CN112521930B - Electrochemiluminescence immunosensor - Google Patents
Electrochemiluminescence immunosensor Download PDFInfo
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- CN112521930B CN112521930B CN202011299411.7A CN202011299411A CN112521930B CN 112521930 B CN112521930 B CN 112521930B CN 202011299411 A CN202011299411 A CN 202011299411A CN 112521930 B CN112521930 B CN 112521930B
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- 229960003180 glutathione Drugs 0.000 claims abstract description 63
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Abstract
The invention discloses an electrochemiluminescence immunosensor, and belongs to the field of detection. It includes from bottom to top: electrode, graphene, silver simple substance and Ru (bpy) 3 2+ AgNPs GSH complex, antibody/antigen; the adjacent components are connected with each other through physical adsorption or chemical bonds; the AgNPs-GSH compound is a compound of nano silver AgNPs and reduced glutathione GSH. Compared with the prior art, the invention improves the electrochemiluminescence signal intensity, thereby improving the sensitivity and accuracy of detection.
Description
Technical Field
The invention belongs to the field of detection.
Background
Electrochemiluminescence, ECL for short, is a chemiluminescent phenomenon that occurs near the surface of an electrode under potential excitation. Bipyridine ruthenium Ru (bpy) 3 2+ Is the most widely used and successful electrochemiluminescence material and contains Ru (bpy) 3 2+ Electrochemiluminescence sensors of (c) have been reported for the detection of a variety of substances.
Han et al by inserting Ru (bpy) 3 2+ Binding with carcinoembryonic antibody, and binding with carcinoembryonic antigen when voltage is scanned within 0 to-1.0V, ru (bpy) 3 2+ Generating a luminescent signal, i.e. allowing detection of carcinoembryonic antigen (Potential-resolved electroche)miluminescence for determination of two antigens at the cell surface.analytical chemistry,2014, 86 (14): 6896-6902). Liu Yuanyuan reports an electrochemiluminescence sensor for detecting Prostate Specific Antigen (PSA), in particular divided into 2 parts: (1) Ru (bpy) 3 2+ Fixed to the electrode, ru (bpy) 3 2+ Connecting PSA antibody, and obtaining electrode-Ru (bpy) 3 2+ -an antibody; (2) Polydopamine nanomicrospheres (PDANS) surface-labeled PSA antibodies. In use, after the antibody of part (1) captures PSA, part (2) is added to form [ electrode-Ru (bpy) 3 2+ -antibodies]-PSA- [ antibody-PDANS]While PDANS vs Ru (bpy) 3 2+ Has a quenching effect, the luminescence signal of which is inversely related to the concentration of PSA, and the difference between the luminescence signal of the PSA sample and that of a blank (no PSA added) can reflect the concentration of PSA (study of disease marker sensor constructed based on carbon-rich nanomaterial, university of Jinan, 2018). However, the initial luminescence signal is weak, so that the quenching effect caused by capturing the antigen to be detected is not obvious, and the detection sensitivity is not high enough.
Disclosure of Invention
The invention aims to solve the problems that: an electrochemiluminescence immunosensor with strong luminescence signals is provided.
The technical scheme of the invention is as follows:
enhanced Ru (bpy) 3 2+ Complex of electrochemiluminescence signal AgNPs: GSH, characterized in that:
the complex is a complex of nano silver AgNPs and reduced glutathione GSH.
Complex AgNPs as described above: GSH, characterized in that:
the preparation method of the compound comprises the following steps:
mixing AgNPs and GSH, and fully reacting at 2-8 ℃ to obtain the catalyst; preferably, the reaction temperature is 4 ℃;
and/or the ratio of AgNPs to GSH is 1.07:100 to 1.07:1800; preferably 1.07:200.
An electrochemiluminescence immunosensor comprising, from bottom to top:electrode, graphene layer, silver simple substance layer, ru (bpy) adsorption on upper surface of silver simple substance layer 3 2+ ，Ru(bpy) 3 2+ Further adsorbing the complex AgNPs of claim 1 or 2: GSH, agNPs: GSH binds to bait proteins via Ag;
the decoy protein is an antigen or antibody for immunological binding to the antibody or antigen to be detected.
As in the previous sensor, at AgNPs not bound to bait protein: ag of GSH is bound with a capping reagent; preferably, the blocking agent is BSA.
A preparation method of an electrochemiluminescence immunosensor comprises the following steps:
(1) Uniformly coating graphene on the surface of the electrode, and drying to form a graphene layer;
(2) Covering the surface of the graphene layer with a silver simple substance;
(3) In Ru (bpy) 3 2+ Adsorbing Ru (bpy) on the surface of silver simple substance in the solution 3 2+ ；
(4) In Ru (bpy) 3 2+ Surface modification of the complex AgNPs of claim 1 or 2: GSH;
(5) In complex AgNPs: antigen or antibody is modified on silver particles of GSH.
Further, the step (2) is to convert silver ions into silver simple substances through electrodeposition and cover the surface of the graphene layer;
preferably, the silver ion concentration is 10mmol/L, the electrodeposition potential is-0.2V, and the time is 100s.
Further, the adsorption time in the step (3) is 8min;
and/or Ru (bpy) 3 2+ Ru (bpy) in solution 3 2+ The concentration was 1mmol/L.
A method for detecting a protein, comprising the steps of:
1) Incubating the sample to be detected on the sensor according to claim 3 or 4, and if the sample contains the target protein, the target protein is in immune binding with the bait protein;
2) Washing the non-immune bound protein;
3) The sensor is placed in a buffer solution, and the luminescence signal is measured by an electrochemiluminescence analyzer.
Further, the pH of the buffer was 8.
The invention has the beneficial effects that: the immunosensor of the invention can enhance Ru (bpy) by means of glutathione GSH aggregation AgNPs (GSH-AgNPs) 3 2+ The sensitivity, precision and accuracy of electrochemiluminescence detection are greatly improved. In addition, the immunosensor disclosed by the invention takes less than 10 minutes for detection, is small in size and easy to carry, and is very suitable for clinical rapid detection.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
Fig. 1: the 8-OhdG immunosensor layer-by-layer self-assembly construction schematic diagram.
Fig. 2: agNPs and AgNPs: GSH morphology characterization HRTEM images and particle size distribution and potential maps (a) AgNPs electron microscopy images, (B) AgNPs: GSH electron microscopy, (C) AgNPs particle size distribution profile, (D) Zeta potential map of AgNPs, (E) AgNPs: GSH particle size distribution profile, (F) AgNPs: zeta potential map of GSH.
Fig. 3: electrode assembly signal response trend graph. (a) GCE/Nafion-GO, (b) GCE/Nafion-GO/AgNPs/Ru (bpy) 3 2+ ，(c)GCE/Nafion-GO/AgNPs/Ru(bpy) 3 2+ /GSH-AgNPs，(d)GCE/Nafion-GO/AgNPs/Ru(bpy) 3 2+ /GSH-AgNPs/anti-8-OHdG，(e)GCE/Nafion-GO/AgNPs/Ru(bpy) 3 2+ /GSH-AgNPs/anti-8-OHdG/BSA。
Fig. 4: potential-luminescence intensity (E-I) response plot (A) and standard curve (B) of gradient concentration 8-OHDG detection.
Fig. 5: four electrodes with different Ag + Ion immobilization method contrast (AgNO) 3 Concentration is 10mmol/L, ru (bpy) 3 2+ The soaking time of (2) was 10 minutes, the initial concentration was 5mmol/L, the initial pH of the phosphate buffer was 7.5, and the deposition potential was-0.2V).
Fig. 6: agNPs electrodeposited turns optimization (AgNO) 3 Concentration is 10mmol/L, ru (bpy) 3 2+ The soaking time of (2) was 10 minutes, ru (bpy) 3 2+ The initial concentration was 5mmol/L, the initial pH of the phosphate buffer was 7.5, and the deposition potential was-0.2V).
Fig. 7: bipyridine ruthenium modification time optimization (c) Ru ＝5mmol/L)。
Fig. 8: the ruthenium bipyridine modification concentration is optimized.
Fig. 9: agNPs: GSH complex ratio optimization.
Fig. 10: agNPs, GSH, agNPs: GSH complex signal comparison.
Fig. 11: and (5) screening the optimal pH value.
Detailed Description
Example 1 an electrochemiluminescence immunosensor and method of using the same
The sensor of this embodiment uses octahydroxydeoxyguanosine (8-OHdG) as a detection target. 8-OhdG is an oxidative adduct produced by the attack of an active oxygen radical such as a hydroxyl radical, singlet oxygen, etc. on the 8 th carbon atom of a guanine base in a DNA molecule, and is a product of oxidative damage to DNA, and has been used as an index for various inflammatory diseases.
1. Preparation of reagents
Preparation of Phosphate Buffer (PBS): preparing PBS buffer solution with the concentration of 0.1mol/L, weighing 7.098g of disodium hydrogen phosphate, 6.805g of monopotassium hydrogen phosphate and 3.728g of potassium chloride, placing into a beaker, adding a certain amount of deionized water, placing into an ultrasonic vibration instrument for ultrasonic treatment, stirring until the solid is fully dissolved, then completely transferring into a 500mL measuring flask, and fixing the volume to 500mL.
Preparation of graphene substrate: 200. Mu.L of absolute ethanol and 300. Mu.L of 5% Nation (perfluorosulfonic acid) were mixed in a 1.5mL centrifuge tube, 0.1g of graphene solid was added, and then the system was sonicated until it was fully diffused.
10mmol/L AgNO 3 Preparation of the solution: accurately weigh 0.425g AgNO 3 Particles, 250mL deionized water is added to prepare 10mmol/L AgNO 3 The solution is preserved in shade and kept in the shade for 15 days, and is reconstituted after expiration.
Bipyridine ruthenium (Ru (bpy) 3 2+ ): accurately weighing bipyridine ruthenium solid according to the optimized concentration of 1.0mmol/L, dissolving with deionized water, placing into a 10mL centrifuge tube, preserving at-20 ℃, and after expiration, re-preparing after the service life is 15 days.
Preparation of reduced Glutathione (GSH): the preparation concentration is 0.2mol/L, 6.146g of GSH solid is accurately weighed in a 50mL beaker, 10mL of ionized water is added for dissolution, the mixture is preserved at 4 ℃, the service life is 15 days, and the mixture is reconstituted after expiration.
Preparation of nano silver (AgNPs): before the synthesis preparation, all glassware was cleaned and soaked with chromic acid to ensure that the vessels used were free of impurities mixed into the synthesis process. Preparing 10mmol/L sodium borohydride 75mL, placing the sodium borohydride in a beaker, placing the beaker in a magnetic stirrer, setting 1200r/min, and simultaneously dropwise adding 10mmol/L AgNO prepared in advance 3 The solution was 9mL in total. And finally, placing the liquid in a refrigerator for preservation at 4 ℃ after the liquid turns from green to yellow-green. Used within 30 days, and synthesized again after expiration. The AgNPs concentration was about 1.07mmol/L in terms of Ag atoms.
Standard 8-OHdG antibody: the standard 8-OHdG antibody used in this example was an enzyme-labeled antibody in an 8-OHdG ELISA kit (human 8 hydroxydeoxyguanosine ELISA kit, shanghai Ji Biotechnology Co., ltd.) and was diluted at a dilution ratio of 1:300 to 1:500 according to the experimental requirement concentration of 1.5 mg/mL. The kit is placed in a refrigerator for preservation at the temperature of minus 20 ℃.
Preparation of Bovine Serum Albumin (BSA): the solid medicine is put in a refrigerator to be preserved at the temperature of minus 20 ℃, when in use, 0.1g is weighed, 100mL of deionized water is added, after the solid medicine is fully dissolved, 10mL of the solution is added with 90mL of deionized water to prepare a 0.01% BSA solution system for sealing 8-OhdG antibody protein. The service life is 7 days, and the medicine is reconstituted after expiration.
Preparation of hepatoma cell (HepG 2) extract: the example used was HepG2 passaged cells cultured in a laboratory. Placing the HepG2 cell culture flask in an ultrasonic instrument with the ultrasonic frequency of 40+/-5 kHz, and setting the ultrasonic time to be 10 minutes to ultrasonically break walls, so that adherent cells in the culture flask are fallen off and broken. Repeatedly flushing PBS buffer solution for 5 times along the bottom surface of the culture bottle by using a pipetting gun, 1mL each time, transferring all the liquid to a centrifuge tube, and preparing the DNA suspension of HepG2 by using a high-speed centrifuge with the rotating speed of 6000r/min and the time of 6 min. To the suspension, 2mL of protein lysate was added and incubated overnight at 55℃and then 10mL of phenol-chloroform (25:24:1) was added, followed by centrifugation for 10min, and then 1mL of sodium acetate and 10mL of isopropanol were added to precipitate DNA, followed by washing with 20mL of 70% ethanol, centrifugation and discarding the supernatant. HepG2 passage cells used in this example had a total cell count of 1X 10 7 After washing with PBS buffer, the flask was filled with 9mL, and finally the DNA pellet was added to 1mL deionized water for detection, and the total dilution was 10mL, so that the cell concentration in the flask was 1X 10 6 And each mL.
8-OHdG gradient concentration standard preparation using liquid: the standard 8-OHdG antigen used in this example was the standard antigen in an ELISA kit for 8-OHdG (8 hydroxydeoxyguanosine ELISA kit for human, shanghai Ji Biotechnology Co., ltd.). Taking 10 μl of 8-OHDG standard solution from ELISA kit, and making into 1000 μg/mL (1×10) with constant volume of 10mL 6 ng/mL 8-OHdG standard formulation. Then stepwise dilution is carried out to prepare the mixture with the concentration of 1 multiplied by 10 -6 ng/mL、1×10 -5 ng/mL、1×10 -4 ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL of 8-OHdG gradient concentration standard use solution.
2. Sensor construction method
Before each experiment, the working electrode was treated as follows: with 0.5 μm and 50nm Al 2 O 3 Polishing powder and deionized water are mixed to polish the glassy carbon electrode (GCE, phi=4mm) for 300 circles each; then alternately ultrasonically cleaning for 3 minutes according to the sequence of deionized water-absolute alcohol-deionized water; finally, the electrode is left at room temperature for airing for use.
The principle of the sensor construction method is shown in fig. 1. In the experiment, 6. Mu.L of the graphene substrate was used to coat the electrode surface until the graphene substrate was uniformly dispersed and completely covered (film forming property by Nation), and the graphene was dried and fixed at room temperature for 5 minutes. Then submerging the modified electrode into AgNO 3 In the method, nano Ag particles are deposited on the graphene layer by adopting an electrodeposition method under the potential of-0.2V, and the deposition time is 100s. Then inserting the electrode into the electrode holder with Ru (bpy) 3 2+ In the centrifuge tube of the solution, the modified electrode head is immersed under the liquid surface, so that the surface of the modified electrode head is fully adsorbed (more than 8 min) and has positive charges. Finally, agNPs (1.07 mmol/L) and GSH (0.2 mol/L) are mixed according to the volume ratio of 1:1, and the-NH of the metal Ag and GSH is utilized 2 Covalent bonding of-SH (reaction conditions: mixing and stirring at 4 ℃ C. For 30 min), agNPs were prepared: GSH complex (negatively charged, zeta potential-33.89 mV). And (3) covering 100 mu L of the compound on the surface of the modified electrode (3-4 hours of low-temperature bubble absorption at 4 ℃), and modifying the AgNPs-GSH nano compound on the surface of the electrode by means of electrostatic combination of positive and negative charges. AgNPs described above: GSH aggregates the nano material surface, utilizes Ag to covalently combine with antibody amino (mixed incubation for 10h at 4 ℃) to modify 8-OHdG antibody. And then taking out the modified electrode, washing to remove non-specific adsorption, and soaking the modified electrode in 0.1% BSA blocking solution at 4 ℃ for 30 minutes.
3. Sensor using method
(1) Standard curve
Adding 8-OHdG with different concentrations onto sensor, incubating at 37deg.C for 60min, washing with PBS, placing the sensor in 0.1mol/L PBS (pH 8.0) solution, and respectively measuring ECL signal intensity I with electrochemiluminescence analyzer i The method comprises the steps of carrying out a first treatment on the surface of the At the same time, incubating a sensor without 8-OhdG antigen as a blank, and determining ECL signal intensity I 0 The method comprises the steps of carrying out a first treatment on the surface of the With I 0 Subtracting I i A standard curve is made of the value (Δi) of (a) versus concentration.
(2) Detection of
Adding the sample onto a sensor, incubating at 37deg.C for 60min, washing with PBS, placing the sensor in 0.1mol/L PBS (pH 8.0) solution, and respectively measuring ECL signal intensity I with electrochemiluminescence analyzer j The concentration of 8-OHdG in the sample can be obtained by taking the standard curve.
In the above steps (1) and (2), the electrochemiluminescence analyzer parameters are set as follows:
the potential setting parameters are: forward scanning, initial potential 0.2V, low potential 0.2V and high potential 1.25V.
The luminescence setting parameters are: amplification stage number 3, photomultiplier high voltage 800V.
The beneficial effects of the sensor of example 1 are further described below in the form of experimental examples.
Experimental example 1 characterization of synthetic nanomaterial
The appearance of AgNPs (FIG. 2-A) and the composite after GSH aggregation of AgNPs (FIG. 2-B) were characterized using High Resolution Transmission Electron Microscopy (HRTEM).
As can be seen from FIG. 2-A, agNPs are distributed in a spot-like manner under a transmission electron microscope, and as can be seen from FIG. 2-C, FIG. 2-D shows that the dominant distribution particle size is 10.10nm (23.41%), and the dominant distribution potential is-5.06 mV. FIG. 2-B is AgNPs: GSH complex, in the form of a bead-like agglomerate distribution, has a dominant particle size of 68.06nm (31.57%) and a dominant potential distribution of-33.89 mV, as can be seen from FIGS. 2-E and 2-F.
Demonstrating that GSH successfully aggregates AgNPs by covalent binding, and that the negative charge increases, enhancing the complex with Ru (bpy) 3 2+ Is a static electricity adsorption of (a).
Experimental example 2 ECL Signal Strength comparison after superposition of materials of each layer of sensor
On the basis of the embodiment 1, after graphene modification in the sensor construction process, electrodepositing nano silver and soaking and absorbing luminophor Ru (bpy) 3 2+ Post-incubation on AgNPs: electrodes after GSH complex, anti-8-OHdG incubation, BSA blocking were used to detect 8-OHdG, respectively.
The results are shown in FIG. 3. Curve a in the figure is ECL signal after bare electrode modification of Nation dispersed graphene. Curve b is an electrode surface electrodeposited with a layer of nano silver particles and soaking and absorbing luminophor Ru (bpy) 3 2+ Post ECL signal. Continued incubation with AgNPs: GSH complex (curve c) significantly improved ECL intensity. When the incubated anti-8-OhdG blocks electrode surface electron transfer (Curve d), and BSA was blockedAfter the excess nanosilver sites (curve e), ECL signal decreases successively.
The results of this experimental example demonstrate that AgNPs: the GSH compound can improve ECL signals, can ensure that the electrodes can still generate strong ECL signals after the antibody and BSA are combined, and improves detection sensitivity.
Experimental example 3 Standard Curve
The ECL signal value obtained by taking the sensor of example 1 and detecting the antigen-free buffer was 2421 (as a blank). Diluting 8-OHdG standard formulation to 1×10 -6 ng/mL、1×10 -5 ng/mL、1×10 -4 After ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL and 1000ng/mL, signal values of all concentrations are detected by the same electrode from low concentration to high concentration, and a standard curve is drawn by taking the concentration of the 8-OHdG standard solution as an abscissa and the ECL signal difference delta I before and after incubation of the 8-OHdG as an ordinate. FIG. 4-A is a graph of potential versus luminous intensity (E-I) for various concentrations of the standard curve (where the black line is the electrode ECL response signal prior to incubation with 8-OhdG antigen), and it can be seen that as the standard sample concentration increases, its corresponding ECL signal value gradually decreases; this is because after the binding of the 8-OHdG antigen, electron transfer to the electrode surface is hindered, resulting in a decrease in luminescence. FIG. 4-B is a standard curve, the concentration of 8-OhdG is positively correlated with the difference in electrochemiluminescence signal ΔI, and the linear range is 1×10 -5 ng/mL-10ng/mL, the linear equation is ΔI=2149+342.75 lgc, and the correlation coefficient can reach 0.9836.
Experimental example 4 detection limit
The ECL signal before incubation of the antigen was measured using the sensor of example 1 and is designated I B The method comprises the steps of carrying out a first treatment on the surface of the Then, 0ng/mL of 8-OhdG equivalent antigen (100. Mu.L) buffer was incubated as a blank, and ECL signals were recorded as I 0 The method comprises the steps of carrying out a first treatment on the surface of the The lowest detection concentration of the antigen to be detected is marked as c min The ECL signal was recorded as I 1 The method comprises the steps of carrying out a first treatment on the surface of the By the formula [12，13，15] ：
And judging and calculating the detection limit.
Experimental results: ECL Signal I prior to incubation of 8-OhdG antigen B Buffer blank signal I without antigen =2465 0 =2343, when the incubated 8-OHdG concentration was 1×10 -5 At the time of ng/mL, the concentration of the catalyst in the sample is, therefore-> (in terms of 8-OHdG molecular weight 299.24 g/mol).
Table 2 shows comparison of detection limits of the method with other methods (An Lingling, lu Maofeng, deng Lingling, etc. electrochemical and DNA oxidative damage detection of 8-hydroxydeoxyguanosine on chitosan/graphene modified electrode [ J ]. Applied chemistry,2014, 31 (2): 200-205; liu Hui. 8-hydroxydeoxyguanosine optical sensing methods based on aptamer research [ D ]. University of south China, 2014 ]) for 8-OhdG detection.
TABLE 2 comparison of detection limits of 8-OhdG with other methods by ECL immunosensor of target
The result of the experimental example shows that the sensor has extremely high detection sensitivity.
Experimental example 5 precision
Using the sensor and method of example 1, the concentration of 8-OhdG in hepatoma cell extracts (preparation method see example 1) was measured. The results are shown in Table 3, and the relative standard deviation between the measured means of the three tests is less than 5%, which indicates that the sensor and the method of the invention have good precision.
TABLE 3 precision of target immunosensor determination of mean 8-OHDG signal
Experimental example 6 accuracy
8-OHdG standard solution was added to the detection base solution (PBS, pH 8.0) and diluted to 0.01ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL and 3mL each, and labeled recovery detection was performed using the sensor and method of example 1. The results of the three assays were shown in Table 4, after subtraction of the base signal of the incubation with 0ng/mL 8-OHDG buffer, with ECL signals 1446, 1845 and 2176, respectively. It can be seen that the recovery rate is between 85% and 130% when the scalar is added to 0.01ng/mL and 0.1ng/mL, and between 90% and 120% when the scalar is added to 0.2ng/mL to 10 ng/mL.
TABLE 4 labeled recovery detection of target ECL immunosensors
The results of this experimental example illustrate:
the sensor has better accuracy in the detection range. Compared with other methods (Li Dong, zhang Qin, zhang Shenghu, etc.), self-made mixed type small column purification-high performance liquid chromatography-tandem mass spectrometry method for simultaneously measuring organic phosphate metabolites and 8-hydroxy-2' -deoxyguanosine [ J ] chromatograms in urine, 2020, 38 (6): 647-654; yang Mingqi, yuan Yue, ren Jianwei, etc., isotope dilution-hydrophilic interaction chromatography tandem mass spectrometry method for rapidly measuring 8-hydroxy deoxyguanosine and cotinine in urine, university of Sichuan university report (medical edition), 2020, 51 (1): 74-80), the sensor has better accuracy (the addition standard recovery rate is closer to 100%) in a higher concentration detection range of 0.2ng/mL-10 ng/mL.
Experimental example 7 actual sample detection
Using the sensor and method of example 1, the concentration of 8-OhdG in hepatoma cell HepG2 extract (preparation method see example 1) was measured.
The ECL signal values obtained were 419, 447, 445, respectively, and the difference ΔI between the ECL signal before incubation of the antigen and after subtraction of the buffer blank was 2266, 2269, 2262, respectively. Since the dilution was 10mL during extraction, the standard curve equation ΔI=2149+342.751 was taken to give a three-pass measurement of c 1 ＝2.195×10＝21.95ng/mL，c 2 ＝2.239×10＝22.39ng/mL，c 3 = 2.136 ×10=21.36 ng/mL. The average of the three measurements was c=21.90 ng/mL.
The SPSS software is used for carrying out single-sample t test on the three detection results and the 8-OHdG content in normal cells, the obtained double-side test P value is 0.001, and then P is less than 0.05, which shows that the difference between the detection result and the normal value is statistically significant, and the 8-OHdG content in HepG2 cells detected by the immunosensor is far higher than the normal cell content of 12.03+/-4.58 ng/mL.
The DNA damage degree in liver cancer cells is higher than that in normal cells, and the content of 8-OHdG in the liver cancer cells is higher than that in normal cells, so that the detection result of the experimental example accords with the common sense in the field.
Experimental example 8 method parameter optimization
In this experimental example, unspecified parameters were the same as in example 1 except that they were already specified. In the experimental example, 4 electrodes are selected for parallel test, and the electrode numbers are respectively as follows: 4031. 4032, GCE1, GCE6.
1. Condition optimization of electrodeposited silver finish
Experiments compare the effect of the two methods of soaking adsorption and electrodeposition on the immobilized AgNPs. The soak adsorption time was set at 80s, the number of electrodeposits was 4 (8seg=80 s), and the other conditions were the same.
FIG. 5 is a comparison of the signal results of two modification methods. The response signals of the four groups of electrodes are higher than those of the electrodes by using the soaking adsorption method, so that the effect of the modified silver is better than that of the silver by using the soaking adsorption method.
Based on the experimental data, the number of turns of electrodeposited AgNPs was optimized. The optimal series of electrodeposited turns (1 turn=2 seg=20 s) was set to 3 turns (6 seg), 4 turns (8 seg), 5 turns (10 seg), 6 turns (12 seg), 7 turns (14 seg) for detection, and the results are shown in fig. 6. From the four electrode signal modification results, when the number of turns (time) of the electrodeposited AgNPs is 5 turns, namely the electrodepositing time is 100s, the electroluminescent intensity of the four electrodes reaches an inflection point.
The experimental results in this section show that: when AgNPs modification is carried out on the electrode, electrodeposition is superior to soaking adsorption, and the optimal number of turns (time) of the electrode electrodeposited AgNPs is 5.
2. Bipyridine ruthenium (Ru (bpy) 3 2+ ) Modification time and concentration optimization
Ru (bpy) 3 2+ The soaking and adsorbing time gradient is 2min, 4min, 6min, 8min, 10min, and 12min. Ru (bpy) of four groups of electrodes 3 2+ The modification time optimization results are shown in fig. 7, and the electroluminescence intensity of the modified electrode increases with the increase of the soaking time. When Ru (bpy) 3 2+ When the soaking modification time is 8min, the electroluminescence intensity of the four groups of electrodes reaches the maximum. After the soaking adsorption time is more than 8min, the electroluminescent intensity is basically unchanged.
On the basis, for Ru (bpy) 3 2+ The concentration optimization gradient is set to be 0.05mmol/L, 0.1mmol/L, 0.5mmol/L, 1.0mmol/L and 5.0mmol/L. The results are shown in FIG. 8. Ru (bpV) passing through four groups of electrodes 3 2+ As a result of optimizing the concentration of (C), ru (bpy) 3 2+ The signal value increases slowly in the process of soaking modification concentration of 0.05mmol/L to 0.5mmol/L, and the signal intensity reaches the peak value when the concentration is 1.0mmol/L, and then the signal decreases.
The results of this section demonstrate: ru (bpy) 3 2+ The optimal time for soaking and adsorbing modification is 8min, and the optimal concentration for soaking and modifying is 1.0mmol/L.
3. AgNPs: optimization of GSH Complex Synthesis ratio
0.2mol/L of reduced Glutathione (GSH) and 1.07mmol/L of AgNP were formulated for use. Mixing a certain amount of AgNPs material and a certain amount of reduced glutathione at 4 ℃ and stirring for reaction for 30min to obtain a compound, and then using the compound and an electrode to perform cold storage incubation at 4 ℃ for 3-4 hours, wherein the incubation amount of the compound is 100 mu L. This experiment was performed as AgNPs: GSH volume ratios of 1:9, 1:4, 1:2, 1:1, 2:1 (the ratio of the amounts of the substances is 1.07:1800, 1.07:800, 1.07:400, 1.07:200, 1.07:100 in order) were optimized, and four groups of electrodes were used for multiple experiments under the same conditions, and the experimental results are shown in FIG. 9.
From the results, when AgNPs: at a GSH volume ratio of 1:1, ECL signal enters plateau, so AgNPs are determined: the volume ratio of GSH complex is 1:1.
At the same time, the modified material of the layer is selected in comparison, namely Ru (bpy) 3 2+ On the basis of modification of only AgNPs, modification of only GSH and modification of AgNPs: comparison between GSH complexes (antibody and BSA continue to be added subsequently, still as in example 1). According to the AgNPs described above: as a result of optimizing the ratio of GSH complex, the effect was found to be optimal when the ratio of the two was 1:1, and the concentrations of AgNPs and GSH were half of those before mixing. Therefore, when the comparison of the modification materials is carried out, agNPs are modified according to the volume ratio of 1:1 under the same modification conditions: GSH composite material (curve a); agNPs and GSH original concentrations (1.07 mmol/L and 0.2mol/L, respectively) were maintained for the separately modified materials (curves b and d); agNPs and GSH were diluted to half the original concentration, respectively, and the modified material alone (curves c and e). The trend of the results of the experiments with multiple groups of modified electrodes is shown in figure 10. As can be seen from the graph, the overall signal of curve d (modified original concentration AgNPs) is lower than that of curve b (modified original concentration GSH), and the overall signal value of curve a (modified AgNPs: GSH complex) is higher than that of curves b and d, and in the three modification methods, agNPs are modified according to the ratio of 1:1: the method of GSH compound has the strongest ECL signal and better effect.
The results of this section demonstrate: GSH aggregation AgNPs can significantly induce anode ECL signal enhancement.
4. Detection of base fluid pH optimization
The pH value influences the solutionThe charging property of each substance further affects Ru (bpy) 3 2+ Redox reaction of the luminophore. The pH of the phosphate buffer used for ECL signal detection was 7.0, 7.5, 8.0, 8.5, and 9.0. The results of FIG. 11 were obtained by performing multiple experiments on both sets of electrodes (electrode numbers: GCE1, GCE 6). It can be seen that a signal peak is obtained when the pH of the buffer solution is 8.0 at the time of ECL signal detection.
The experiment in this section shows that: the buffer with ph=8 is the most suitable ECL signal detection buffer.
In conclusion, the immunosensor of the invention can enhance Ru (bpy) by means of glutathione GSH aggregation AgNPs (GSH-AgNPs) 3 2+ The sensitivity, precision and accuracy of electrochemiluminescence detection are greatly improved. In addition, the immunosensor disclosed by the invention takes less than 10 minutes for detection, is small in size and easy to carry, and is very suitable for clinical rapid detection.
It will be appreciated that the 8-OHdG antibody in the immunosensor of the present invention can be replaced with other antibodies, and can be used to detect other substances as well, with similar results.
Claims (9)
1. An electrochemiluminescence immunosensor, characterized in that: it includes from bottom to top: electrode, graphene layer, silver simple substance layer, ru (bpy) adsorption on upper surface of silver simple substance layer 3 2+ ，Ru(bpy) 3 2+ Then the composite AgNPs of nano silver AgNPs and reduced glutathione GSH is further adsorbed,
AgNPs, GSH is combined with bait protein through Ag;
the decoy protein is an antigen or an antibody and is used for being in immunological binding with the antibody or the antigen to be detected;
the preparation method of the nano silver AgNPs and reduced glutathione GSH compound AgNPs comprises the following steps:
mixing AgNPs and GSH, and fully reacting at 2-8 ℃ to obtain the catalyst;
the ratio of AgNPs to GSH is 1.07:100 to 1.07:1800.
2. The sensor of claim 1, wherein: blocking agents are bound to AgNPs-GSH Ag which are not bound to bait proteins.
3. The sensor of claim 2, wherein: the blocking agent is BSA.
4. A preparation method of an electrochemiluminescence immunosensor is characterized in that: the method comprises the following steps:
(1) Uniformly coating graphene on the surface of the electrode, and drying to form a graphene layer;
(2) Covering the surface of the graphene layer with a silver simple substance;
(3) In Ru (bpy) 3 2+ Adsorbing Ru (bpy) on the surface of silver simple substance in the solution 3 2+ ；
(4) In Ru (bpy) 3 2+ Surface modified nano silver AgNPs and reduced glutathione GSH compound AgNPs are GSH;
(5) Modifying antigen or antibody on silver particles of the complex AgNPs: GSH;
the preparation method of the nano silver AgNPs and reduced glutathione GSH compound AgNPs comprises the following steps:
mixing AgNPs and GSH, and fully reacting at 2-8 ℃ to obtain the catalyst;
the ratio of AgNPs to GSH is 1.07:100 to 1.07:1800.
5. The method of claim 4, wherein: and (2) converting silver ions into silver simple substances through electrodeposition, and covering the surface of the graphene layer.
6. The method of claim 5, wherein: the concentration of silver ions is 10mmol/L, the electrodeposition potential is-0.2V, and the time is 100s.
7. The method of claim 4, wherein: the adsorption time in the step (3) is 8min;
and/or Ru (bpy) 3 2+ Ru (bpy) in solution 3 2+ The concentration was 1mmol/L.
8. A method for detecting a protein, comprising the steps of:
1) Incubating a sample to be detected with the sensor according to any one of claims 1-3, wherein the target protein is immunologically bound to the bait protein if the sample contains the target protein;
2) Washing the non-immune bound protein;
3) The sensor is placed in a buffer solution, and the luminescence signal is measured by an electrochemiluminescence analyzer.
9. The method as recited in claim 8, wherein: the pH of the buffer was 8.
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