CN115932266A - Electrochemiluminescence immunosensor for non-small cell lung cancer CYFRA21-1 detection and detection method - Google Patents
Electrochemiluminescence immunosensor for non-small cell lung cancer CYFRA21-1 detection and detection method Download PDFInfo
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Abstract
The invention provides an electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1, which takes AuNPs as a sensing interface and CS @ La-TCPP/Au/Ab 2 For signaling probes, through Ab 1 、Ab 2 And the specific combination of the protein and CYFRA21-1 successfully constructs an ECL immunosensor. Compared with the traditional CYFRA21-1 detection method, the method has the advantages of high sensitivity, strong specificity, quick detection, convenient operation, low equipment material price and no pollution, thereby providing a new analysis method for the detection of CYFRA 21-1.
Description
Technical Field
The invention relates to the technical field of electrochemical detection, in particular to an electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1 and a detection method.
Background
Lung cancer is the most malignant tumor with the highest morbidity and mortality in the world and is one of the most serious threats to human health worldwide. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for over 80% of all lung cancer cases. Of these, approximately 75% of NSCLC patients have been diagnosed at an advanced stage with low survival rate. Therefore, there is a need to develop an economical, sensitive, rapid method for detecting non-small cell lung cancer that will deliver correct, timely treatment early in the development of cancer, thereby inhibiting further development of cancer. Among the numerous biomarkers for NSCLC, cytokeratin fragment antigen 21-1 (CYFRA 21-1) has received much attention. CYFRA21-1 is a 36kDa fragment of cytokeratin 19, located in the cytoskeleton of epithelial cells, highly expressed in the serum of NSCLC patients, and is considered to be one of the most important candidates for diagnosing NSCLC. At present, the conventional CYFRA21-1 detection method comprises histological and serological detection methods, wherein the histological detection method comprises Immunohistochemistry (IHC), fluorescence In Situ Hybridization (FISH) and the like, but the methods belong to morphological detection and are greatly influenced by subjectivity, and meanwhile, the histological detection needs to obtain a pathological sample, so that the CYFRA21-1 state of a patient who does not obtain cancer histopathology cannot be judged, and the repeated detection is difficult.
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.
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 is characterized in that the construction method of the electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 comprises the following steps:
soaking in piranha lotion, and processing with Al 2 O 3 The glassy carbon electrode with mirror surface polished by powder is 0.5M H 2 SO 4 Carrying out electrochemical activation, then washing with ultrapure water, and drying to obtain a dried glassy carbon electrode; immersing the dried glassy carbon electrode in 1% 4 Depositing the solution for 30-35S, drying at room temperature, and dripping Ab 1 Incubating the solution at 4-5 deg.C for 10-12h, adding 1% Bovine Serum Albumin (BSA) solution dropwise, incubating at 4-5 deg.C for 0.5-1h, adding target cytokeratin antigen 21-1 (CYFRA 21-1) solution dropwise, incubating at 4-5 deg.C for 1-1.5h, and adding signaling probe CS @ La-TCPP/Au/Ab dropwise 2 And incubating the solution at 4-5 ℃ for 1.5-2h to obtain the electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA 21-1.
The signal probe CS @ La-TCPP/Au/Ab 2 The preparation method of the solution comprises the following steps: ab is added to CS @ LaTCPP/Au dispersion 2 Stirring the solution in ice bath for 10-12h, centrifuging, washing the precipitate with water, and re-dispersing the precipitate in ultrapure water to obtain CS @ La-TCPP/Au/Ab 2 The signaling probe solution of (1).
The preparation method of the CS @ LaTCPP/Au dispersion comprises the following steps: adding AuNPs into CS @ La-TCPP dispersion liquid, stirring for 7-8h at normal temperature, centrifuging, washing precipitate, and dispersing the precipitate in ultrapure water to obtain CS @ La-TCPP/Au dispersion liquid.
The preparation method of the CS @ La-TCPP dispersion liquid comprises the following steps: dissolving chitosan in glacial acetic acid solution (0.1 wt%), dispersing uniformly, taking La-TCPP, dispersing in ultrapure water, adding CS solution, and magnetically stirring for 10-12h to obtain uniformly dispersed CS @ La-TCPP solution; centrifuging, washing the precipitate with ultrapure water, and dispersing the precipitate in ultrapure water to obtain CS @ La-TCPP dispersion liquid.
The preparation steps of the La-TCPP are as follows: lanthanum nitrate hexahydrate (La (NO) 3 ) 3 ·6H 2 Dissolving O) and TCPP in N, N-Dimethylformamide (DMF) solution, respectively ultrasonic treating at room temperature for 10-12min, heating TCPP solution to 100-130 deg.C in silicon oil bath, and adding La (NO) 3 ) 3 ·6H 2 And O solution, reacting the mixture for 5-6h at 100-130 ℃ under continuous magnetic stirring, cooling to room temperature, centrifuging, washing the precipitate with pure water for three times, and drying in an oven at 50-60 ℃ to obtain black La-TCPP.
The preparation steps of the Au nano-particles are as follows: 1% of HAuCl 4 Adding the solution into ultrapure water, boiling, adding 1% trisodium citrate solution, boiling for 15min, cooling, and recovering to original volume with ultrapure water to obtain transparent wine red solution, i.e. gold nanoparticles (AuNPs).
An electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1, comprising the following steps:
(1) Preparing a signal probe;
1) La-TCPP: 16.42mg of lanthanum nitrate hexahydrate (La (NO) 3 ) 3 ·6H 2 O), 30mg TCPP were dissolved in 4mL and 1ml of N, N-Dimethylformamide (DMF) solutions, respectively, sonicated at room temperature for 10min, then the TCPP solution was heated to 120 ℃ in a silicone oil bath, and La (NO) was slowly added 3 ) 3 ·6H 2 And (4) O solution. The mixture was kept at this temperature for 6h with continuous magnetic stirring. Cooling to room temperature, centrifuging, washing with pure water for three times, and drying in an oven at 60 ℃ to obtain a black La-TCPP material;
2) CS @ La-TCPP dispersion: weighing 10mg of Chitosan (CS) and dissolving the Chitosan (CS) in 10mL of glacial acetic acid solution (0.1 wt%), uniformly dispersing, weighing 1mg of La-TCPP prepared in the step 1) and dispersing in 1mL of ultrapure water, adding into 1mL of CS solution, and magnetically stirring for 12h to obtain uniformly dispersed CS @ La-TCPP solution; centrifuging, washing with ultrapure water, and dispersing the precipitate in 1mL of ultrapure water to obtain CS @ La-TCPP dispersion liquid;
3) Au nanoparticles: 1mL of HAuCl 4 Adding the solution into 100mL of ultrapure water, boiling, then quickly adding 2.5mL of 1% trisodium citrate solution, continuously boiling for 15min, cooling, recovering the volume to the original volume by using the ultrapure water to obtain a transparent wine red solution, namely the gold nanoparticles (AuNPs), and storing at 4 ℃.
4) CS @ La-TCPP/Au: adding 450 mu L of AuNPs prepared in the step 3) into the CS @ La-TCPP dispersion liquid prepared in the step 2), stirring for 7h at normal temperature, centrifuging, washing, and dispersing the precipitate in 1mL of ultrapure water to obtain CS @ La-TCPP/Au dispersion liquid;
5)CS@La-TCPP/Au/Ab 2 : adding 100 μ L Ab with a concentration of 10 μ g/mL to 1mL of CS @ LaTCPP/Au dispersion prepared in step 4) 2 Stirring the solution for 12h in an ice bath, centrifuging, washing with water, and redispersing the precipitate in 1mL of ultrapure water to obtain CS @ La-TCPP/Au/Ab 2 The signaling probe solution of (4) and stored at 4 ℃ until use.
(2) The construction of the electrochemiluminescence immunosensor for the detection of the non-small cell lung cancer CYFRA 21-1:
1) Antigen (CYFRA 21-1), antibody (Ab) were treated (diluted) with 0.1mM PBS (pH = 7.4) buffer at room temperature 1 、Ab 2 ) Storing at 4 ℃ for later use;
2) Piranha washing solution (98% H) for glassy carbon electrode 2 SO 4 /30％ H 2 O 2 Soaking for 30min under the condition of = 3;
3) Respectively using Al of 0.3 mu m and 0.05 mu m for the electrode obtained in the step 2) 2 O 3 Polishing the powder to form a mirror surface, then respectively carrying out ultrasonic treatment on the electrode according to the sequence of ultrapure water, absolute ethyl alcohol and ultrapure water, and drying for later use;
4) The electrode obtained in the step 3) is placed in a position of 0.5M H 2 SO 4 Performing electrochemical activation, washing with ultrapure water, and drying;
5) Immersing the glassy carbon electrode cleaned in step 4) in 1mL of HAuCl 4 Depositing the solution for 30S, and drying at room temperature;
6) Add 10. Mu.L of Ab 1 Dropwise adding the solution on the electrode prepared in the step 5) and incubating for 12h at 4 ℃;
7) Dripping 10 mu L of 1% Bovine Serum Albumin (BSA) solution on the electrode obtained in the step 6), and incubating for 0.5h at 4 ℃;
8) Dripping 10 μ L of target cytokeratin fragment antigen 21-1 (CYFRA 21-1) solution on the electrode obtained in step 7), and incubating at 4 deg.C for 1.5h;
9) 10 μ L of CS @ La-TCPP/Au/Ab prepared in step (1) 2 Dripping the signal probe solution on the electrode prepared in the step 8) and incubating for 2h at 4 ℃ to obtain the electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA 21-1.
The invention also provides a method for detecting the non-small cell lung cancer CYFRA21-1 by using the electrochemiluminescence immunosensor.
The method for detecting the non-small cell lung cancer CYFRA21-1 by using the electrochemiluminescence immunosensor is characterized by comprising the following steps:
1) Dripping different concentrations of target cytokeratin fragment antigen 21-1 (CYFRA 21-1) onto the electrode of the electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA 21-1;
2) The electrode was placed in a chamber containing 5mM K 2 S 2 O 8 In 0.1M PBS (pH = 7.0), and the luminescence thereof was measuredA strength value;
3) Drawing a working curve according to the linear relation between the luminous intensity obtained in the step 2) and the concentration logarithm value of CYFRA 21-1;
4) Detecting a sample to be detected by using the electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1, and calculating the obtained current value through the working curve prepared in the step 3) to obtain the concentration of the 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 CYFRA21-1 have the prominent characteristics that:
the invention prepares a lanthanide series metal organic framework (La-TCPP) as a luminophor loaded Au nano-particle (AuNPs), and then loads a large amount of secondary antibodies (Ab) through the bonding effect of the metal nano-particle and amino 2 ) Finally, CS @ La-TCPP/Au/Ab is prepared 2 The signaling probe solution of (1). The La-TCPP is used as the luminophor in the ECL system for the first time, the ECL signal response and the signal stability of the single material TCPP are obviously improved, and the signal amplification is realized, so that the sensitivity of the sensor is improved. TCPP is also a luminophor, and due to water solubility, a luminescent signal is weak and unstable, but an organic framework (La-TCPP) formed by the TCPP and lanthanide series metal can obviously improve an ECL signal of the luminophor and stability of the ECL signal, so that an optical signal generated by a sensor is amplified, and then AuNPs are assembled to serve as an intermediate connector and a secondary antibody (Ab) 2 ) Crosslinking to form tracer label, increasing Ab 2 The amount of the supported. In addition, gold nanoparticles (AuNPs) are used as a sensing interface in the invention to capture a large amount of primary antibody (Ab) specifically bound with CYFRA21-1 1 ) Further realizing signal amplification. Therefore, in the invention, auNPs is used as a sensing interface, CS @ La-TCPP/Au/Ab 2 For signaling probes, through Ab 1 、Ab 2 And the specific combination of the protein and CYFRA21-1 successfully constructs the ECL immunosensor. Compared with the traditional CYFRA21-1 detection method, the method has the advantages of high sensitivity, strong specificity, quick detection, convenient operation, low equipment material price and no pollution, thereby providing a new analysis method for the detection of CYFRA 21-1.
The beneficial effects of the invention are:
1) The characteristics of low luminous efficiency and poor luminous stability of the novel lanthanide series metal organic framework (La-TCPP) luminous body can be greatly improved, so that the performance of the sensor is improved, the signal amplification effect is realized, and the detection sensitivity is improved.
2) The AuNPs have good conductivity, can improve the conductivity of the electrode, and simultaneously load an antibody through Jin Anjian;
3) The antibody has high specificity for recognizing a target substance (antigen), and can improve the selectivity of the sensor, thereby providing a new research direction and an analysis method for detecting trace CYFRA 21-1.
4) The related materials can be synthesized under the laboratory condition, and the method has the advantages of simple operation, low price of raw materials, low toxicity, environmental protection, small using amount each time and reduced experiment cost.
5) The whole detection and analysis method has clear and simple steps, high sensitivity and rapid signal response.
6) The electrochemical immunosensor prepared by the method can provide a new method for the detection of CYFRA 21-1; the electrochemiluminescence immunosensor prepared by the method can also be applied to the aspects of measurement of other biological samples, monitoring of food, medicine and environment and the like.
Drawings
FIG. 1 shows different modified electrodes at 0.1M PBS (pH 7.0) and with 5mM K 2 S 2 O 8 In 0.1M PBS (pH 7.0), from 0 to-1.2V at a scan rate of 200 mV/s.
FIG. 2 shows different modified electrodes at 5mM K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]A cyclic voltammogram (A) and an impedance profile (B) obtained at a scan rate of 100mV/s at a voltage in the solution ranging from-0.2 to 0.6V.
FIG. 3 is the result of the detection of CYFRA21-1 at various concentrations by the sensor of the present invention, in which panel A is at 5mM K 2 S 2 O 8 0.1M PBS (pH 7.0) sensor pairs 0,0.00001,0.0001,0.001,0.01,0.1,1, 10 and 10, respectivelyTime-electrochemiluminescence intensity profile of 0ng/mL CYFRA21-1 scan; and the graph B is a calibration curve of the electrochemical luminescence intensity of the sensor and logarithmic values of different concentrations CYFRA 21-1.
FIG. 4 is a graph of sensor stability, time-electrochemiluminescence intensity after 15 consecutive scans of sensors incubated with 1ng/mL CYFRA21-1 and 100ng/mL CYFRA, respectively;
FIG. 5 shows the results of the intra-and inter-batch differences obtained after scanning the sensor with 1ng/mL CYFRA21-1 incubated with different glassy carbon electrodes simultaneously and for five consecutive days under the same conditions.
FIG. 6 is a specific assay diagram of a CYFRA21-1 immunosensor in which the interferents are ascorbic acid (AA, 10 ng/mL), glucose (Glu, 10 ng/mL), dopamine (DA, 10 ng/mL), human serum albumin (HSA, 10 ng/mL) and prostate specific antigen (PSA, 10 ng/mL).
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
The main chemical reagents used in the examples of the present invention are as follows: cytokeratin 19 fragment (CYFRA 21-1) antigen and antibody are purchased from Wuhanyun clone technology limited; lanthanum nitrate hexahydrate (La (NO) 3 ) 3 ·6H 2 O) purchased from alatin biochemical technology, ltd (shanghai, china); tetrakis (4-carboxyphenyl) porphine (TCPP) is available from Shanghai-derived PhylloBiotech, inc.; chitosan (CS) was purchased from cologne (chinese chengdu); chloroauric acid (HAuCl) 4 ) Purchased from Sigma (USA); n, N-Dimethylformamide (DMF) was purchased from Michelin Biochemical technology Ltd (Shanghai, china); bovine Serum Albumin (BSA) and potassium persulfate (K) 2 S 2 O 8 ) From J&K Scientific Ltd (Beijing, china).
The equipment and technical parameters used are as follows: the instrument comprises the following steps: time/voltage-electrochemiluminescence intensity measurements were performed using an MPI-E type electrochemiluminescence workstation (seian, china). Cyclic Voltammetry (CV) and impedance method (EIS) measurements were performed using a Metrohm Autolab b.v. electrochemical workstation (switzerland Modular instrument). The electrochemiluminescence detection adopts a three-electrode system: modified glassy carbon batteryThe electrode (diameter 4 mm) was used as the working electrode, the platinum wire as the counter electrode and silver-silver chloride (saturated KCl) as the reference electrode. The electrochemical detection adopts a three-electrode system: the modified glassy carbon electrode (diameter 4 mm) is used as a working electrode, a platinum wire is used as a counter electrode, and a Saturated Calomel Electrode (SCE) is used as a reference electrode. The pH meter monitors the pH value (S210 SevenCompact, mettler-toledo, shanghai, china). Electrochemiluminescence three-electrode system containing 5mM K 2 S 2 O 8 In 0.1M PBS (pH 7.0) at 200 mV/s. Electrochemical three-electrode system at 5mM K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]The solution was scanned at 100 mV/s.
Example 1
The electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 is prepared and operated according to the following steps:
(1) Preparing a signal probe;
1) La-TCPP: 16.42mg of lanthanum nitrate hexahydrate (La (NO) 3 ) 3 ·6H 2 O), 30mg TCPP were dissolved in 4mL and 1ml of N, N-Dimethylformamide (DMF) solutions, respectively, sonicated at room temperature for 10min, then the TCPP solution was heated to 120 ℃ in a silicone oil bath, and La (NO) was slowly added 3 ) 3 ·6H 2 And (4) O solution. The mixture was kept at this temperature for 6h with continuous magnetic stirring. Cooling to room temperature, centrifuging, washing with pure water for three times, and drying in an oven at 60 ℃ to obtain a black La-TCPP material;
2) CS @ La-TCPP dispersion: weighing 10mg of Chitosan (CS) to be dissolved in 10mL of glacial acetic acid solution (0.1 wt%), uniformly dispersing, weighing 1mg of La-TCPP prepared in the step 1) to be dispersed in 1mL of ultrapure water, adding into 1mL of CS solution, and magnetically stirring for 12h to obtain uniformly dispersed CS @ La-TCPP solution; centrifuging, washing with ultrapure water, and dispersing the precipitate in 1mL of ultrapure water to obtain CS @ La-TCPP dispersion liquid;
3) Au nanoparticles: adding 1mL of HAuCl4 solution into 100mL of ultrapure water, boiling, rapidly adding 2.5mL of 1% trisodium citrate solution, boiling for 15min, cooling, recovering to original volume with ultrapure water to obtain transparent wine red solution, i.e. nanogold (AuNPs), and storing at 4 deg.C.
4) CS @ La-TCPP @ Au: adding 450 mu L of AuNPs prepared in the step 3) into the CS @ La-TCPP dispersion liquid prepared in the step 2), stirring for 7h at normal temperature, centrifuging, washing, and dispersing the precipitate into 1mL of ultrapure water to obtain the CS @ La-TCPP/Au dispersion liquid;
5)CS@La-TCPP/Au/Ab 2 : 1mL of CS @ La-TCPP/Au dispersion prepared in step 4) was added with 100. Mu.l of Ab at a concentration of 10. Mu.g/mL 2 Stirring the solution for 12h in an ice bath, centrifuging, washing with water, and redispersing the precipitate in 1mL of ultrapure water to obtain CS @ La-TCPP/Au/Ab 2 The signal probe solution of (4) and stored at 4 ℃ until use.
(2) The construction of the electrochemiluminescence immunosensor for the detection of the non-small cell lung cancer CYFRA 21-1:
1) Antigen (CYFRA 21-1), antibody (Ab) were treated (diluted) with 0.1mM PBS (pH = 7.4) buffer at room temperature 1 、Ab 2 ) Storing at 4 deg.C;
2) Piranha washing solution (98% H) for glassy carbon electrode 2 SO 4 /30％ H 2 O 2 1, v/v) soaking for 30min, and then washing with ultrapure water for later use;
3) Respectively using Al with the thickness of 0.3 mu m and Al with the thickness of 0.05 mu m for the electrode obtained in the step 2) 2 O 3 Polishing 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) The electrode obtained in the step 3) is placed in a position of 0.5M H 2 SO 4 Performing electrochemical activation, washing with ultrapure water, and drying;
5) Immersing the glassy carbon electrode cleaned by step 4) in 1mL of HAuCl 4 Depositing the solution for 30S, and drying at room temperature;
6) Add 10. Mu.L of Ab 1 Dropwise adding the mixture on the electrode prepared in the step 5) and incubating for 12h at 4 ℃;
7) Dripping 10 mu L of 1% Bovine Serum Albumin (BSA) solution on the electrode obtained in the step 6) and incubating for 0.5h at 4 ℃;
8) Dripping 10 μ L of target cytokeratin fragment antigen 21-1 (CYFRA 21-1) on the electrode obtained in step 7), and incubating at 4 deg.C for 1.5h;
9) 10 μ L of CS @ La-TCPP/Au/Ab prepared in step (1) 2 Dripping the signal probe solution on the electrode prepared in the step 8) and incubating for 2h at 4 ℃ to obtain the electrochemiluminescence immunosensor for detecting CYFRA 21-1.
Example 2
Detection of CYFRA21-1 using electrochemiluminescence immunosensor
The electrochemiluminescence immunosensor constructed in example 1 was used to detect CYFRA21-1, and the following procedure was followed:
1. drawing a working curve
1) The electrode of step 4) of the construction procedure of the electrochemiluminescence immunosensor of example 1 was placed in a container containing 5mM K 2 S 2 O 8 In 0.1M PBS (pH = 7.0); dropwise adding 10. Mu.L of La-TCPP solution to the electrode of step 4) of example 1 in 0.1M PBS (pH = 7.0) and 5mM K, respectively 2 S 2 O 8 Was characterized in 0.1M PBS (pH = 7.0) and the results are shown in figure 1: (a) GCE (PBS + K) 2 S 2 O 8 )；(b)La-TCPP(PBS)；(c)La-TCPP(PBS+K 2 S 2 O 8 )。
2) The modified electrodes of steps 4) to 8) in the construction steps of the electrochemiluminescence immunosensor of example 1 were placed at 5mM K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ]CV and EIS characterization was performed in solution. The current response signal is measured, and the result is shown in fig. 2A: (a) GCE; (b) depositing gold; (c) Ab is added dropwise 1 A solution; (d) BSA blocking; (e) dropwise adding antigen CYFRA21-1 solution. The impedance response signal is measured, and the result is shown in fig. 2B: (a) GCE; (b) depositing gold; (c) Ab is added dropwise 1 A solution; (d) BSA blocking; (e) dropwise adding antigen CYFRA21-1 solution.
3) To the electrode of the immunosensor prepared in example 1, 10. Mu.L of the target CYFRA21-1 was dropped at different concentrations, and the emission intensity was measured. As shown in fig. 3A: 1 → 8 in the order: 0.00001,0.0001,0.001,0.01,0.1,1, 10 and 100ng/mL.
4) The working curve was plotted according to the linear relationship between the obtained luminescence intensity values and the log values of CYFRA21-1 concentration (as shown in fig. 3B). The measurement result shows that the response value of the luminous intensity and the concentration logarithm value of CYFRA21-1 are in good linear relation in the range of 10fg/mL-100ng/mL, the linear correlation coefficient is 0.9968, and the detection limit is 0.71fg/mL; the results are shown in FIG. 3B.
2. And (3) testing the stability of the sensor: after incubation of the sensor prepared in example 1 at different concentrations of CYFRA21-1 (1 ng/mL and 100 ng/mL) under optimal conditions for 15 consecutive ECL measurements, the Relative Standard Deviation (RSD) of the luminescence intensity was 2.64% and 0.98%, respectively (as shown in FIG. 4), indicating good stability of the sensor.
3. And (3) testing intra-batch difference and inter-batch difference of the sensors: after ECL measurement was performed on the sensor prepared in example 1 by incubating CYFRA21-1 (1 ng/mL) with the same concentration by using five different glassy carbon electrodes (as shown in FIG. 5), the Relative Standard Deviation (RSD) of the intra-batch difference and the inter-batch difference was 1.34% and 1.41%, respectively, indicating that the sensor has good reproducibility.
4. And (3) sensor specificity test: to study the specificity of the proposed adaptive sensors, interferents that may be present in serum were used: ascorbic acid (AA, 10 ng/mL), glucose (Glu, 10 ng/mL), dopamine (DA, 10 ng/mL), human serum albumin (HSA, 10 ng/mL) and prostate specific antigen (PSA, 10 ng/mL) at the same concentration and under the same conditions with 5mM K 2 S 2 O 8 In 0.1M PBS (pH = 7.0). The results show (as shown in FIG. 6) that the Ab-based antibody was proposed 1 -CYFRA21-1-Ab 2 The immunosensor has good specificity.
4. Application of actual sample analysis
To evaluate the practical applicability and accuracy of the proposed electrochemiluminescence immunosensor, CYFRA21-1 (shown in table 1) was added to a sample of 50-fold diluted healthy human serum at various concentrations, and then the electrochemical immunosensor prepared in example 1 was used for detection. The detection results are shown in Table 1, the relative standard deviation range is 2.64% to 5.57%, and the recovery rate is 92.43% to 106.49%. The result shows that the immunosensor prepared by the invention is feasible for detecting CYFRA21-1 and can meet the requirement of practical analysis.
Table 1 electrochemiluminescence immunosensor assay CYFRA21-1 (n = 3) of healthy human serum samples
Claims (4)
1. An electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1 is characterized in that the construction method of the electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 comprises the following steps:
soaking in piranha lotion, and processing with Al 2 O 3 The glassy carbon electrode with mirror surface polished by powder is 0.5M H 2 SO 4 Carrying out electrochemical activation, then washing with ultrapure water, and drying to obtain a dried glassy carbon electrode; immersing the dried glassy carbon electrode into 1% 4 Depositing the solution for 30-35S, drying at room temperature, and dripping Ab 1 Incubating the solution at 4-5 deg.C for 10-12h, adding 1% Bovine Serum Albumin (BSA) solution dropwise, incubating at 4-5 deg.C for 0.5-1h, adding target cytokeratin antigen 21-1 (CYFRA 21-1) solution dropwise, incubating at 4-5 deg.C for 1-1.5h, and adding signaling probe CS @ La-TCPP/Au/Ab dropwise 2 Incubating the solution at 4-5 ℃ for 1.5-2h to obtain the electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA 21-1;
the signal probe CS @ La-TCPP/Au/Ab 2 The preparation method of the solution comprises the following steps: ab is added to CS @ LaTCPP/Au dispersion 2 Stirring the solution in ice bath for 10-12h, centrifuging, washing the precipitate with water, and re-dispersing the precipitate in ultrapure water to obtain CS @ La-TCPP/Au/Ab 2 The signal probe solution of (1);
the preparation method of the CS @ LaTCPP/Au dispersion comprises the following steps: adding AuNPs into CS @ La-TCPP dispersion liquid, stirring for 7-8h at normal temperature, centrifuging, washing precipitate, and dispersing the precipitate in ultrapure water to obtain CS @ La-TCPP/Au dispersion liquid;
the preparation method of the CS @ La-TCPP dispersion liquid comprises the following steps: dissolving chitosan in glacial acetic acid solution (0.1 wt%), dispersing uniformly, taking La-TCPP, dispersing in ultrapure water, adding CS solution, and magnetically stirring for 10-12h to obtain uniformly dispersed CS @ La-TCPP solution; centrifuging, washing the precipitate with ultrapure water, and dispersing the precipitate in ultrapure water to obtain CS @ La-TCPP dispersion liquid;
the preparation steps of the La-TCPP are as follows: lanthanum nitrate hexahydrate (La (NO) 3 ) 3 ·6H 2 Dissolving O) and TCPP in N, N-Dimethylformamide (DMF) solution, respectively ultrasonic treating at room temperature for 10-12min, heating TCPP solution to 100-130 deg.C in silicon oil bath, and adding La (NO) 3 ) 3 ·6H 2 And O solution, reacting the mixture for 5-6h at 100-130 ℃ under continuous magnetic stirring, cooling to room temperature, centrifuging, washing the precipitate with pure water for three times, and drying in an oven at 50-60 ℃ to obtain black La-TCPP.
2. The method of claim 2, wherein: the preparation steps of the Au nano-particles are as follows: 1% of HAuCl 4 Adding the solution into ultrapure water, boiling, adding 1% trisodium citrate solution, boiling for 15min, cooling, and recovering to original volume with ultrapure water to obtain transparent wine red solution, i.e. gold nanoparticles (AuNPs).
3. An electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1, comprising the following steps:
(1) Preparing a signal probe;
1) La-TCPP: 16.42mg of lanthanum nitrate hexahydrate (La (NO) 3 ) 3 ·6H 2 O), 30mg TCPP were dissolved in 4mL and 1ml of N, N-Dimethylformamide (DMF) solutions, respectively, sonicated at room temperature for 10min, then the TCPP solution was heated to 120 ℃ in a silicone oil bath, and La (NO) was added 3 ) 3 ·6H 2 Keeping the mixture to react for 6 hours at the temperature under continuous magnetic stirring by using an O solution, cooling to room temperature, centrifuging, washing with pure water for three times, and drying in an oven at 60 ℃ to obtain a black La-TCPP material;
2) CS @ La-TCPP dispersion: weighing 10mg of Chitosan (CS) and dissolving the Chitosan (CS) in 10mL of glacial acetic acid solution (0.1 wt%), uniformly dispersing, weighing 1mg of La-TCPP prepared in the step 1) and dispersing in 1mL of ultrapure water, adding into 1mL of CS solution, and magnetically stirring for 12h to obtain uniformly dispersed CS @ La-TCPP solution; centrifuging, washing with ultrapure water, and dispersing the precipitate in 1mL of ultrapure water to obtain CS @ La-TCPP dispersion liquid;
3) Au nanoparticles: 1mL of HAuCl 4 Adding the solution into 100mL of ultrapure water, boiling, then quickly adding 2.5mL of 1% trisodium citrate solution, continuously boiling for 15min, cooling, recovering the volume to the original volume by using the ultrapure water to obtain a transparent wine red solution, namely gold nanoparticles (AuNPs), and storing at 4 ℃;
4) CS @ La-TCPP/Au: adding 450 mu L of AuNPs prepared in the step 3) into the CS @ La-TCPP dispersion liquid prepared in the step 2), stirring for 7h at normal temperature, centrifuging, washing, and dispersing the precipitate in 1mL of ultrapure water to obtain CS @ La-TCPP/Au dispersion liquid;
5)CS@La-TCPP/Au/Ab 2 : adding 100 μ L Ab with concentration of 10 μ g/mL into 1mL of CS @ LaTCPP/Au dispersion prepared in step 4) 2 Stirring the solution for 12h in ice bath, centrifuging, washing with water, and redispersing the precipitate in 1mL of ultrapure water to obtain CS @ La-TCPP/Au/Ab 2 Storing the signal probe solution at 4 ℃ for later use;
(2) The construction of the electrochemiluminescence immunosensor for the detection of the non-small cell lung cancer CYFRA 21-1:
1) Antigen (CYFRA 21-1), antibody (Ab) treatment with 0.1mM PBS (pH = 7.4) buffer at room temperature 1 、Ab 2 ) Storing at 4 ℃ for later use;
2) The piranha washing solution for glassy carbon electrodes (98%) 2 SO 4 /30％H 2 O 2 1, v/v) soaking for 30min, and then washing with ultrapure water for later use;
3) Respectively using Al with the thickness of 0.3 mu m and Al with the thickness of 0.05 mu m for the electrode obtained in the step 2) 2 O 3 Polishing 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) The electrode obtained in the step 3) is placed in a position of 0.5M H 2 SO 4 Performing electrochemical activation, washing with ultrapure water, and drying;
5) Immersing the glassy carbon electrode cleaned in step 4) in 1mL of HAuCl 4 Depositing the solution for 30S, and drying at room temperature;
6) Add 10. Mu.L of Ab 1 Dropwise adding the solution on the electrode prepared in the step 5), and incubating for 12h at 4 ℃;
7) Dripping 10 mu L of 1% Bovine Serum Albumin (BSA) solution on the electrode obtained in the step 6) and incubating for 0.5h at 4 ℃;
8) Dripping 10 μ L of target cytokeratin fragment antigen 21-1 (CYFRA 21-1) solution on the electrode obtained in step 7), and incubating at 4 deg.C for 1.5h;
9) 10 μ L of CS @ La-TCPP/Au/Ab prepared in step (1) 2 Dripping the signal probe solution on the electrode prepared in the step 8) and incubating for 2h at 4 ℃ to obtain the electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA 21-1.
4. A method for detecting non-small cell lung cancer CYFRA21-1 using the electrochemiluminescence immunosensor according to any one of claims 1 to 3, comprising the steps of:
1) Dripping different concentrations of the cytokeratin fragment antigen 21-1 (CYFRA 21-1) of the target substance onto the electrode of the electrochemiluminescence immunosensor for detecting non-small cell lung cancer CYFRA21-1 according to any one of claims 1 to 3;
2) The electrode was placed in a chamber containing 5mM K 2 S 2 O 8 In 0.1M PBS (pH = 7.0), and measuring the luminescence intensity value thereof;
3) Drawing a working curve according to the linear relation between the luminous intensity obtained in the step 2) and the concentration logarithm value of CYFRA 21-1;
4) Detecting a sample to be detected by using the electrochemiluminescence immunosensor for detecting the non-small cell lung cancer CYFRA21-1 according to any one of claims 1 to 3, and calculating the obtained current value through the working curve prepared in the step 3) to obtain the concentration of the CYFRA21-1 in the sample to be detected.
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