standard addition method, and bone-derived alkaline phosphatase with different concentrations was added to diluted serum, and the average recovery rate of bone-derived alkaline phosphatase in the sample was determined, and the results are shown in Table 1.
TABLE 1 Standard addition method for detecting bone derived alkaline phosphatase in serum samples.
The detection results in table 1 show that the recovery rate of the bone source alkaline phosphatase detection results in the sample is 95.7% -101.3%, which indicates that the electrochemiluminescence immunosensor can be applied to detection of actual biological samples, and the results are accurate and reliable.
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof, but rather as providing for the use of additional embodiments and advantages of all such modifications, equivalents, improvements and similar to the present invention are intended to be included within the scope of the present invention as defined by the appended claims.
Claims (10)
1. The preparation method of the luminescent carbon nanomaterial is characterized by comprising the following steps of: mixing a raw material for preparing nitrogen-doped carbon dots with Dimethylformamide (DMF) to form DMF containing the nitrogen-doped carbon dots; and carrying out coordination aggregation on the DMF containing the nitrogen-doped carbon dots and the zirconium oxygen clusters to obtain zirconium coordinated carbon dot dendritic macromolecules (Zr-CD DRs), namely the luminescent carbon nanomaterial.
2. The method of manufacture of claim 1, wherein: the nitrogen-doped carbon point is synthesized by taking citric acid and hydrazine hydrate as raw materials and adopting a solvothermal method; the solvothermal method for synthesizing the nitrogen-doped carbon dot specifically comprises the following steps of:
dissolving citric acid in Dimethylformamide (DMF), and then adding hydrazine hydrate dropwise to the DMF solution under stirring to form a mixture;
transferring the mixture into a high-pressure reaction kettle with polytetrafluoroethylene lining for solvothermal reaction, cooling to room temperature after the reaction is completed, and centrifuging to obtain an upper solution.
3. The preparation method according to claim 1, wherein the coordination aggregation of the nitrogen-doped carbon dots and zirconium oxygen clusters specifically comprises the following steps:
ZrOCl 2 ·8H 2 O is dissolved in DMF containing nitrogen-doped carbon dots, and is obtained by heating and cooling to room temperature and then separating.
4. A luminescent carbon nanomaterial characterized in that it is prepared by the method of claim 2.
5. An electrochemiluminescence immunosensor, characterized in that: comprises an immunosensor containing Zr-CD DRs and a probe containing nickel-phenol coordination nanospheres (Ni-PCS); the Zr-CD DRs are electrochemical luminescence signal labels and electrode modification materials, and the Ni-PCS is a signal quenching material; the Zr-CD DRs are luminescent carbon nanomaterial of claim 4; the immunosensor containing Zr-CD DRs is a BSA/Ab1/AgNPs/Zr-CD DRs/CS immunosensor; the probe of the nickel-phenol-containing coordination nanosphere (Ni-PCS) is an Ab2-Pt@Ni-PCS quenching probe.
6. The electrochemiluminescence immunosensor of claim 5, wherein: the BSA/Ab1/AgNPs/Zr-CD DRs/CS immunosensor is prepared by the following steps:
s1, dispersing Zr-CD DRs in a chitosan solution, and then dropwise adding the dispersed Zr-CD DRs onto the surface of a pretreated glassy carbon electrode to obtain a Zr-CD DRs/CS modified electrode;
s2, silver nano particles (AgNPs) solution is dripped on the surface of the Zr-CD DRs/CS modified electrode, and the AgNPs/Zr-CD DRs/CS modified electrode is obtained;
s3, dripping the BALP antibody (Ab 1) to the surface of the AgNPs/Zr-CDDRs/CS modified electrode obtained in the step S2 to obtain an Ab1/AgNPs/Zr-CD DRs/CS modified electrode;
s4, dropwise adding a blocking agent BSA solution to the surface of the electrode modified by the Ab1/AgNPs/Zr-CDDRs/CS obtained in the step S3 to obtain the BSA/Ab1/AgNPs/Zr-CD DRs/CS immunosensor.
7. The electrochemiluminescence immunosensor of claim 5, wherein the Ab2-pt@ni-PCS quenching probe is prepared by:
synthesizing Ni-PCS by formaldehyde auxiliary metal ligand crosslinking method;
dispersing the Ni-PCS solution in deionized water containing polyvinylpyrrolidone (PVP), mixing, adding H 2 PtCl 6 The solution is slightly stirred and then added with a reducing agent NaBH 4 The reaction and separation are carried out to obtain platinum nanoparticle functionalized nickel phenol coordination nanospheres (Pt@Ni-PCS); and re-dispersing the Pt@Ni-PCS in PBS, and sequentially adding a BALP antibody (Ab 2) and a BSA solution to obtain the Ab2-Pt@Ni-PCS quenching probe.
8. A method for detecting bone-derived alkaline phosphatase, comprising the steps of:
connecting the electrochemiluminescence immunosensor of any one of claims 5 to 7 as a working electrode to an electrochemiluminescence detector;
taking Phosphate Buffer Solution (PBS) as a detection base solution, and adding a coreactant Triethylamine (TEA) into the PBS for detection;
and detecting the intensity of the electrochemiluminescence signal generated by the BALP with different concentrations by an electrochemiluminescence method under the preset condition of the electrochemiluminescence detector.
9. The method for detecting bone-derived alkaline phosphatase according to claim 8, wherein: the electrochemiluminescence detector is an ultra-weak electrochemiluminescence detector.
10. The method for detecting bone-derived alkaline phosphatase according to claim 8, wherein: the preset conditions comprise that the high voltage of the photomultiplier is 800V, the cyclic voltammetry scanning potential range of the electrochemical workstation is 0.2-0.8V, and the scanning rate is 0.2V/s.
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