CN114235907A - Electrochemical luminescence immunosensor for non-small cell lung cancer CYFRA21-1 detection and detection method - Google Patents
Electrochemical luminescence immunosensor for non-small cell lung cancer CYFRA21-1 detection and detection method Download PDFInfo
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Abstract
The invention provides an electrochemical luminescence immunosensor for non-small cell lung cancer CYFRA21-1 detection, which is characterized in that a MXene-PEI-NiMn LDH novel nano composite material is prepared as a substrate material of the sensor, a La-MOF @ ZIF-67 bi-metal organic frame is used for carrying Luminol as a luminescent substance, and the electrochemical luminescence sensor for detecting CYFRA21-1 is constructed by utilizing a sandwich method. The substrate materials MXene-PEI-NiMn LDH and the bimetallic organic framework La-MOF @ ZIF in the invention-67 as co-reaction promoter, co-catalysing H2O2The generated hydroxyl free radical sensor not only has stronger signal amplification effect, but also has the advantages of wide linear range, strong specificity, short analysis time and the like, and can provide a new method for diagnosing the non-small cell lung cancer. The detection method is simple to operate and high in practicability.
Description
Technical Field
The invention belongs to the technical field of electrochemical luminescence immunosensors and nano composite materials, and relates to an electrochemical luminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1 and a detection method.
Background
Lung cancer is one of the most common malignancies in the world and has become a leading cause of cancer-related death worldwide. Lung cancer is classified into small cell lung cancer and non-small cell lung cancer, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all lung cancers, and approximately 75% of NSCLC patients are already in an advanced stage when they are found. The existing diagnosis method has the defects of high price, complex and time-consuming process, large body injury of a patient and the like. Therefore, for the non-small cell lung cancer, the method has important clinical significance for exploring a disease diagnosis method which is low in cost, simple, convenient and quick and has small body damage. CYFRA21-1 is a soluble fragment of cytokeratin 19 (CK 19). It is a common biomarker of non-small cell lung cancer, and the level of the biomarker is closely related to the type and the severity of a tumor, so that the detection of CYFRA21-1 has important significance for the diagnosis of the lung cancer.
At present, the detection method of CYFRA21-1 mainly comprises the traditional methods such as fluorimetry, surface plasma resonance analysis, immunoradiometric method and the like. Although the sensitivity is high, the defects of time consumption, high cost, expensive equipment, complex operation and the like exist, so that the development of a simple, sensitive, rapid and economic method for detecting the clinical specimen CYFRA21-1 is necessary.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA 21-1. The invention constructs a sandwich type electrochemical luminescence immunosensor which takes a bimetallic organic framework as a carrier and a nano composite material of MXene and NiMn LDH as a substrate and synergistically promotes Luminol luminescence, and provides a new diagnosis approach for detecting non-small cell lung cancer.
Except for special description, the parts are parts by weight, and the percentages are mass percentages.
In order to achieve the purpose, the technical scheme of the invention is as follows:
an electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1, which is characterized in that,
an electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 is constructed by a signal probe and a substrate material.
The method for constructing the electrochemical luminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 comprises the following steps: the substrate material MXene-PEI-NiMn LDH solution is dripped on the surface of a clean glassy carbon electrode, then the AuNPs solution is dripped on the surface of the glassy carbon electrode, and Ab dissolved by PBS buffer solution at room temperature is added1Dropwise adding the mixture on a glassy carbon electrode, and incubating for 12h at 4 ℃; then dropwise adding 1% BSA solution on the electrode, and incubating for 30min at 4 ℃; and finally, dripping CYFRA21-1 dissolved by PBS buffer solution at room temperature on a glassy carbon electrode, and incubating for 1.5h at 4 ℃ to obtain the electrochemical luminescence immunosensor for detecting CYFRA 21-1.
The signal probe consists of La-MOF @ ZIF-67@ AuNPs @ Luminol solution and Ab2Stirring at room temperature for 12 hours, centrifuging, and washing the precipitate to obtain a signal probe.
The preparation method of the La-MOF @ ZIF-67@ AuNPs @ Luminol solution comprises the following steps:
1) preparation of ZIF-67 dispersion: mixing Co (NO)3)2·6H2Dissolving O and 2-methylimidazole in methanol, stirring for 3 hours at room temperature, centrifuging, washing with methanol and water, drying at 60 ℃ to obtain brown powder, and dispersing the brown powder in ultrapure water to obtain ZIF-67 dispersion liquid with the concentration of 1 mg/mL;
2) preparation of La-MOF Dispersion La (NO)3)3Adding 2-amino terephthalic acid into a DMF solution, stirring at room temperature for 10 minutes, then transferring into a stainless steel autoclave lined with polytetrafluoroethylene, reacting at 150 ℃ for 6 hours, cooling to room temperature, washing with methanol and water, centrifuging, collecting precipitate, and vacuum drying at 60 ℃ for 12 hours; finally, dispersing the dried precipitate in ultrapure water to prepare a La-MOF dispersion liquid with the concentration of 1 mg/ml;
3) preparation of AuNPs solution: 100mL of 0.01% HAuCl4SolutionThe solution was boiled under stirring, then 2.5mL of 1% sodium citrate solution was added, kept boiling for 15min and then cooled to room temperature. Finally, the solution turned purple-red in color, indicating successful synthesis of AuNPs.
4) Preparation of La-MOF @ ZIF-67@ AuNPs @ Luminol: firstly, adding 1mL of 1mg/mL La-MOF dispersion liquid into 1mL of 1mg/mL ZIF-67 dispersion liquid, stirring for 12 hours at room temperature, and centrifugally washing to obtain La-MOF @ ZIF-67 nanocomposite dispersion liquid; then adding the AuNPs solution into the nano composite material dispersion liquid, keeping the mixture uniformly stirred for 4 hours at room temperature, washing with water, centrifuging to obtain precipitate La-MOF @ ZIF-67@ AuNPs, and dissolving with ultrapure water to obtain La-MOF @ ZIF-67@ AuNPs solution; then adding Luminol, stirring for 4h, centrifuging, and dissolving the precipitate with ultrapure water to obtain a La-MOF @ ZIF-67@ AuNPs @ Luminol solution.
The substrate material MXene-PEI-NiMn LDH solution is prepared by mixing MXene dispersion and 1% PEI solution, stirring the mixture at room temperature for 2 hours, adding NiMn LDH dispersion, stirring for 8 hours, centrifuging and washing the precipitate to obtain the MXene-PEI-NiMn LDH substrate material; and then dissolving the MXene-PEI-NiMn LDH substrate material in ultrapure water to obtain a substrate material MXene-PEI-NiMn LDH solution for later use at 4 ℃.
The preparation method of the NiMn LDH dispersion liquid is characterized in that MnSO is added4·4H2O、 Ni(NO3)2·6H2O and NH4Dissolving F in ultrapure water, carrying out ultrasonic treatment to obtain a uniform solution, and dropwise adding NH3·H2O, then stirring for 4 hours at room temperature, aging for 12 hours, centrifuging the product, washing the precipitate with ultrapure water, and drying in a vacuum oven at 60 ℃ for 12 hours; and finally dispersing the dried precipitate in ultrapure water to prepare a NiMn LDH dispersion liquid with the concentration of 1 mg/ml.
An electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1 is characterized in that the preparation process comprises the following steps:
(1) preparation of a signal probe:
1) preparation of ZIF-67 dispersion: 1.455g of Co (NO)3)2·6H2O and 1.65g of 2-methylimidazole were dissolved in 30mL of each solutionAnd methanol, and then the mixture was stirred at room temperature for 3 hours, and then the mixture was centrifuged to obtain a brown powder, which was washed with methanol and water, dried at 60 ℃, and dispersed in ultrapure water to prepare a ZIF-67 dispersion having a concentration of 1 mg/mL.
2) Preparation of La-MOF Dispersion 2mmol of La (NO)3)3Adding 2-amino terephthalic acid into 30mL of DMF solution, carrying out ultrasonic treatment until the solution is uniformly dispersed, stirring the solution at room temperature for 10 minutes, then transferring the solution into a stainless steel autoclave lined with polytetrafluoroethylene, reacting the solution at 150 ℃ for 6 hours, cooling the solution to the room temperature, washing the solution with methanol and water, centrifuging the solution, collecting precipitates, and carrying out vacuum drying at 60 ℃ for 12 hours; finally, the precipitate was dispersed in ultrapure water to prepare a dispersion of La-MOF at a concentration of 1 mg/ml.
3) Preparation of AuNPs solution: 100mL of 0.01% HAuCl4The solution was boiled under stirring, then 2.5mL of 1% sodium citrate solution was added, kept boiling for 15min and then cooled to room temperature. Finally, the solution turned purple-red in color, indicating successful synthesis of AuNPs.
4) La-MOF @ ZIF-67@ AuNPs @ Luminol solution: firstly, adding 1mL of 1mg/mL La-MOF dispersion liquid into 1mL of 1mg/mL ZIF-67 dispersion liquid, stirring for 12 hours at room temperature, centrifuging, and dissolving precipitate with ultrapure water to obtain La-MOF @ ZIF-67 nanocomposite dispersion liquid; and then adding the AuNPs solution into the nano composite material dispersion liquid, stirring for 4 hours at room temperature, washing with water, centrifuging, dissolving the precipitate with ultrapure water to obtain La-MOF @ ZIF-67@ AuNPs solution, adding 1mL of 0.01M Luminol, stirring for 4 hours, centrifuging, and dissolving the precipitate with ultrapure water to obtain La-MOF @ ZIF-67@ AuNPs @ Luminol solution.
5) Preparation of a signal probe: taking 1mL of La-MOF @ ZIF-67@ AuNPs @ Luminol solution prepared in the step 4) and 100 mu L of Ab2And stirring at room temperature for 12 hours, centrifuging, and washing precipitates to obtain the signal probe.
(2) Preparation of the base material:
1) NiMn LDH 0.3mmol of MnSO4·4H2O、0.3 mmol Ni(NO3)2·6H2O and 1.8mmol NH4F was dissolved in 25mL of ultrapure water. Ultrasonic treatmentAfter forming a homogeneous solution, 2mL NH was added dropwise3·H2And O, stirring and mixing for 4 hours at room temperature, aging for 12 hours, centrifuging the product, washing the precipitate with ultrapure water, drying in a vacuum oven at 60 ℃ for 12 hours, and finally dispersing the dried precipitate in the ultrapure water to obtain the NiMn LDH dispersion liquid with the concentration of 1 mg/ml.
2) MXene-PEI-NiMn LDH: mixing 1mL of MXene dispersion (1mg/mL) with 500. mu.L of 1% PEI solution, stirring at room temperature for 2 hours, adding 1mg of NiMn LDH dissolved in 1mL of ultrapure water, stirring for 8 hours, and centrifuging to obtain a precipitated MXene-PEI-NiMn LDH composite material. The prepared MXene-PEI-NiMn LDH composite material is dissolved in 1mL of ultrapure water for later use at 4 ℃.
(3) The construction process of the electrochemical luminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 comprises the following steps:
1) ab was dissolved in 0.1M PBS (pH = 7.4) buffer at room temperature1And CYFRA21-1, stored for later use;
2) piranha washing solution (98% H) for glassy carbon electrode2SO4/30% H2O2= 3:1, v/v) soaking for 30min, and then washing with ultrapure water for standby;
3) respectively using Al of 0.3 mu m and 0.05 mu m for the electrode obtained in the step 2)2O3Polishing the powder to form a mirror surface, then respectively carrying out ultrasonic treatment on the electrodes according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying for later use;
4) subjecting the electrode obtained in step 3) to a temperature of 0.5M H2SO4Performing electrochemical activation, washing with ultrapure water, and drying;
5) dripping 10 mu L of substrate material solution on the surface of the glassy carbon electrode cleaned in the step 4), and drying at room temperature;
6) dripping 10 mu L of AuNPs solution on the surface of the glassy carbon electrode in the step 5), and drying at room temperature;
7) mu.L of Ab prepared in the step 1)1Dropwise adding a buffer salt solution on the electrode prepared in the step 6), and incubating for 12h at 4 ℃;
8) dripping 6 mu L of 1% BSA solution on the electrode obtained in the step 7), and incubating for 30min at 4 ℃;
9) dripping 10 mu L of CYFRA21-1 buffer salt solution prepared in the step 1) on the electrode prepared in the step 8), and incubating for 1.5h at 4 ℃ to obtain the electrochemical luminescence immunosensor for detecting CYFRA 21-1.
The invention also provides a method for detecting the non-small cell lung cancer CYFRA21-1 by using the electrochemical luminescence immunosensor.
A method for detecting non-small cell lung cancer CYFRA21-1 by using an electrochemical luminescence immunosensor is characterized by comprising the following steps:
(1) dripping a signal probe solution on the surface of an electrode of the electrochemical luminescence immunosensor, and incubating for 4 hours at 4 ℃;
(2) washing the electrode prepared in the step 1) with ultrapure water, airing, and placing the electrode in a container containing 5mM H2O2Measuring the change value of the electrochemiluminescence response signal of CYFRA21-1 with different concentrations in 0.1M PBS solution;
(3) drawing a standard curve according to the linear relation between the logarithm values of different concentrations and the change value of the electrochemical luminescence response signal;
(4) and detecting a sample to be detected by using the electrochemical luminescence immunosensor to obtain an electrochemical luminescence response signal value, and converting through a standard curve to obtain the concentration of CYFRA21-1 in the sample to be detected.
Compared with the prior art, the preparation method and the application of the electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 have the prominent characteristics that:
the method prepares the MXene-PEI-NiMn LDH nano composite material finally formed by the Polyethyleneimine (PEI) functionalized nitrogen-doped MXene modified NiMn LDH nano material as the sensitive interface of the sensor; the loading capacity of AuNPs on unit area is synergistically improved by utilizing larger specific surface area and good conductivity of the novel nano composite; then Ab1Immobilized on the surface of the electrode through Au-N bond, CYFRA21-1 and Ab1And finally, the specific binding of the signaling probe and the target probe with different concentrations causes different changes of an electrochemiluminescence signal, so that the quantitative detection of the CYFRA21-1 is realized. The prepared electrochemiluminescence immunizationThe sensor was successfully used for ultrasensitive detection of CYFRA 21-1. Compared with the traditional CYFRA21-1 detection method, the method has the advantages of high sensitivity, strong specificity, rapid detection, convenient operation, low equipment material price and no pollution, thereby providing a new analysis method for the detection of CYFRA 21-1.
Has the advantages that:
(1) by functionalizing MXene with polyethyleneimine, the conductivity of NiMn LDH is greatly improved, the MXene is effectively prevented from being aggregated, and can be uniformly spread on the surface of an electrode, so that the excellent physical and chemical properties of the MXene can be fully exerted.
(2) The prepared MXene-PEI-NiMn LDH has larger specific surface area and is used as a binding site for fixing metal, and the MXene-PEI-NiMn LDH and the binding site are synergistic to improve the load capacity of AuNPs on a unit area and further improve the Ab1Thereby improving the sensitivity of the sensor, and providing a new research direction and an analysis method for detecting the trace CYFRA 21-1.
(4) The related materials can be synthesized under the laboratory condition, the operation is simple, the raw materials are low in price, the using amount is very small each time, and the experiment cost is reduced.
(5) The whole detection and analysis method has clear and simple steps, high sensitivity and rapid signal response.
(6) The electrochemical biosensor prepared by the method can provide a new method for detecting the non-small cell lung cancer CYFRA 21-1; the electrochemical sensor prepared by the invention can also be applied to the aspects of diagnosis, analysis, detection and the like of diseases.
Drawings
FIG. 1: the sensor of the invention has the following detection results on different concentrations CYFRA 21-1: panel A shows a sample containing 5mM H2O2ECL profile of sensor scanning for different concentrations of CYFRA21-1 in 0.1M PBS buffer; graph B is a calibration curve of various concentrations CYFRA21-1 log values versus sensor ECL response values.
FIG. 2: the stability, reproducibility and specificity of the sensor of the invention are as follows: FIG. A shows the results of the stability test of the sensor of the present invention: two different concentrations of CYFRA21-1(10 pg/mL and 10) will be incubated0 pg/mL) was scanned for 15 ECL plots; and B: the detection result of the reproducibility of the sensor of the invention is as follows: for the same batch of 5 different electrodes containing 5mM H2O2ECL profile scanned in 0.1M PBS; panel C is a bar graph of ECL response values measured after incubation of CYFRA21-1 (100 pg/mL) and an interferent (1 ng/mL), such as Ascorbic Acid (AA), Dopamine (DA), glucose (Glu), Human Serum Albumin (HSA), carcinoembryonic antigen (CEA), blank solution (blank) under the same conditions.
Detailed Description
The present invention is described in detail below with reference to specific examples, which are given for the purpose of further illustrating the invention and are not to be construed as limiting the scope of the invention, and the invention may be modified and adapted by those skilled in the art in light of the above disclosure.
The raw materials and reagents used in the invention are all commercial products.
MXene was ordered from the premier nanomaterial science and technology limited (nanjing, china). MnSO4·4H2O and N, N-Dimethylformamide (DMF) were purchased from michelin biochemical limited, shanghai, china. Bovine Serum Albumin (BSA) was purchased from J&K Scientific Ltd (Beijing, China). Gold chloride hydrate (HAuCl)4) And 2-aminoterephthalic acid were purchased from Sigma-Aldrich Chemical Co., USA. Polyethyleneimine (PEI) was purchased from Alfa Aesar co. Methanol was purchased from Chongqing Chuandong chemical group Co Ltd, Ni (NO)3)2·6 H2O、NH4F、La(NO3)3·6H2O、Co(NO3)2·6H2O, 2-methylimidazole, luminol were purchased from Aladdin (USA). Hydrogen peroxide (H)2O2) Purchased from Chongqing Kelong chemical group Co. CYFRA21-1 antigen and CYFRA21-1 antibody (Ab) used in the present invention1And Ab2) Purchased from Cloud-Clone limited (Wuhan, China).
The equipment and technical parameters used are as follows:
at an electrochemical workstation (In Autolab PGSTSAT 302N, Metrohm China Ltd), Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) measurements were performed using a three-electrode system, which included a platinum wire (counter electrode), a saturated calomel electrode (SCE, reference electrode), and a modified glassy carbon electrode (GCE, working electrode). ECL was performed by a workstation (Xi' an Remax Electronic High-Tech Ltd, China) using a three-electrode system containing 5mM H2O2In 0.1M PBS.
Example 1
Preparation of composite nano material and construction of electrochemical luminescence immunosensor
The method comprises the following steps (constructing a schematic diagram as shown in figure 1):
step 1: ZIF-67 dispersion: 1.455g of Co (NO)3)2·6H2O and 1.65g of 2-methylimidazole were dissolved in 30mL of methanol, respectively. After the ultrasonic treatment, the above solution was mixed and stirred at room temperature for 3 hours. The mixture was centrifuged to give a brown powder, which was washed with methanol and water and dried at 60 ℃ for further use. Finally, the brown powder was dispersed in ultrapure water to prepare a ZIF-67 dispersion having a concentration of 1 mg/ml.
Step 2: La-MOF Dispersion 2mmol of La (NO)3)3And 2-aminoterephthalic acid was slowly added to 30mL of DMF solution. After the ultrasonic treatment until the mixture is uniformly dispersed, the mixture is stirred for 10 minutes at room temperature, then transferred to a stainless steel autoclave lined with polytetrafluoroethylene, reacted for 6 hours at 150 ℃, cooled to room temperature, washed by methanol and water, centrifuged to collect precipitate, and dried in vacuum for 12 hours at 60 ℃. Finally, the precipitate is dispersed in ultrapure water to prepare a La-MOF dispersion solution with the concentration of 1 mg/ml.
And step 3: preparation of AuNPs solution: 100mL of 0.01% HAuCl4The solution was boiled under vigorous stirring, then 2.5mL of 1% sodium citrate solution was added rapidly to the boiling solution, the solution was kept boiling for 15min and cooled to room temperature. Finally, the solution turned purple-red in color, indicating successful synthesis of AuNPs.
And 4, step 4: preparation of La-MOF @ ZIF-67@ AuNPs @ Luminol solution: 1mL of 1mg/mL La-MOF dispersion was added to 1mL of 1mg/mL ZIF-67 dispersion and stirred at room temperature for 12 hours. Centrifuging, washing the precipitate to obtain a La-MOF @ ZIF-67 nano composite material, dispersing the La-MOF @ ZIF-67 by using ultrapure water, adding AuNPs solution, uniformly stirring for 4 hours at room temperature, and centrifuging to obtain the precipitate La-MOF @ ZIF-67@ AuNPs. Dissolving La-MOF @ ZIF-67@ AuNPs in ultrapure water, adding 1mL of 0.01M Luminol, stirring for 4 hours, centrifuging,
dissolving the precipitate with ultrapure water to obtain a La-MOF @ ZIF-67@ AuNPs @ Luminol solution.
And 5: preparing a signal probe, namely taking 1mL of the La-MOF @ ZIF-67@ AuNPs @ Luminol solution prepared in the step 4) and 100 mu L of Ab2Stirring at room temperature for 12 hours, centrifuging, and washing precipitates to obtain a signal probe.
Step 6: NiMn LDH 0.3mmol of MnSO4·4H2O、0.3 mmol Ni(NO3)2·6H2O and 1.8mmol NH4F was dissolved in 25mL of ultrapure water. After being treated by ultrasonic to form a uniform solution, 2mL of NH is added dropwise under vigorous stirring3·H2And O. The dispersion was then mixed with a magnetic stirrer at room temperature for 4 hours. After aging for 12 hours, the product was centrifuged, and the precipitate was washed with ultrapure water and dried in a vacuum oven at 60 ℃ for 12 hours. And finally, dispersing the dried precipitate in ultrapure water to obtain the NiMn LDH dispersion liquid with the concentration of 1 mg/ml.
And 7: 1mL of MXene dispersion (1mg/mL) was mixed with 500. mu.L of 1% PEI solution, and the mixture was stirred at room temperature for 2 hours. 1mg of NiMn LDH dissolved in 1mL of ultrapure water was added to the above dispersion with vigorous stirring and kept stirring for 8 hours. Centrifuging to obtain the precipitate MXene-PEI-NiMn LDH composite material (substrate material). The prepared MXene-PEI-NiMn LDH composite material is dissolved in 1mL of ultrapure water and is reserved at 4 ℃.
And 8: ab was dissolved in 0.1M PBS (pH = 7.4) buffer at room temperature1And CYFRA21-1, stored for use.
And step 9: piranha washing solution (98% H) for glassy carbon electrode2SO4/30% H2O2= 3:1, v/v) soaking for 30min, and washing with ultrapure water for later use.
Step 10: will be provided withThe electrodes obtained in step 9 were coated with 0.3 μm and 0.05 μm Al, respectively2O3Polishing the powder to form a mirror surface, then respectively carrying out ultrasonic treatment on the electrodes according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying for later use.
Step 11: the electrode obtained in step 10 was set at 0.5M H2SO4Then washing with ultrapure water and drying.
Step 12: and (3) dripping 10 mu L of the base material solution prepared in the step (7) onto the surface of the glassy carbon electrode cleaned in the step (11), and drying at room temperature.
Step 13: 10 μ L of AuNPs solution was added dropwise to the glassy carbon electrode surface of step 12 and dried at room temperature.
Step 14: mu.L of Ab prepared in step 81The buffered saline solution was dropped on the electrode prepared in step 13, and incubated at 4 ℃ for 12 hours.
Step 15: dripping 6 μ L of 1% BSA solution on the electrode obtained in step 14, incubating at 4 deg.C for 30min,
step 16: dripping 10 μ L of CYFRA21-1 buffer salt solution obtained in step 8 on the electrode obtained in step 15, and incubating at 4 deg.C for 1.5h to obtain the electrochemical luminescence immunosensor for detecting CYFRA 21.
Example 2
Detection of non-small cell lung cancer CYFRA21-1 by electrochemical luminescence immunosensor
The electrochemiluminescence immunosensor constructed in example 1 was used to detect CYFRA21-1, and the following procedures were performed:
(1) drawing a standard curve:
in the immunosensor constructed in example 1, 10. mu.L of the signal probe prepared in step 5 was dropped onto the surface of the electrode, and the electrode was placed in a solution containing 5mM H2O2Was characterized in 0.1M PBS solution, and current values were measured for various concentrations of CYFRA 21-1. A standard curve is drawn according to the log values of different concentrations CYFRA21-1 and ECL response signals, and the detection results show that the two are in a good linear relation in a concentration range of 100 fg/mL-100 ng/mL, the linear correlation coefficient is 0.9980, the detection limit is 85.20 fM, and the results are shown in detail in figure 1.
(2) And (3) detecting the stability of the sensor:
the prepared sensor respectively scans two different concentrations of CYFRA21-1 for 15 circles continuously, and then the ECL signal changes little, and the RSD is respectively 1.53% and 1.77%, and the result is detailed in figure 2 (A), and the data show that the sensor has acceptable stability.
(3) Sensor reproducibility detection:
under the same conditions, when CYFRA21-1 (100 pg/mL) was measured using 5 different electrodes of the same batch prepared according to the present invention, the results are shown in detail in fig. 2(B), and the Relative Standard Deviation (RSD) of the current response values was 1.43%, indicating that the reproducibility of the sensor was good.
(4) And (3) specific detection of a sensor:
in order to test the specificity of the sensor of the present invention, the following were tested under the same conditions: CYFRA21-1 (100 pg/mL) and interferents AA, DA, Glu, HSA, CEA, blank (1 ng/mL) in a medium containing 5mM H2O2The results of the ECL response values in 0.1M PBS of (1) are shown in FIG. 2(C), which indicate that the biosensor of the present invention has satisfactory specificity.
Claims (7)
1. An electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1, comprising: constructing an electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 through the signaling probe and the substrate material; the method for constructing the electrochemical luminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 comprises the following steps: the substrate material MXene-PEI-NiMn LDH solution is dripped on the surface of a clean glassy carbon electrode, then the AuNPs solution is dripped on the surface of the glassy carbon electrode, and Ab dissolved by PBS buffer solution at room temperature1Dropwise adding the mixture on a glassy carbon electrode, and incubating for 12h at 4 ℃; then dropwise adding 1% BSA solution on the electrode, and incubating for 30min at 4 ℃; and finally, dripping CYFRA21-1 dissolved by PBS buffer solution at room temperature on a glassy carbon electrode, and incubating for 1.5h at 4 ℃ to obtain the electrochemical luminescence immunosensor for detecting CYFRA 21-1.
2. The electrochemiluminescence immunosensor of claim 1, wherein: said signalThe probe consists of La-MOF @ ZIF-67@ AuNPs @ Luminol solution and Ab2Stirring at room temperature for 12 hr, centrifuging, and washing the precipitate.
3. The electrochemiluminescence immunosensor of claim 2, wherein: the preparation method of the La-MOF @ ZIF-67@ AuNPs @ Luminol solution comprises the following steps:
1) preparation of ZIF-67 dispersion: mixing Co (NO)3)2·6H2Dissolving O and 2-methylimidazole in methanol, stirring for 3 hours at room temperature, centrifuging, washing with methanol and water, drying at 60 ℃ to obtain brown powder, and dispersing the brown powder in ultrapure water to obtain ZIF-67 dispersion liquid with the concentration of 1 mg/mL;
2) preparation of La-MOF Dispersion La (NO)3)3Adding 2-amino terephthalic acid into a DMF solution, stirring at room temperature for 10 minutes, then transferring into a stainless steel autoclave lined with polytetrafluoroethylene, reacting at 150 ℃ for 6 hours, cooling to room temperature, washing with methanol and water, centrifuging, collecting precipitate, and vacuum drying at 60 ℃ for 12 hours; finally, dispersing the dried precipitate in ultrapure water to prepare a La-MOF dispersion liquid with the concentration of 1 mg/ml;
3) preparation of AuNPs solution: 100mL of 0.01% HAuCl4Boiling the solution under stirring, adding 2.5mL of 1% sodium citrate solution, keeping boiling for 15min, and cooling to room temperature;
4) preparation of La-MOF @ ZIF-67@ AuNPs @ Luminol: firstly, adding 1mL of 1mg/mL La-MOF dispersion liquid into 1mL of 1mg/mL ZIF-67 dispersion liquid, stirring for 12 hours at room temperature, and centrifugally washing to obtain La-MOF @ ZIF-67 nanocomposite dispersion liquid; then adding the AuNPs solution into the nano composite material dispersion liquid, keeping the mixture uniformly stirred for 4 hours at room temperature, washing with water, centrifuging to obtain precipitate La-MOF @ ZIF-67@ AuNPs, and dissolving with ultrapure water to obtain La-MOF @ ZIF-67@ AuNPs solution; then adding Luminol, stirring for 4h, centrifuging, and dissolving the precipitate with ultrapure water to obtain a La-MOF @ ZIF-67@ AuNPs @ Luminol solution.
4. The electrochemiluminescence immunosensor of any one of claims 1-3, wherein: the substrate material MXene-PEI-NiMn LDH solution is prepared by mixing MXene dispersion and 1% PEI solution, stirring the mixture at room temperature for 2 hours, adding NiMn LDH dispersion, stirring for 8 hours, centrifuging and washing the precipitate to obtain the MXene-PEI-NiMn LDH substrate material; and then dissolving the MXene-PEI-NiMn LDH substrate material in ultrapure water to obtain the MXene-PEI-NiMn LDH substrate material solution.
5. The electrochemiluminescence immunosensor of claim 4, wherein: the preparation method of the NiMn LDH dispersion liquid is characterized in that MnSO is added4·4H2O、Ni(NO3)2·6H2O and NH4Dissolving F in ultrapure water, carrying out ultrasonic treatment to obtain a uniform solution, and dropwise adding NH3·H2O, then stirring for 4 hours at room temperature, aging for 12 hours, centrifuging the product, washing the precipitate with ultrapure water, and drying in a vacuum oven at 60 ℃ for 12 hours; and finally dispersing the dried precipitate in ultrapure water to prepare a NiMn LDH dispersion liquid with the concentration of 1 mg/ml.
6. An electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1 is characterized in that the preparation process comprises the following steps:
(1) preparation of a signal probe:
1) preparation of ZIF-67 dispersion: 1.455g of Co (NO)3)2·6H2Dissolving O and 1.65g of 2-methylimidazole in 30mL of methanol respectively, mixing, stirring at room temperature for 3 hours, centrifuging the mixture to obtain brown powder, washing with methanol and water, drying at 60 ℃, dispersing the brown powder in ultrapure water, and preparing a ZIF-67 dispersion solution with the concentration of 1 mg/mL;
2) preparation of La-MOF Dispersion 2mmol of La (NO)3)3Adding 2-amino terephthalic acid into 30mL of DMF solution, carrying out ultrasonic treatment until the solution is uniformly dispersed, stirring the solution at room temperature for 10 minutes, then transferring the solution into a stainless steel autoclave lined with polytetrafluoroethylene, reacting the solution at 150 ℃ for 6 hours, cooling the solution to the room temperature, washing the solution with methanol and water, centrifuging the solution, collecting precipitates, and carrying out vacuum drying at 60 ℃ for 12 hours; finally dispersing the precipitatePreparing a La-MOF dispersion with the concentration of 1mg/ml in ultrapure water;
3) preparation of AuNPs solution: 100mL of 0.01% HAuCl4Boiling the solution under stirring, adding 2.5mL of 1% sodium citrate solution, keeping boiling for 15min, and cooling to room temperature;
4) La-MOF @ ZIF-67@ AuNPs @ Luminol solution: firstly, adding 1mL of 1mg/mL La-MOF dispersion liquid into 1mL of 1mg/mL ZIF-67 dispersion liquid, stirring for 12 hours at room temperature, centrifuging, and dissolving precipitate with ultrapure water to obtain La-MOF @ ZIF-67 nanocomposite dispersion liquid; adding the AuNPs solution into the nano composite material dispersion liquid, stirring for 4 hours at room temperature, washing with water, centrifuging, dissolving the precipitate with ultrapure water to prepare La-MOF @ ZIF-67@ AuNPs solution, adding 1mL of 0.01M Luminol, stirring for 4 hours, centrifuging, dissolving the precipitate with ultrapure water to prepare La-MOF @ ZIF-67@ AuNPs @ Luminol solution;
5) preparation of a signal probe: taking 1mL of the La-MOF @ ZIF-67@ AuNPs @ Luminol solution prepared in the step 4) and 100 mu LAb2Stirring for 12 hours at room temperature, centrifuging, and washing a precipitate to obtain a signal probe;
(2) preparation of the base material:
1) NiMn LDH 0.3mmol of MnSO4·4H2O、0.3mmol Ni(NO3)2·6H2O and 1.8mmol NH4F is dissolved in 25mL of ultrapure water; after being treated by ultrasonic to form uniform solution, 2mLNH is added dropwise3·H2O, stirring and mixing for 4 hours at room temperature, aging for 12 hours, centrifuging the product, washing the precipitate with ultrapure water, drying in a vacuum oven at 60 ℃ for 12 hours, and finally dispersing the dried precipitate in the ultrapure water to obtain a NiMn LDH dispersion liquid with the concentration of 1 mg/ml;
2) MXene-PEI-NiMn LDH: mixing 1mL of MXene dispersion (1mg/mL) with 500 μ L of 1% PEI solution, stirring at room temperature for 2 hours, adding 1mg of NiMn LDH dissolved in 1mL of ultrapure water, stirring for 8 hours, and centrifuging to obtain a precipitated MXene-PEI-NiMn LDH composite material; dissolving the prepared MXene-PEI-NiMn LDH composite material in 1mL of ultrapure water at 4 ℃ for later use;
(3) the construction process of the electrochemical luminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 comprises the following steps:
1) ab was dissolved in 0.1M PBS (pH 7.4) buffer at room temperature1And CYFRA21-1, stored for later use;
2) piranha washing solution (98% H) for glassy carbon electrode2SO4/30％H2O2Soaking for 30min at a ratio of 3:1, v/v), and washing with ultrapure water for later use;
3) respectively using Al of 0.3 mu m and 0.05 mu m for the electrode obtained in the step 2)2O3Polishing the powder to form a mirror surface, then respectively carrying out ultrasonic treatment on the electrodes according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying for later use;
4) subjecting the electrode obtained in step 3) to a temperature of 0.5M H2SO4Performing electrochemical activation, washing with ultrapure water, and drying;
5) dripping 10 mu L of substrate material solution on the surface of the glassy carbon electrode cleaned in the step 4), and drying at room temperature;
6) dripping 10 mu of LAuNPs solution on the surface of the glassy carbon electrode in the step 5), and drying at room temperature;
7) mu.L of Ab prepared in the step 1)1Dropwise adding a buffer salt solution on the electrode prepared in the step 6), and incubating for 12h at 4 ℃;
8) dripping 6 mu L of 1% BSA solution on the electrode obtained in the step 7), and incubating for 30min at 4 ℃;
9) dripping 10 mu L of CYFRA21-1 buffer salt solution prepared in the step 1) on the electrode prepared in the step 8), and incubating for 1.5h at 4 ℃ to obtain the electrochemical luminescence immunosensor for detecting CYFRA 21-1.
7. A method for detecting non-small cell lung cancer CYFRA21-1 using the electrochemiluminescence immunosensor according to any one of claims 1-6, comprising the steps of:
(1) dripping a signal probe solution on the surface of an electrode of the electrochemical luminescence immunosensor, and incubating for 4 hours at 4 ℃;
(2) washing the electrode prepared in the step 1) with ultrapure water, airing, and placing the electrode in a container containing 5mM H2O2In 0.1M PBS, the electrochemiluminescence response of different concentrations of CYFRA21-1 was measuredA signal change value;
(3) drawing a standard curve according to the linear relation between the logarithm values of different concentrations and the change value of the electrochemical luminescence response signal;
(4) and detecting a sample to be detected by using the electrochemical luminescence immunosensor to obtain a current value, and converting through a standard curve to obtain the concentration of CYFRA21-1 in the sample to be detected.
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