CN114354583A - Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof - Google Patents
Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof Download PDFInfo
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Abstract
The invention discloses an electrochemiluminescence lung cancer detection kit based on a metal-free light ATRP signal amplification strategy, a use method and application thereof, wherein the kit comprises the following raw materials: BMP, gold electrode, MPA, EDC, NHS, Ab1, BSA, Ab2, Me6TREN, EY, NAS, luminol, ultrapure water, H2O2DMSO, ethanol. The invention utilizes a metal-free photoinduction atom transfer radical polymerization (photo-ATRP) strategy, avoids the use of nano materials and biological enzymes in the current common signal amplification strategy, amplifies signals by times, and improves the sensitivity, stability and reproducibility of detection. Meanwhile, the use of a heavy metal ion catalyst in the traditional ATRP reaction is avoided, the heavy metal ions do not need to be recovered and removed, the interference of the inherent color of the heavy metal ions on the detection result is overcome, the biotoxicity is not generated, and the environment is more environment-friendly.
Description
Technical Field
The invention relates to an electrochemiluminescence lung cancer detection kit based on a metal-free photoinduced atom transfer radical polymerization (photo-ATRP) signal amplification strategy, a use method and application, and belongs to the technical field of bioanalysis.
Background
CYFRA21-1 is considered to be a tumor marker mainly used for detecting lung cancer, and has important value particularly for diagnosing non-small cell lung cancer (NSCLC). If there is an unclear circumferential shade in the lung, with serum CYFRA21-1 concentrations >30ng/mL, the likelihood of primary bronchopulmonary carcinoma is very high. The positive detection rate of the CYFRA21-1 to various non-small cell lung cancers is 70 to 85 percent. The serum concentration level of CYFRA21-1 is positively correlated with the clinical stage of tumor, and can be used as effective index for tracking early recurrence after lung cancer operation and radiotherapy and chemotherapy. Therefore, the CYFRA21-1 test is of great significance for the clinical diagnosis of NSCLC. Several methods of detecting CYFRA21-1 have been reported so far: enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), chemiluminescent immunoassay, chemiluminescent enzyme immunoassay, and the like. However, the detection process still has the disadvantages of false positive, long time consumption, special instrument and professional operators and the like.
The detection performance of the sensor can be improved by utilizing signal amplification technologies such as chain hybridization reaction, roller amplification reaction, nano material and polymerization reaction. Wherein, Atom Transfer Radical Polymerization (ATRP) has the advantages of wide monomer raw material, controllable polymerization reaction and the like and is widely applied to the field of biosensing. However, the traditional ATRP reaction requires a large amount of heavy metal (e.g., copper) ions as a catalyst due to the slow polymerization rate, the heavy metal ions have certain biological toxicity, complicated removal and recovery processes are required, and the color of the heavy metal ions interferes with the detection result of the sensor. Compared with the traditional metal catalyst, the light-induced ATRP has the following advantages: 1) no heavy metal catalyst is needed to be added; 2) the reaction condition is mild; 3) the photoinduced polymerization process is controllable. Therefore, the research of the ultra-sensitive electrochemical sensing platform for CYFRA21-1 detection based on the light ATRP signal amplification strategy is of great significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an electrochemiluminescence lung cancer detection kit based on a metal-free photoinduced atom transfer radical polymerization (photo-ATRP) signal amplification strategy, a use method and application thereof, so that the use of a heavy metal ion catalyst in the traditional ATRP reaction is avoided, the signal is amplified in multiples, and the detection sensitivity, stability and reproducibility are improved.
In order to achieve the above object, one of the technical solutions of the present invention is:
an electrochemiluminescence lung cancer detection kit based on a metal-free light ATRP signal amplification strategy comprises the following raw materials: BMP, gold electrode, MPA, EDC, NHS, Ab1, BSA, Ab2, Me6TREN, EY, NAS, luminol, ultrapure water, H2O2DMSO, ethanol.
Further, when a part of the raw materials were used, it was necessary to prepare a solution in which the BMP solution concentration was 3mM, the MPA solution concentration was 10mM, the NHS solution for activating BMP concentration was 3mM, the EDC solution concentration was 3mM, the EDC and NHS mixed solution for activating MPA concentrations were 0.2M and 0.05M, respectively, the Ab1 solution concentration was 1. mu.g/mL, the BSA solution concentration was 1%, the Ab2 solution concentration was 1. mu.g/mL, Me6The concentration of TREN solution is 10mM, the concentration of EY solution is 5mM, the concentration of NAS solution is 12mM, and the concentration of luminol solution is 15 mM.
One of the technical schemes of the invention is as follows: an application method of an electrochemiluminescence lung cancer detection kit comprises the following steps:
(1) ab2-BMP (Ab 2)
Dissolving BMP in an ethanol solution to obtain a BMP solution;
adding NHS solution and EDC solution into BMP solution, stirring to obtain BMP solution activated by carboxyl;
③ adding the BMP solution activated by carboxyl into the Ab2 solution, and stirring to obtain Ab2 solution;
(2) electrode pretreatment
Polishing the bare gold electrode to obtain a polished mirror surface;
(3) electrode modification
Soaking the pretreated electrode in MPA solution, washing and drying;
soaking the electrode in the step I in mixed solution of EDC and NHS;
directly dripping the Ab1 solution on the surface of the electrode in the step II, reacting, and then soaking in the BSA solution;
dripping the sample to be detected on the surface of the electrode in the step (c), reacting and washing;
dripping Ab2 solution on the surface of the electrode in the step (iv) for reaction;
sixthly, the electrode in the step five is placed on Me6TREN solution, EY solution, NAS solution, H2Reacting in a mixed solution consisting of O and DMSO;
seventhly, placing the electrode in the step (c) in luminol solution for reaction;
eighthly, placing the electrode in the step seven on a H2O2The luminol luminescence intensity was measured.
Further, the concentration of the ethanol solution in the step (1) is 50-80% (v/v); the volume ratio of the BMP solution to the NHS solution to the EDC solution is 1:1: 1; the concentration of the carboxy-activated BMP solution was 1mM, the concentration of the Ab2 solution was 1. mu.g/mL, and the volume ratio of the carboxy-activated BMP solution to the Ab2 solution was 1: 1.
Further, the stirring temperature in the step (1) is 37 ℃, and the time is 1-3 h.
Further, the soaking temperature in the step (2) is 37 ℃, and the soaking time is 2-8 hours; the soaking temperature is 37 ℃, and the time is 1-2 hours; the reaction temperature of the third step is 37 ℃, and the reaction time is 1-3 hours; the reaction temperature is 37 ℃, and the reaction time is 1-3 h; the reaction temperature is 37 ℃ and the reaction time is 1-3 h; sixthly, the reaction condition is room temperature, 470nm light irradiation, 2-5 h; the reaction temperature is 37 ℃ for 1-3 h.
Further, Me6TREN solution, EY solution, NAS solution, H2The volume ratio of O to DMSO is 5:5:1000:5000: 3990.
One of the technical schemes of the invention is as follows: an application of the detection kit in CYFRA21-1 detection.
One of the technical schemes of the invention is as follows: an application of the detection kit in lung cancer detection.
The schematic diagram of the detection method of the invention is shown in figure 1.
The invention employs photo-ATRP and ECL signal amplification strategies. First, the MPA probe was attached to the electrode surface via a self-assembled polar covalent bond, and Ab1 was attached to the electrode surface as a capture probe via an amino condensation reaction. After the residual binding sites were closed with Bovine Serum Albumin (BSA), CYFR21-1 and Ab2 were bound by antigen-antibody interaction*Are sequentially connected on the surface of the gold electrode. The bromine group in the BMP is recognized by the double bond of the NAS, and a certain amount of NAS is grafted to the electrode surface by photo-ATRP. The monolithic NAS of photo-ATRP can provide many binding sites for the luminescent material luminol, thereby significantly amplifying the ECL signal. Finally, many luminol molecules are tightly attached to a large number of NAS's by amino condensation reactions.
The metal-free light ATRP process takes BMP as an initiator, EY as a catalyst and ME6TREN is a ligand, and polymerization of NAS was performed under 470nm light irradiation. The reductive quenching pathway starts under excitation of blue light of a specific wavelength, and as photons are absorbed, electrons transition from a ground state (EY) to an excited state (EY)*). Then EY*In the absence of electron donor (D, Me)6TREN) while generating EY·-And D·+Simultaneously generate D·+. Based on three catalysts (EY, EY)*And EY·-) A reversible cycle was established to regulate the reduction and quenching pathways of the photo-ATRP. Activation of the initiator (BMP) of the photo-ATRP with the concomitant production of a primary radical (PO. cndot.), of a halogen atom (X)-) And EY. Starting free radical addition reaction through double bonds of monomers, carrying out chain transfer to generate a polymeric chain, and achieving activation/deactivation balance. By the above-mentioned reduction quenching reaction, a large amount of the monomer is grafted to the electrode surface. The monomer was then combined with luminol for ECL detection. Hydrogen peroxide is an efficient co-reaction in the luminol ECL reactionSubstance, which tends to decompose into superoxide radicals (OH)·) And superoxide anion radical (O)2 ·-). Under the condition of applying a certain voltage, luminol and hydrogen peroxide as a co-reactant are oxidized at the same time, then the hydrogen peroxide is rapidly decomposed to generate a high-energy free radical intermediate, the high-energy free radical intermediate participates in the oxidation reaction of the luminol, the luminol is enabled to be in an excited state, and then the luminol returns to a ground state from the excited state and generates light with corresponding wavelength at the same time.
The invention utilizes a metal-free photoinduction atom transfer radical polymerization (photo-ATRP) strategy, avoids the use of nano materials and biological enzymes (which are easily influenced by external environment, temperature and the like) in the current common signal amplification strategy, multiplies the signals, and improves the sensitivity, stability and reproducibility of detection.
The invention adopts a metal-free photoinduction atom transfer radical polymerization (photo-ATRP) strategy, avoids the use of a heavy metal ion catalyst in the traditional ATRP reaction, does not need to recover and remove the heavy metal ions, overcomes the interference of the inherent color of the heavy metal ions on the detection result, does not generate biotoxicity, and is more environment-friendly.
Drawings
FIG. 1 is a schematic diagram of the detection method of the present invention.
FIG. 2A shows the electrochemiluminescence intensity under different conditions. Wherein, curve a is MPA-free, curve b is Ab 1-free, curve c is CYFRA 21-1-free, curve d is Ab 2-free, curve e is NAS-free, curve f is no light, curve g is EY-free, curve h is Me-free6TREN, curve i luminol/NAS/Ab 2 ×/CYFRA21-1/Ab 1/MPA/Au.
FIG. 2B is the evolution of the impedance curve of the electrode after modification from each step of the bare gold electrode (curve a → h), and the inset is the equivalent circuit diagram of EIS. RsFor solution resistance, CPE is a constant phase element, ZwIs Warburg impedance and RctIs a charge transfer resistance.
FIG. 2C is a CV curve of the electrode after modification from the bare gold electrode per step (curve a → h).
FIG. 3 is an atomic force microscope photograph of the electrode before and after surface modification of NAS. Wherein A is before modifying NAS, and B is after modifying NAS.
Fig. 4 is a photograph of contact angles of electrode surfaces in different modification states. Wherein A is before photo-ATRP and B is after photo-ATRP.
FIG. 5 shows the optimization of photo-ATRP reaction time (A), the optimization of NAS concentration (B), the optimization of luminol reaction time (C), and the optimization of luminol concentration (D).
FIG. 6 is a graph (A) of Electrochemiluminescence (ECL) intensity versus CYFRA21-1 concentration and the corresponding linear correlation curve (B).
FIG. 7A is a comparison of the electroluminescence intensity of 10pg/mL CYFRA21-1 and BSA, CEA, cTnI at the same concentration under the same detection conditions.
FIG. 7B is the signal intensity of different concentrations of CYFRA21-1 in PBS and 10% serum samples.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Ab1, Ab2 and CYFRA21-1 are all purchased from Beijing Zhongji health science and technology Limited, and the product numbers are CY11N005, CY11N007 and CS-CY211 respectively.
Example 1 kit
An electrochemiluminescence detection kit based on a metal-free light ATRP signal amplification strategy comprises the following raw materials: 2-Bromobutyric acid (BMP), gold electrode, 3-mercaptopropionic acid (MPA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Ab1, BSA, Ab2, Me6TREN, Eosin Y (EY), N-acryloxysuccinimide (NAS), luminol, ultrapure water, H2O2Dimethyl sulfoxide (DMSO), ethanol.
When part of the raw materials are used, a solution is prepared, wherein the concentration of a BMP solution is 3mM, the concentration of an MPA solution is 10mM, the concentration of an NHS solution for activating the BMP is 3mM, the concentration of an EDC solution is 3mM, the concentrations of EDC and NHS in a mixed solution of EDC and NHS for activating the MPA are 0.2M and 0.05M respectively, the concentration of an Ab1 solution is 1 mu g/mL, the mass concentration of a BSA solution is 1%, the concentration of an Ab2 solution is 1 mu g/mL, and Me is6Concentration of TREN solution 10mM, EThe concentration of the Y solution is 5mM, the concentration of the NAS solution is 12mM, and the concentration of the luminol solution is 15 mM.
Example 2 method of Using the kit
(1) Ab2-BMP (Ab 2)
Dissolving BMP in 60% (v/v) ethanol solution to obtain 3mM BMP solution;
② adding 150. mu.L NHS solution (3mM) and 150. mu.L EDC solution (3mM) into 150. mu.L BMP solution, stirring for 1h at 37 ℃ to obtain 1mM carboxyl activated BMP solution;
③ adding the carboxyl activated BMP solution into Ab2 solution (1. mu.g/mL) (1: 1 volume ratio), and stirring at 37 ℃ for 1h to obtain Ab2 solution.
(2) Electrode pretreatment
The bare gold electrode (diameter 2mm) was polished to obtain a polished mirror surface.
(3) Electrode modification
Soaking the pretreated electrode in 150 mu L of MPA solution (10mM) at 37 ℃ for 8h, washing with ultrapure water, and drying with nitrogen;
soaking the electrode obtained in the step I in 150 mu L of mixed solution of EDC (0.2M) and NHS (0.05M) for 1h at 37 ℃;
③ directly dripping 10 mu L of Ab1 solution (1 mu g/mL) on the surface of the electrode (MPA/Au) in the step II, reacting for 1h in a constant temperature incubator at 37 ℃, and then soaking for 1h in 150 mu L of 1% BSA solution;
dripping 10 mu L of a sample to be detected (containing CYFRA21-1) on the surface of the electrode (Ab1/MPA/Au) in the step (c), reacting for 2h at 37 ℃, and washing with ultrapure water;
dripping 10 uL Ab2 solution on the surface of the electrode (CYFRA21-1/Ab1/MPA/Au) in the step (r), and reacting for 1h at 37 ℃;
sixthly, the electrode (Ab 2X/CYFRA 21-1/Ab1/MPA/Au) of the fifth step is placed on 5 microliter Me6TREN solution (10mM), 5. mu.L EY solution (5mM), 1mL NAS solution (12mM), 5mL H2O and 3990. mu.L DMSO, and reacting for 3h (photo-ATRP) under the irradiation of 470nm light at room temperature;
seventhly, placing the electrode (NAS/Ab 2/CYFRA 21-1/Ab1/MPA/Au) in the step (sixth) into 150 mu L of luminol solution (15mM) and reacting for 1.5h at 37 ℃;
eighthly, placing the electrode (luminol/NAS/Ab 2)/CYFRA 21-1/Ab1/MPA/Au) in the step (seventhly) in 6mL H2O2The luminol luminescence intensity was measured in an MPI-E electrochemiluminescence detector (10mM, pH 7.4 in PBS buffer).
Example 3 feasibility verification
To demonstrate the feasibility of establishing the assay of the invention, 9 blank experiments were performed and ECL (enhanced chemiluminescence) reactions were recorded. As shown in FIG. 2A, in the absence of MPA (curve a), Ab1 (curve b), CYFRA21-1 (curve c), Ab2 (curve d), NAS (curve e), illumination (curve f), EY (curve g), Me6In the case of TREN (curve h) or luminol (curve i), the ECL signal was weak, and a significant ECL response was clearly observed for the fully modified electrodes (curve j), and taken together, the above experimental results indicate the necessity of the presence of each modification step and the feasibility of the method to detect CYFRA 21-1.
Example 4 characterization
Electrochemical Impedance Spectroscopy (EIS) can sensitively monitor microscopic changes from the electrode surface to the electrolyte for each step of verifying electrode modification. When the electrode is unmodified, R is shown in FIG. 2BctRelatively low (-240 Ω); when the small molecule MPA is combined with the gold electrode, RctIncreasing (-401 omega), and forming a layer of thin film on the surface of the electrode; after modification of Ab1, BSA, CYFRA21-1 and Ab2, RctThis continues to rise, which is associated with the protein macromolecular structure causing an ever-increasing thickness of the previously formed membrane. After photo-ATRP, RctA significant increase (-5726 Ω) which is the result of a large number of successful NAS grafts onto the initiator to form polymer chains. Finally, when the last step is completed, RctA further increase (-6875 Ω) is due to the spatial resistance increased by the connection of luminol. These results indicate that each of the connections of the present invention was successful.
The surface characteristics of the electrode were monitored for each fixation step using Cyclic Voltammetry (CV) (fig. 2C). When the MPA is connected to the bare electrode by self-assembly, the peak current is reduced compared to the bare gold electrode. When Ab1, BSA, CYFRA21-1, Ab2, NAS, and luminol were modified in this order on the electrode surface, the peak current was decreased in this order, and the results were consistent with the EIS results. The results show that the preparation of the full electrode of the invention is successful.
The electrode surface was characterized by Atomic Force Microscopy (AFM) and Water Contact Angle (WCA). The thicknesses of the front and rear electrode surfaces of the modified NAS were 12.2nm and 30.5nm, respectively, indicating the success of the photo-ATRP reaction (FIG. 3). The wettability of the electrode surface changes with the reaction and can therefore be characterized by WCA (fig. 4). The WCA of FIG. 4A was 96.6 before polymerization occurred and 99.9 after polymerization of FIG. 4B. These characterization results demonstrate the success of the full electrode construction of the present invention.
Example 5 optimization of assay conditions
To improve the sensitivity of the assay, the invention investigated the effect of photo-ATRP reaction time and NAS concentration and luminol reaction time and concentration on the reaction.
(1) Optimization of photo-ATRP reaction time
FIG. 5A shows that the ECL signal increases almost linearly with reaction time over the first 150 min. Gradually, the growth rate slowed down and eventually reached zero after 180 min. This is due to the effect of poor electron transfer efficiency or radical termination. Thus, the optimum reaction polymerization time for the photo-ATRP is 180 min.
(2) NAS concentration optimization
FIG. 5B shows the change in NAS concentration and luminescence intensity, with the electroluminescent signal increasing with concentration and reaching a maximum at 12mM, confirming that the NAS is saturated. Therefore, 12mM was selected as the optimum concentration for NAS.
(3) Reaction time and concentration of luminol
As shown in fig. 5C, the electroluminescence signal gradually increased with the luminol reaction time within 60min and stopped increasing at 90 min. Therefore, the optimal time for the luminol reaction is chosen to be 90 min. Figure 5D shows that the electroluminescent signal increases with increasing luminol concentration within 15 mM. Subsequently, the electroluminescent signal gradually levels off with further increase in luminol concentration. Therefore, the concentration of luminol was set to 15mM in the experiment.
Example 6 analytical Properties
The detection Limit (LOD) and linear response range of the CYFRA21-1 assay kit were evaluated by ECL under optimal conditions. As shown in FIG. 6A, ECL signal increased linearly when the concentration of CYFRA21-1 was within the interval of 10fg/mL to 1 ng/mL. FIG. 6B further shows IECLThe log value of (A) is in positive correlation with the concentration of CYFRA 21-1. The corresponding linear equation is I1458 log (C)CYFRA 21-1/pg mL-1)+3320(R20.9914), the calculated LOD was 5.8fg/mL (S/N-3).
Compared with other methods, the method for detecting CYFRA21-1 has relatively low detection limit due to the combination of the sensitivity of ECL and the advantage of metal-free light ATRP signal amplification (the following table). The kit has potential application value in early detection of lung cancer.
Example 7: selectivity, interference rejection and stability of electroluminescent sensors
To verify the selectivity of this signal amplification method, the present invention selects different detection substances, including cardiac troponin I (cTnI, 10pg/mL), carcinoembryonic antigen (CEA, 10pg/mL) and BSA (10pg/mL), and performs the selectivity test under the same experimental conditions (CYFRA21-1, 10 pg/mL). As can be seen from FIG. 7, the optical signal intensities of BSA, CEA and cTnI were 6.16%, 6.78% and 9.61% of CYFRA21-1, respectively. Since Ab1 on the electrode can be specifically recognized by CYFRA21-1, this allows Ab1 to be recognized and bound to CYFRA 21-1. This resulted in differences in the binding efficiency of different antigens to Ab1 in the experiment, indicating that the method of the invention can effectively distinguish CYFRA 21-1.
To verify the stability of this signal amplification method, 5 fully modified electrodes (luminol/NAS/Ab 2;/CYFRA 21-1/Ab1/MPA/Au) were selected and stored in a refrigerator at 4 ℃. After three weeks, the intensity of ECL signal reached around 91% of the initial signal. These results show that the electrode (luminol/NAS/Ab 2 x/CYFRA 21-1/Ab1/MPA/Au) has good stability during storage.
To test the anti-interference effect of the kit, ECL was used to detect different concentrations of CYFRA21-1(1ng/mL, 10pg/mL, 100fg/mL) in PBS and 10% serum samples. As shown in FIG. 7B, the signal intensities of 1ng/mL, 10pg/mL, 100fg/mL CYFRA21-1 in the 10% serum sample were 96.89%, 96.28% and 82.63% in PBS, respectively. These results indicate that the method of the invention has good anti-interference ability in the serum of complex patients and has significant clinical application potential.
Claims (10)
1. An electrochemiluminescence lung cancer detection kit based on a metal-free light ATRP signal amplification strategy is characterized by comprising the following raw materials: BMP, gold electrode, MPA, EDC, NHS, Ab1, BSA, Ab2, Me6TREN, EY, NAS, luminol.
2. The electrochemiluminescence lung cancer detection kit of claim 1, further comprising ultrapure water, H2O2DMSO, ethanol.
3. The electrochemiluminescence lung cancer assay kit of claim 1, wherein a part of the raw materials are used to prepare a solution, wherein the concentration of the BMP solution is 3mM, the concentration of the MPA solution is 10mM, the concentration of the NHS solution for activating BMP is 3mM, the concentration of the EDC solution is 3mM, the concentrations of EDC and NHS in the mixed solution of EDC and NHS for activating MPA are 0.2M and 0.05M, respectively, the concentration of the Ab1 solution is 1 μ g/mL, the concentration of the BSA solution is 1%, the concentration of the Ab2 solution is 1 μ g/mL, Me is 2 μ g/mL, and Me is6The concentration of TREN solution is 10mM, the concentration of EY solution is 5mM, the concentration of NAS solution is 12mM, and the concentration of luminol solution is 15 mM.
4. The method of using the electrochemiluminescence lung cancer detection kit of claim 1, comprising the steps of:
(1) ab2-BMP (Ab 2)
Dissolving BMP in an ethanol solution to obtain a BMP solution;
adding NHS solution and EDC solution into BMP solution, stirring to obtain BMP solution activated by carboxyl;
③ adding the BMP solution activated by carboxyl into the Ab2 solution, and stirring to obtain Ab2 solution;
(2) electrode pretreatment
Polishing the bare gold electrode to obtain a polished mirror surface;
(3) electrode modification
Soaking the pretreated electrode in MPA solution, washing and drying;
soaking the electrode in the step I in mixed solution of EDC and NHS;
directly dripping the Ab1 solution on the surface of the electrode in the step II, reacting, and then soaking in the BSA solution;
dripping the sample to be detected on the surface of the electrode in the step (c), reacting and washing;
dripping Ab2 solution on the surface of the electrode in the step (iv) for reaction;
sixthly, the electrode in the step five is placed on Me6TREN solution, EY solution, NAS solution, H2Reacting in a mixed solution consisting of O and DMSO;
seventhly, placing the electrode in the step (c) in luminol solution for reaction;
eighthly, placing the electrode in the step seven on a H2O2The luminol luminescence intensity was measured.
5. The electrochemiluminescence lung cancer detection kit of claim 4, wherein the concentration of the ethanol solution in step (1) is 50% to 80% (v/v); the volume ratio of the BMP solution to the NHS solution to the EDC solution is 1:1: 1; the concentration of the carboxy-activated BMP solution was 1mM, the concentration of the Ab2 solution was 1. mu.g/mL, and the volume ratio of the carboxy-activated BMP solution to the Ab2 solution was 1: 1.
6. The electrochemiluminescence lung cancer detection kit as claimed in claim 4, wherein the stirring temperature in step (1) is 37 ℃ and the stirring time is 1-3 h.
7. The use method of the electrochemiluminescence lung cancer detection kit according to claim 4, wherein the soaking temperature of the first step in the step (2) is 37 ℃ and the time is 2-8 hours; the soaking temperature is 37 ℃, and the time is 1-2 hours; the reaction temperature of the third step is 37 ℃, and the reaction time is 1-3 hours; the reaction temperature is 37 ℃, and the reaction time is 1-3 h; the reaction temperature is 37 ℃ and the reaction time is 1-3 h; sixthly, the reaction condition is room temperature, 470nm light irradiation, 2-5 h; the reaction temperature is 37 ℃ for 1-3 h.
8. The use method of the electrochemiluminescence lung cancer detection kit according to claim 4, wherein Me is6TREN solution, EY solution, NAS solution, H2The volume ratio of O to DMSO is 5:5:1000:5000: 3990.
9. Use of the test kit of claim 1 in the CYFRA21-1 test.
10. Use of the test kit of claim 1 for lung cancer detection.
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