CN113533476B - ECL sensing chip and preparation method and application thereof - Google Patents
ECL sensing chip and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an ECL sensing chip for rapidly detecting cysteine proteinase-3 and a preparation method and application thereof, and relates to the technical field of bioelectrochemical sensors. The invention takes PCN-333(Al) as a carrier of a luminescent reagent Ru, and covalently bonds carboxyl Ru and a coreactant PEI to prepare a Ru-PEI @ PCN-333(Al) composite material; modifying the composite material and AuNPs to the surface of the SPE working electrode by using CS as an adhesive; and modifying the ferrocene polypeptide on the electrode through an Au-S bond to construct the ECL sensing chip. Due to the proximity of Fc, ECL signal of Ru is quenched; ECL signaling was restored upon addition of Caspase-3. The sensor chip can effectively monitor Caspase-3 activity in the process of apoptosis and has wide application prospect in the aspects of drug efficacy evaluation, cancer treatment monitoring and the like.
Description
Technical Field
The invention belongs to the technical field of bioelectrochemical sensors, and particularly relates to an ECL (electron cyclotron resonance) sensing chip for rapidly detecting cysteine protease-3 as well as a preparation method and application thereof.
Background
Apoptosis is a gene-mediated cell suicide behavior and plays an important role in maintaining the environmental homeostasis of organisms and eliminating abnormal cells. Abnormal apoptotic processes can lead to a number of serious diseases such as cancer, neurodegenerative diseases, and the like. Caspases (Caspases) are a class of caspase enzymes that are closely associated with apoptosis. Caspase-3 is the most important and most studied protein in the Caspase family, and is located at the core position of the apoptotic network. The level of apoptosis in a cell may be indicated by Caspase-3 expression. Therefore, monitoring the activity of Caspase-3 is of great significance for understanding the role of Caspase-3 in the apoptosis process and diagnosing and treating related diseases.
At present, methods for detecting Caspase-3 activity mainly comprise a colorimetric method, a fluorescence method, a chemiluminescence method, an electrochemical method and the like. The electrochemical luminescence (ECL) method is a technology combining electrochemistry and chemiluminescence, and has the advantages of high sensitivity, wide dynamic range, simple operation, low background signal and the like. However, due to the lack of a good-performance electrochemiluminescence reagent and a reasonably designed biosensing mechanism, the method is rarely reported to be used for detecting Caspase-3 activity.
Disclosure of Invention
In view of the above, the invention aims to provide an ECL sensing chip for rapidly detecting cysteine protease-3, and a preparation method and an application thereof, and the MOFs immobilized Ru luminescent material is applied to the ECL sensing field of Caspase-3 for the first time, so that high sensitivity and stability are obtained, the activity of Caspase-3 in the apoptosis process can be effectively monitored, and the ECL sensing chip has a wide application prospect in the aspects of drug efficacy evaluation, cancer treatment monitoring and the like.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a luminescent material Ru-PEI @ PCN-333(Al), which comprises the following steps: will [ Ru (bpy)2(mcpbpy)]2+Mixing the solution and polyethyleneimine to perform covalent binding reaction to obtain a Ru-PEI complex;
and mixing and stirring the Ru-PEI complex and PCN-333(Al) for 24 hours to obtain the luminescent material Ru-PEI @ PCN-333 (Al).
Preferably, the preparation method of the Ru-PEI complex comprises the following steps: will [ Ru (bpy)2(mcpbpy)]2+And mixing the Ru-PEI complex with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for 20-40 min, and further mixing the obtained mixed solution with polyethyleneimine for 2-4 h to obtain the Ru-PEI complex.
The invention also provides the luminescent material Ru-PEI @ PCN-333(Al) prepared by the preparation method.
The invention also provides application of the luminescent material Ru-PEI @ PCN-333(Al) in preparation of an electrochemical luminescence chip.
Preferably, the electrochemiluminescence chip comprises an ECL sensing chip.
The invention also provides an ECL sensing chip which comprises a screen printing electrode substrate, and luminescent materials Ru-PEI @ PCN-333(Al), AuNPs and ferrocene polypeptide which are adhered to the surface of the screen printing electrode substrate through chitosan.
Preferably, the polypeptide sequence of the ferrocene polypeptide comprises amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 3.
The invention also provides a preparation method of the ECL sensing chip, which comprises the following steps:
mixing chitosan, luminescent material Ru-PEI @ PCN-333(Al) and AuNPs to obtain modified slurry;
dripping the modified slurry on the surface of the screen printing electrode to obtain a modified electrode semi-finished product;
and modifying the ferrocene polypeptide on the modified electrode semi-finished product through an Au-S bond to obtain the ECL sensing chip.
The invention also provides an application of the luminescent material Ru-PEI @ PCN-333(Al) or the ECL sensing chip in preparation of at least one tool as follows:
(a) means for monitoring apoptosis;
(b) means for screening for caspase inhibitors;
(c) means for evaluating caspase inhibitors;
(d) a means for screening antitumor drugs;
(e) means for evaluating the effectiveness of a drug treatment;
(f) a tool for cancer treatment detection.
Has the advantages that: the invention provides a luminescent material Ru-PEI @ PCN-333(Al) and a preparation method thereof, wherein an MOFs material PCN-333(Al) is used as a carrier of a luminescent reagent Ru, and a carboxyl Ru and a coreactant PEI are covalently bonded to prepare a Ru-PEI @ PCN-333(Al) composite material; modifying the composite material and AuNPs to the surface of an SPE working electrode (carbon) by using Chitosan (CS) as an adhesive; and modifying the ferrocene polypeptide on the electrode through an Au-S bond to construct a novel ECL sensing chip. Due to the proximity of Fc, ECL signal of Ru is quenched; when Caspase-3 was added, the recognition site Asp-Glu-Val-Asp of Caspase-3 in Fc-polypeptide (Fc-peptide) was specifically cleaved, and the Fc-containing peptide fragment was detached from the electrode surface, thereby recovering the ECL signal (FIG. 8). According to the invention, the MOFs immobilized Ru luminescent material is applied to the ECL sensing field of Caspase-3 for the first time, and higher sensitivity and stability are obtained. The sensor chip can effectively monitor the activity of Caspase-3 in the process of apoptosis and has wide application prospect in the aspects of drug efficacy evaluation, cancer treatment monitoring and the like.
Drawings
FIG. 1 is a graph depicting the morphology of PCN-333(Al), Ru-PEI @ PCN-333(Al), and AuNPs;
FIG. 2 shows the results of electrode uniformity tests, wherein A and B are SEM images of SPE electrodes and ECL sensor chips, respectively, and C, D, E and F show the results of EDS mapping tests, respectively;
FIG. 3 shows the electrodes at 5mM K3[Fe(CN)6]And cyclic voltammogram (A) in 0.1M KCl (pH7.4), square wave voltammogram (B) in 0.1M PBS (pH7.4), and at 1mM K3[Fe(CN)6]、1mM K4[Fe(CN)6]And an alternating current impedance spectrum (C) in 0.1M KCl (pH7.4), wherein the wave a is a naked SPE electrode, the wave b is an AuNPs/Ru-PEI @ PCN-333(Al)/CS membrane modified SPE electrode, and the wave C is an Fc-peptide/AuNPs/Ru-PEI @ PCN-333(Al)/CS membrane modified SPE electrode;
FIG. 4 shows the signal variation of ECL sensor chip, wherein (A) shows the electron emission signals of different electrodes, (B) shows the stability of ECL signal of material modified electrode, and (C) shows the reproducibility of ECL signal of material modified electrode;
FIG. 5 shows the optimization results of ECL sensor chip (A) the relationship between blank signal intensity and Fc-peptide self-assembly time of ECL sensor chip; (B) the relation between the signal intensity and the cut-off time of the ECL sensing chip at 100pg/ml Caspase-3;
FIG. 6 shows the analytical performance of ECL sensor chip, (A) ECL signal is gradually enhanced with increasing Caspase-3 concentration and is in linear relationship in a certain range; (B) the linear equation is that Δ I is 1722.3lg [ Caspase-3] +2768.1, r is 0.9988; (C) selectivity of ECL sensor chip for multiple proteins;
FIG. 7 is a graph showing the results of Caspase-3 assay using different methods, wherein (A) Caspase-3 activity is determined using ECL sensor (top) and colorimetric (bottom) assays after incubation with apoptosis-inducing agents for 0, 2, 4, 6, 8, 12, 16, and 24 hours, respectively, and Caspase-3 activity is determined using a Caspase-3 colorimetric kit; (B) ECL determination of inhibition of Caspase-3 by different concentrations of Ac-DEVD-pNA; (C) concentration dependence of drugs (daunorubicin and doxorubicin) on Caspase-3 activity;
fig. 8 shows a method for manufacturing the ECL sensor chip and a signal generation mechanism according to the present invention.
Detailed Description
The invention provides a preparation method of a luminescent material Ru-PEI @ PCN-333(Al), which comprises the following steps: will [ Ru (bpy)2(mcpbpy)]2+Mixing the solution and polyethyleneimine to perform covalent binding reaction to obtain a Ru-PEI complex;
and mixing and stirring the Ru-PEI complex and PCN-333(Al) for 24 hours to obtain the luminescent material Ru-PEI @ PCN-333 (Al).
The preparation method of the Ru-PEI complex preferably comprises the following steps: will [ Ru (bpy)2(mcpbpy)]2+And mixing the Ru-PEI complex with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) for 20-40 min, and further mixing the obtained mixed solution with Polyethyleneimine (PEI) for 2-4 h to obtain the Ru-PEI complex. In the present invention, the [ Ru (bpy)2(mcpbpy)]2+Preferably consisting of [ Ru (bpy)2(mcpbpy)]Cl2Providing, mixing said [ Ru (bpy)2(mcpbpy)]Cl2Preferably, EDC and NHS are mixed and stirred for 30min to activate carboxyl group for covalent bond reaction with PEI amide bond. The present invention is said [ Ru (bpy)2(mcpbpy)]Cl2The molar ratio of EDC to NHS is preferably (0.02-0.03): (1.3-1.4): (0.08-0.15). In the present example, it is preferable that 14.8mg of [ Ru (bpy)2(mcpbpy)]Cl2Dissolved in 4mL of ultrapure water, 57.6mg of EDC and 11.6mg of NHS were added thereto with vigorous stirring, and the mixture was stirred for 30min to activate the carboxyl group. The invention is described as vigorously stirredThe stirring speed is preferably 800-1200 rpm. The PEI of the present invention is preferably present in the form of an aqueous solution, and the mass percent of PEI in the aqueous PEI solution is preferably 1%. In the present example, the PEI is preferably added in an amount of 2 mL. The rate of continued stirring according to the invention is preferably 600 rpm.
The invention is about the [ Ru (bpy)2(mcpbpy)]Cl2The sources of EDC, NHS and PEI are not particularly limited and may be those conventionally available in the art.
After the Ru-PEI complex is obtained, the Ru-PEI complex is preferably further mixed with PCN-333(Al) and continuously stirred for 24 hours, and centrifugal concentration is carried out after dialysis to obtain the luminescent material Ru-PEI @ PCN-333 (Al).
The source and production method of the PCN-333(Al) are not particularly limited, and in the examples of the present invention, the PCN-333(Al) is preferably produced by a method described in d.feng, t.f.liu, j.su, m.bosch, z.wei, w.wan, et Al, Stable metal-organic frame connecting single-molecule tracks for enzyme encapsulation, Nature commu.6 (2015)5979. The PCN-333(Al) of the present invention and the [ Ru (bpy)2(mcpbpy)]Cl2Is preferably 10: 14.8. The rotation speed of the continuous stirring in the present invention is preferably 600 rpm. The dialysis of the invention preferably comprises the step of dialyzing the obtained mixed solution for 24 hours by using a dialysis bag, wherein the dialysis bag preferably comprises a substance with a dialysis molecular weight of 8000-14000. The centrifugal concentration of the invention preferably comprises the steps of centrifuging at the rotating speed of 600rpm, and collecting precipitates to obtain the luminescent material Ru-PEI @ PCN-333 (Al). The luminescent material Ru-PEI @ PCN-333(Al) is preferably dispersed in 1mL of ultrapure water and stored at 4 ℃ in a dark place.
The invention also provides the luminescent material Ru-PEI @ PCN-333(Al) obtained by the preparation method.
In the present invention, the PCN-333(Al) is in an octahedral shape, and the Ru-PEI @ PCN-333(Al) surface compounded with Ru-PEI becomes rough, but the octahedral configuration is still maintained. The XRD single crystal diffraction simulation of the PCN-333(Al) and the Ru-PEI @ PCN-333(Al) shows that the peak positions in an XRD pattern are good in inosculation, and the crystalline form of the PCN-333(Al) is not influenced by the luminescent material Ru-PEI @ PCN-333 (Al).
The invention also provides application of the luminescent material Ru-PEI @ PCN-333(Al) in preparation of an electrochemical luminescence chip. The electrochemical luminescence chip of the invention preferably comprises an ECL sensing chip.
The invention also provides an ECL sensing chip which comprises a screen printing electrode substrate, and luminescent materials Ru-PEI @ PCN-333(Al), AuNPs and ferrocene polypeptide which are adhered to the surface of the screen printing electrode substrate through Chitosan (CS).
The amino acid sequence of the ferrocene polypeptide of the present invention preferably comprises Fc-SEQ ID NO.1(Fc-GGDEVDGC), Fc-SEQ ID NO.2(Fc-GGGDEVDGC) or Fc-SEQ ID NO.3 (Fc-GGGGDEVDGC). In the system of the embodiment of the invention, preferably, 0.5mL of Ru-PEI @ PCN-333(Al), 0.4mL of LAuNPs and 0.1mL of 0.1% Chitosan (CS) solution are reacted with 5-20 μ L of 10 μ M ferrocene polypeptide.
According to the ECL sensing chip, an Fc peptide segment of an Fc-peptide/AuNPs/Ru-PEI @ PCN-333(Al)/CS membrane ON an SPE electrode is modified through Caspase-3 cracking, an ECL signal turn-ON is achieved, the activity of Caspase-3 in the process of apoptosis is monitored, and specifically, due to the fact that Fc is close to, the ECL signal of Ru is quenched; after Caspase-3 is added, the recognition site Asp-Glu-Val-Asp of Caspase-3 in Fc-peptide can be specifically cut off, so that the Fc-containing peptide segment is separated from the surface of the electrode and the ECL signal is recovered.
The invention also provides a preparation method of the ECL sensing chip, which comprises the following steps: mixing chitosan, luminescent material Ru-PEI @ PCN-333(Al) and AuNPs to obtain modified slurry;
dripping the modified slurry on the surface of the screen printing electrode to obtain a modified electrode semi-finished product;
and modifying the ferrocene polypeptide on the modified electrode semi-finished product through an Au-S bond to obtain the ECL sensing chip.
The invention utilizes a Screen Printing Electrode (SPE) to construct a biosensor chip, wherein the SPE is preferably integrated with three electrodes: a working electrode (carbon), a counter electrode (carbon) and a reference electrode (Ag/AgCl)), and the SPE is made of PET.
According to the invention, luminescent materials Ru-PEI @ PCN-333(Al) and AuNPs are modified on the surface of SPE, and preferably, the method comprises the step of mixing and stirring 0.5mL of the luminescent materials Ru-PEI @ PCN-333(Al), 0.4mL of the AuNPs and 0.1mL of 0.1% Chitosan (CS) solution for 30 min. Before the SPE of the present invention is used, it preferably further comprises a pretreatment, and the pretreatment preferably comprises: at 0.05M H2SO4And (3) circularly scanning the solution by using a cyclic voltammetry at a scanning speed of 0.1V/s and a voltage range of 0.5-1.1V until a stable CV curve is obtained, and then sequentially cleaning the solution by using ultrapure water, alcohol, a mixed solution of ultrapure water and alcohol in a ratio of 1:1, and drying the solution for later use. The source and preparation method of the AuNPs are not particularly limited in the present invention.
The modification of the invention preferably comprises the steps of dripping the mixed solution formed by mixing and stirring for 30min onto the SPE working electrode, and naturally drying to obtain the AuNPs/Ru-PEI @ PCN-333(Al)/CS film. The ferrocene polypeptide is modified on the electrode through an Au-S bond, and the preferable method comprises the steps of dripping 5-20 mu L of 10 mu M ferrocene polypeptide on the surface of the modified electrode, and incubating for 30min at 37 ℃.
After the ferrocene polypeptide is modified on the surface of the electrode, the invention preferably further comprises the step of reacting for 1h at room temperature by using 1mM mercapto-1-ethanol (MCH) so as to block the non-specific adsorption sites on the surface of the electrode.
The invention also provides the ECL sensing chip prepared by the preparation method.
The invention also provides an application of the luminescent material Ru-PEI @ PCN-333(Al) or the ECL sensing chip in preparation of at least one tool as follows:
(a) means for monitoring apoptosis;
(b) means for screening for caspase inhibitors;
(c) means for evaluating caspase inhibitors;
(d) a means for screening antitumor drugs;
(e) means for evaluating the effectiveness of a drug treatment;
(f) a tool for cancer treatment detection.
In the invention, the ECL sensing chip can be used for monitoring the generation of Caspase-3 in the process of apoptosis and evaluating the effects of a Caspase-3 inhibitor and an anti-tumor drug on MOLM-13 cell.
The ECL sensor chip for rapid detection of cysteine protease-3 and the preparation method and application thereof provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1
1. Synthesis of PCN-333(Al)
50mg of H3TATB and 200 mgAlCl3·6H2O was dissolved in 10mL of DMF, followed by ultrasonic dissolution by addition of 1.0mL of trifluoroacetic acid, heating in an oven at 135 ℃ for 48 hours, cooling to room temperature and centrifugation to obtain a white precipitate, which was washed three times with DMF to obtain a white powder sample PCN-333 (Al).
2. Synthesis of Ru-PEI @ PCN-333(Al)
14.8mg of [ Ru (bpy)2(mcpbpy)]Cl2Dissolved in 4mL of ultrapure water, 57.6mg of EDC and 11.6mg of NHS were added thereto with vigorous stirring, and the mixture was stirred for 30min to activate the carboxyl group. Then, 2mL of a 1% PEI (Mw 1800) solution was added and stirring was continued for 2h to obtain a Ru-PEI complex. Then 10mg of negatively charged PCN-333(Al) was added and stirring was continued for 24 h. And dialyzing the obtained mixed solution for 24 hours by using a dialysis bag (8000-14000 Da), centrifuging and concentrating, dispersing the final product Ru-PEI @ PCN-333(Al) in 1mL of ultrapure water, and storing in a refrigerator at 4 ℃ in a dark place.
The morphology of PCN-333(Al) and Ru-PEI @ PCN-333(Al) was characterized using SEM, as shown in FIG. 1, with the PCN-333(Al) being octahedral in shape (A in FIG. 1), and the Ru-PEI @ PCN-333(Al) surface with Ru-PEI composited therewith was roughened, but still retained the octahedral configuration (B in FIG. 1). XRD of PCN-333(Al) and Ru-PEI @ PCN-333(Al) is shown as C in figure 1, the XRD pattern obtained by the experiment is well matched with the XRD pattern obtained by single crystal diffraction simulation, and the preparation success of the PCN-333(Al) and the Ru-PEI @ PCN-333(Al) is shown, and the crystal form of the PCN-333(Al) is not influenced by doping the Ru-PEI compound.
3. Synthesis of AuNPs
4.0mL of 1% citric acid tribasic under vigorous stirringSodium was added to boiling 100mL of 0.01% HAuCl4The solution was stirred and boiled for 30 min. After cooling to room temperature, AuNPs with a diameter of about 12nm were obtained and stored in a refrigerator at 4 ℃ protected from light. The AuNPs prepared are characterized by using TEM and UV-Vis absorption spectra, and as shown in D in FIG. 1, the characteristic absorption peak is 520nm, the AuNPs are uniformly distributed in a spherical shape, and the particle size is about 12 nm.
4. Construction of ECL sensor chip
And (3) constructing a biosensor chip by using a Screen Printing Electrode (SPE) integrated with a three-electrode PET material. The electrodes were pretreated at 0.05M H before material modification2SO4And (3) cyclic voltammetry is used in the solution, and the scanning speed is 0.1V/s within the voltage range of 0.5-1.1V, and cyclic scanning is carried out until a stable CV curve is obtained. Sequentially cleaning with ultrapure water, alcohol, a mixed solution of ultrapure water and alcohol in a ratio of 1:1, and drying for later use.
Mixing and stirring 0.5mL of Ru-PEI @ PCN-333(Al), 0.4mL of LUNPs and 0.1mL of 0.1% Chitosan (CS) solution for 30min, dripping 10 mu L of the mixed solution onto an SPE working electrode, and naturally drying to obtain the AuNPs/Ru-PEI @ PCN-333(Al)/CS film. Then 10. mu.L of 10. mu.M Fc-GGGDEVDGC (Fc-SEQ ID NO.2) was added dropwise to the modified electrode surface and incubated for 30 min. Finally, 10. mu.L of 1mM MCH was used for 1 hour at room temperature to block non-specific adsorption sites on the electrode surface. After each step of electrode modification, the electrodes were washed with PBS (0.1M, pH 7.4).
4.1 characterization of the synthetic materials
The SPE working electrode surface was characterized using SEM, and the result is shown in fig. 2, where the bare SPE working electrode was a carbon electrode with carbon particles uniformly distributed on the electrode surface. When the working electrode is modified by using CS as an adhesive and AuNPs and Ru-PEI @ PCN-333(Al), the surface of the electrode is uneven. The distribution condition of the EDS mapping on the electrode is tested, the test area is the electrode boundary, and the carbon is used as reference, so that three elements of Al, Ru and Au are uniformly distributed on the working electrode, and the modified electrode has better uniformity.
4.2 electrochemical characterization
The assembly process of the electrodes was examined using Cyclic Voltammetry (CV), Square Wave Voltammetry (SWV) and alternating current impedance (EIS) techniques. As shown in FIG. 3, compared with a bare carbon electrode (curve A in FIG. 3), the oxidation-reduction peak current of the AuNPs/Ru-PEI @ PCN-333(Al)/CS membrane modified electrode is obviously increased (curve A in FIG. 3), which proves that the nanocomposite material has good conductivity and higher specific surface area and promotes the transfer of electrons. After the Fc-peptide was added dropwise to the electrode, the redox peak current dropped significantly as the polypeptide biomacromolecule prevented electron transfer.
The results of the research on the electrodes before and after the labeling of Fc-peptide by SWV method are shown in B in FIG. 3, and when Fc-peptide was bonded to AuNPs/Ru-PEI @ PCN-333(Al)/CS film, +1.1V Ru2+The current value dropped significantly due to ferrocene for [ Ru (bpy)2(mcpbpy)]2+Has quenching effect, and has characteristic oxidation peak of Fc at +0.5V, which shows that Fc-peptide and AuNPs are fixed on the sensing chip platform through Au-S bond self-assembly. In FIG. 3, C is the EIS spectrum of different modified electrodes. The semicircular portion at higher frequencies in the EIS spectrum corresponds to the electron transfer resistance (Rct). The EIS spectra of the electrodes were similar to CV behavior. The Rct of the bare carbon electrode is 533 omega, the Rct is reduced to 266 omega after the electrode is modified by using an AuNPs/Ru-PEI @ PCN-333(Al)/CS film, and the Rct is obviously increased to 1430 omega after the electrode is self-assembled with Fc-peptide. The EIS results further demonstrate the successful construction of the sensor chip and its binding capacity.
4.3 characterization of ECL sensor chip
The change of the ECL signal of the working electrode during the modification of the sensor chip was examined by cyclic voltammetry, and the result is shown in a of fig. 4, where the ECL signal was almost absent from the bare SPE electrode in the PBS solution (curve a). When AuNPs/Ru-PEI @ PCN-333(Al)/CS was added dropwise to the electrode, the ECL signal increased sharply (curve b) because of Ru2+And the ECL emission is stronger in the coexistence of the co-reactant PEI. It is presumed that the ECL luminescence mechanism of Ru-PEI @ PCN-333(Al) is as follows:
[Ru2+-PEI@PCN-333(Al)]-e-→[Ru3+-PEI@PCN-333(Al)]
PEI-e-→PEI+·
PEI+·→PEI·+H+
[Ru3+-PEI@PCN-333(Al)]+PEI·→[Ru2+-PEI@PCN-333(Al)]*+PEI
[Ru2+-PEI@PCN-333(Al)]*→[Ru2+-PEI@PCN-333(Al)]+hν
when Fc-peptide was modified onto the electrode, there was a significant decrease in ECL signal due to Ru2+The ECL emission of (c) is quenched by the close Fc (curve c). A further drop in ECL signal was observed with the addition of MCH to the electrode (curve d) since MCH prevented electron transport. However, after 100pg/mL Caspase-3 was added dropwise to the electrode for incubation, an obvious increase in ECL signal was observed (curve e), since Fc-peptide contains the recognition site Asp-Glu-Val-Asp of Caspase-3, and the Fc-containing peptide was cleaved from the electrode surface after digestion, thus obtaining a signal recovery state.
Meanwhile, the stability and the reproducibility of the ECL signal of the material modified electrode are examined. As shown in B in FIG. 4, after the same electrode modifies the AuNPs/Ru-PEI @ PCN-333(Al)/CS film material, under continuous cyclic potential scanning for 12 circles, the ECL signal has no obvious change, and the RSD is less than 2%, which proves that the sensing chip has high stability. AuNPs/Ru-PEI @ PCN-333(Al)/CS membrane materials are respectively modified by using different SPE bare electrodes, the prepared electrodes are placed in a dryer to be stored in a dark place for 15 days, ECL signals are not obviously changed, RSD is respectively less than 3.5% and 5%, and the electrodes are proved to have better reproducibility and stability (C in figure 4).
4.4 optimization of the experimental conditions
Two time conditions are involved for the sensor chip: the time for binding of Fc-peptide to AuNPs/Ru-PEI @ PCN-333(Al)/CS membrane and the time for cleavage of peptide by Caspase-3 were optimized to determine the effect on ECL signal.
As shown in A in FIG. 5, as the binding time of Fc-peptide to AuNPs/Ru-PEI @ PCN-333(Al)/CS film was increased, ECL signal gradually decreased and the luminescence intensity became stable after 30min, indicating that the binding reaction could reach the maximum quenching degree at 30 min. The binding time of the fixed Fc-peptide and AuNPs/Ru-PEI @ PCN-333(Al)/CS membrane is 30min, and the time for cleaving the peptide by Caspase-3 is changed. As a result, as shown in B in FIG. 5, using 100pg/mL Caspase-3, the ECL intensity increased with increasing cleavage time and stabilized at 40 min. Therefore, in this experiment, 30min was selected as the optimal Fc-peptide binding time and 40min was selected as the optimal Caspase-3 cleavage time.
4.5 analytical Properties of ECL sensor chip
Under the optimal condition, the prepared ECL sensing chip is incubated by Caspase-3 solutions with different concentrations, and then an ECL signal is detected. As a result, as shown in FIG. 6A, the ECL signal was gradually increased with the increase of Caspase-3 concentration and was linear in a certain range. The linear equation is shown as B in FIG. 6, wherein Δ I is 1722.3lg [ Caspase-3] +2768.1, r is 0.9988, the detection limit is 0.017pg/mL (3 σ), and the sensitivity is high.
In order to evaluate the specificity of the method, the same studies were performed on several proteins such as trypsin, lysozyme, alkaline phosphatase and tyrosinase, and the results are shown in fig. 6C, which do not affect the ECL strength, indicating that the sensor chip has high selectivity.
5. Cell culture and apoptosis-inducing treatment
Removing MOLM-13cell (China-Japan University of Jilin University) in exponential growth phase, centrifuging at 1000rpm for 10min, discarding supernatant, redispersing cells into fresh culture solution, and controlling cell concentration to 5.0 × 10 by using cell counter5cells/mL. Then 4mL of cell solution and 2 mu L of apoptosis inducing reagent A in the apoptosis positive control kit are respectively acted for 0 h, 2h, 4h, 8h, 12h, 16h and 24h to induce apoptosis. Cells were centrifuged from the culture and washed 1 time with PBS. 100 mu of LWestern and IP cell lysate are added and the mixture is lysed for 15min in an ice bath. The mixture is frozen and centrifuged at 13000rpm for 15min at 4 ℃, and the supernatant is transferred to a new centrifugal tube precooled by an ice bath and stored at-70 ℃ for standby.
6. Determination of Caspase-3
And (3) ECL determination: mu.L of Caspase-3 or sample treatments at different concentrations (0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50, 100, 200pg/mL) were added dropwise to the working electrode, reacted at 37 ℃ for 40min, followed by washing the electrode surface with PBS (0.1M, pH7.4) and the SPE was placed in a small beaker containing 10mL of PBS (0.1M, pH7.4) for ECL detection. The voltage of the photomultiplier is 800V, the scanning range is 0.2-1.3V, and the scanning speed is 0.1V/s. Caspase-3 was measured by the increase in ECL intensity.
And (3) colorimetric determination: the experiment was carried out using a commercial Caspase-3 activity detection kit according to the protocol. mu.L of the treated cell sample solution was added to 40. mu.L of the detection buffer, mixed well, and then 10. mu.L of LAc-DEVD-pNA (2mM) was added and mixed well again. Incubate at 37 ℃ for 2h in the dark. The absorbance value was then determined with a spectrophotometer at 405 nm.
The results of ECL measurements are shown in fig. 7, a, with increasing time to induce apoptosis, ECL signal increased and reached equilibrium at 12 h; the result of the colorimetric kit is shown as A in figure 7, and the absorbance at 405nm gradually increases with the prolonging of the action time of the apoptosis inducing reagent and becomes stable after 12 h. The results of the two measurements are identical, which indicates that the ECL sensing chip can be used for monitoring the activation of Caspase-3 in the process of apoptosis.
To further assess the specificity of the ECL sensor chip, Caspase-3 activation during apoptosis was inhibited using Caspase-3 inhibitors (Ac-DEVD-CHO) at various concentrations (25, 50, 100. mu.M). Results as shown in fig. 7B, apoptosis was induced for 12h after 30min of cytostatic pretreatment, and ECL intensity was measured. As the concentration of inhibitor increases, the ECL signal gradually decreases and the colorimetric response measurement matches.
The invention also inspects the influence of two antitumor drugs of daunorubicin and adriamycin on the activity of the acute myeloid leukemia cell Caspase-3. After two drugs are used for acting on cells for 12h, the ECL intensity is measured, and the result is as C vegetarian diet in figure 7, the ECL signal is gradually enhanced along with the increase of the drug concentration, and the Caspase-3 activity is in direct proportion to the drug concentration. Compared with two medicines, the two medicines achieve the same apoptosis degree, and the concentration of daunorubicin is lower than that of adriamycin.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> ECL sensing chip for rapid detection of cysteine protease-3, and preparation method and application thereof
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Claims (3)
1. An ECL sensing chip for detecting cysteine protease-3 comprises a screen printing electrode substrate, and luminescent materials Ru-PEI @ PCN-333(Al), AuNPs and ferrocene polypeptide, wherein the luminescent materials Ru-PEI @ PCN-333(Al), AuNPs and ferrocene polypeptide are bonded to the surface of the screen printing electrode substrate through chitosan;
the polypeptide sequence of the ferrocene polypeptide comprises amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO.3, wherein the amino acid sequence contains a recognition action site Asp-Glu-Val-Asp of cysteine protease-3;
the preparation method of the luminescent material Ru-PEI @ PCN-333(Al) comprises the following steps: will [ Ru (bpy)2(mcpbpy)]2+Mixing the Ru-PEI complex with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for 20-40 min, and further mixing the obtained mixed solution with polyethyleneimine for 2-4 h to obtain the Ru-PEI complex;
and mixing and stirring the Ru-PEI complex and PCN-333(Al) for 24 hours to obtain the luminescent material Ru-PEI @ PCN-333 (Al).
2. The method for preparing an ECL sensor chip for detecting cystatin-3 according to claim 1, comprising the steps of:
mixing chitosan, luminescent material Ru-PEI @ PCN-333(Al) and AuNPs to obtain modified slurry;
dripping the modified slurry on the surface of the screen printing electrode to obtain a modified electrode semi-finished product;
and modifying the ferrocene polypeptide on the modified electrode semi-finished product through an Au-S bond to obtain the ECL sensing chip.
3. Use of an ECL sensor chip according to claim 1 for the detection of cystatin-3 for the preparation of at least one of the following tools:
(a) means for monitoring apoptosis;
(b) means for screening for caspase inhibitors;
(c) means for evaluating caspase inhibitors;
(d) a means for screening antitumor drugs;
(e) means for evaluating the effectiveness of a drug treatment;
(f) a tool for cancer treatment detection.
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