CN113155930A - Electrochemical immunosensing method for detecting leukemia stem cell tumor marker CD123 by using multiple signal amplification technology - Google Patents
Electrochemical immunosensing method for detecting leukemia stem cell tumor marker CD123 by using multiple signal amplification technology Download PDFInfo
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Abstract
The invention discloses an electrochemical immunosensing method for detecting a leukemia stem cell tumor marker CD123 by a multiple signal amplification technology. The electrochemical immunosensing method for detecting the leukemia stem cell tumor marker CD123 is constructed by utilizing a nanogold material, an amino terephthalic acid conductive polymer film and an enzyme catalysis multiple signal amplification technology through CD123 detection research. The experimental result shows that under the optimal experimental condition, the current signal of the immunosensor and the CD123 concentration present a good linear relation in the CD123 concentration range of 0.02-2.5 mug/mL, and the detection limit is as low as 7 ng/mL. The established electrochemical immunosensor is stable, reliable, economical and convenient, has the advantages of accuracy, high sensitivity and high specificity, can be well applied to the detection of CD123, provides a new detection platform for the diagnosis, curative effect judgment and prognosis evaluation of leukemia patients, and has excellent potential clinical application value.
Description
Technical Field
The invention relates to an electrochemical immunosensor method for detecting a leukemia stem cell tumor marker CD123 by using a multiple signal amplification technology.
Background
The nano electrochemical immunosensor is a new biosensor, combines nanotechnology, electrochemical analysis method and immunology technology, has the advantages of simple operation, low cost, high analysis speed, high sensitivity and good selectivity, and becomes the first choice of a plurality of detection technologies. In recent years, electrochemical immunosensors have developed rapidly, for example: many studies on electrochemical immunosensors for detecting markers such as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), Prostate Specific Antigen (PSA), and CA125 have been reported. The nano electrochemical immunosensor takes tumor cells and related markers thereof as research objects, and is widely applied to the aspects of clinical medicine, basic medicine, chemistry, environment, biology, agriculture and the like.
Leukemia (leukemia) is a clonal, heterogeneous disease of malignant proliferation of cells at some stage during the development of hematopoietic stem cells. Among them, Acute Leukemia (AL) is predominant. A part of trace cell groups exist in leukemia patients, the cells have the capacity of unlimited proliferation and self-renewal, are initial cells of leukemia exacerbation, are mostly in a stationary phase, are generally insensitive to chemotherapeutic drugs, can escape from the attack of the chemotherapeutic drugs, and are the root of leukemia refractory and relapse. This population of leukemic cells is known as Leukemic Stem Cells (LSCs), and the interleukin-3 receptor alpha chain (Cluster of Differentiation 123, CD123) is a currently recognized marker for the recognition of LSCs from normal hematopoietic stem cells. Thus, CD123 is a sensitive and specific tumor marker associated with leukemia. CD123 has been reported to be highly expressed in the bone marrow blood of patients with acute myelogenous leukemia, and not expressed in the blood of normal persons. The sensitive detection of CD123 has important significance for diagnosis, treatment and prognosis judgment of leukemia.
At present, the detection methods of leukemia stem cell markers such as CD123 and the like mainly comprise flow cytometry, immunohistochemical technology, enzyme-linked immunosorbent assay, microarray technology, gene amplification technology and the like, but the technologies need large-scale precise instruments or have the defects of high cost, time and labor waste and certain limitations. Therefore, a simple, reliable and cheap detection technology is developed to replace the traditional method, and the method has wide application prospect. Electrochemical immunosensors have attracted extensive attention in immunoassays due to their simple sample pretreatment, ordinary instrument operation, fast test time, and high sensitivity. At present, the electrochemical immunosensor technology is applied to the research of detecting leukemia stem cell tumor markers, and reports are not found yet. Therefore, the research takes the CD123 as a detection target, combines an immunological technology, a nanotechnology and an electrochemical sensing technology, explores and researches from the aspects of functional preparation of a nanomaterial, construction of a novel sensing interface and the like, aims to construct a nano electrochemical immunosensor for rapid, economic, simple and convenient to operate and sensitive detection of the CD123, and establishes a novel method for sensitive, rapid and economic detection of the leukemia stem cell marker CD 123.
The experiment described below combines nanotechnology, immunological technology and electrochemical sensing analysis technology, gold chloride acid is deposited on the surface of a gold electrode by an electrodeposition method, gold nanoparticles are deposited on the surface of the gold electrode by an electrochemical method, then 2-Aminoterephthalic acid (ATA) is polymerized by cyclic voltammetry to form a layer of polymer film on the surface of the gold electrode, the surface of the electrode is activated by a mixed solution of potassium persulfate and hydrogen peroxide to enhance electron transfer, and then 1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide [ (1-Ethyl-3- (3-dimethyllapminopropyl) carbodiimide hydrochloride, EDC is processed]Coupling carboxyl and amino with N-Hydroxysuccinimide (NHS), and tightly connecting amino terephthalic acid (2-amino terephthalic acid, ATA) and CD123 monoclonal antibody, thereby uniformly and firmly connecting the monoclonal antibodyFixedly assembled on the surface of a gold electrode, blocking a non-specific adsorption site by using bovine serum albumin, combining a CD123 antigen through the specific reaction of an antigen antibody, sequentially assembling the CD123 antigen and a biotin-modified CD123 polyclonal antibody on an electrode interface, specifically combining avidin carried by HRP and biotin carried by the CD123 polyclonal antibody, assembling avidin-modified horse radish peroxidase (s-HRP) on the surface of the gold electrode, and finally forming a sandwich structure, wherein the sandwich structure can be used for catalyzing H by HRP2O2TMB substrate reaction, thereby performing rapid and flexible electrochemical detection. The experimental result shows that under the optimal experimental condition, the current signal of the immunosensor and the CD123 concentration present a good linear relation in the CD123 concentration range of 0.02-2.5 mug/mL, and the detection limit is as low as 7 ng/mL. The immunosensor assay exhibits excellent stability, reproducibility, and specificity. The detection result of the CD123 standard substance is basically consistent with the actual concentration thereof. The established electrochemical immunosensor is stable, reliable, economical and convenient, has the advantages of accuracy, high sensitivity and high specificity, can be well applied to the detection of CD123, provides a new detection platform for the diagnosis, curative effect judgment and prognosis evaluation of leukemia patients, is expected to be expanded and applied to the detection of other membrane protein tumor markers, and has excellent potential clinical application value.
Disclosure of Invention
1. The invention aims to construct a sandwich type electrochemical immunosensor based on a multiple signal amplification technology, which is sensitive, rapid and economical, and is used for detecting a leukemia stem cell tumor marker CD 123.
2. The preparation method of the nanometer electrochemical immunosensor for detecting CD123, disclosed by the invention, sequentially comprises the following steps of:
the invention constructs a sensitive, efficient and stable electrochemical immunosensing method for detecting the leukemia stem cell tumor marker CD123 by utilizing a nanogold material, an amino terephthalic acid conductive polymer film and an enzyme catalysis multiple signal amplification technology and through CD123 detection research.
The said multiple signal amplification techniqueThe therapeutic electrochemical immunosensor is used for detecting a leukemia stem cell tumor marker CD123, and is characterized by sequentially comprising the following steps: (1) depositing gold nanoparticles on the surface of the nano gold electrode by using an electrodeposition method; (2) polymerizing 2-amino terephthalic acid (2-Aminoterephthalic acid, ATA) by using a cyclic voltammetry method to form a layer of polymer film on the surface of the nano gold electrode prepared in the step (1); (3) activating the surface of the electrode by combining with a mixed solution of potassium persulfate and hydrogen peroxide to enhance electron transfer, and tightly connecting Amino Terephthalic Acid (ATA) and a CD123 monoclonal antibody by utilizing 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to couple carboxyl and amino, thereby uniformly and firmly assembling the monoclonal antibody on the surface of the electrode; (4) blocking nonspecific adsorption sites by using bovine serum albumin, combining a CD123 antigen through the specific reaction of the antigen antibody, and sequentially assembling the CD123 antigen and a biotin-modified CD123 polyclonal antibody on a nanogold electrode interface; (5) by means of specific combination of avidin carried by HRP and biotin carried by CD123 polyclonal antibody, avidin-modified horse radish peroxidase (s-HRP) is assembled on the surface of the nanogold electrode, and HRP is utilized to catalyze H2O2And (4) carrying out TMB substrate reaction to carry out rapid and flexible electrochemical detection on a leukemia stem cell tumor marker CD 123.
The sandwich type electrochemical immunosensor based on the multiple signal amplification technology is used for detecting a leukemia stem cell tumor marker CD123, and is characterized in that a nano-gold electrode structure is combined with electropolymerization ATA, so that the specific surface area and the conductivity can be effectively increased, and the sensitivity of the sensing method is enhanced.
The sandwich type electrochemical immunosensor based on the multiple signal amplification technology is used for a method for detecting a leukemia stem cell tumor marker CD123, and is characterized in that potassium persulfate containing hydrogen peroxide is used for oxidation, so that an electric signal is further enhanced, high-sensitivity detection of CD123 is realized, and the sensitivity of the sensing method is further enhanced.
Specifically, the sandwich type electrochemical immunosensor based on the multiple signal amplification technology is used for detecting a leukemia stem cell tumor marker CD123, and the method comprises the following steps: (1) ab 1/AuE. mu.L of 50 mug/mL working liquid drop of the CD123 monoclonal capture antibody is coated on the pretreated gold electrode, and the gold electrode is placed at 4 ℃ for incubation for 11h overnight. Washing off the antibody which is not stably combined with the gold electrode and the antibody which is not combined with the gold electrode in the morning next day, and naturally airing at room temperature;
(2) BSA/Ab1/AuE: soaking the gold electrode modified with Ab1 in the step (1) in an EP tube containing 1% BSA blocking solution, carrying out water bath at 37 ℃ for 1h, washing away unbound BSA, and airing at room temperature;
(3) CD123/BSA/Ab1/AuE: and (3) dripping 3 muL of CD123 protein solution with corresponding concentration on the surface of the electrode modified with the capture antibody in the step (2), and incubating at constant temperature for 30 min. Then taking out and washing, and airing at room temperature;
(4) ab2/CD123/BSA/Ab1/AuE, namely dripping 3 mu L of 2 mu g/mL of CD123 polyclonal detection antibody (bio-Ab2) with biotin modification on the surface of the electrode treated in the step (3), reacting at 37 ℃ for 60% of constant temperature and humidity for 60min, taking out, washing and airing at room temperature;
(5) HRP/Ab2/CD123/BSA/Ab 1/AuE. mu.L of s-HRP was dropped on the surface of the electrode, and the electrode was gently washed after reaction at room temperature for 30 min. The liquid drops on the surface of the electrode are thrown off and immediately contain H2O2Electrochemical detection was performed in the TMB probe solution.
Compared with the prior art, the invention has the characteristics and advantages that: the immunosensor assay exhibits excellent stability, reproducibility, and specificity. The detection result of the CD123 standard substance is basically consistent with the actual concentration thereof. The established electrochemical immunosensor is stable, reliable, economical and convenient, has the advantages of accuracy, high sensitivity and high specificity, and has excellent potential clinical application value.
Drawings
FIG. 1 is a schematic diagram of the signal amplification strategy of the sandwich-type electrochemical sensor of the present invention.
FIG. 2 shows the detection results of the electrochemical immunosensor of the present invention by the i-T method.
FIG. 3 is a cyclic voltammogram of various modified electrodes of the invention.
FIG. 4A is a scanning electron micrograph of a blank representation, i.e., a bare gold electrode without any modification.
FIG. 4B is a scanning electron microscope characterization image of a gold electrode modified by nano-gold obtained by electrodeposition using an i-t method.
FIG. 5 is an EDS map of ATA electrodes.
FIG. 6 is a linear relationship graph of the current response value (I) and different concentrations of CD123 protein (the concentrations of the CD123 standard substance are 0.02, 0.05, 0.1, 1, 2, 2.5 mug/mL respectively).
FIG. 7 is a linear relationship graph of the current response difference (difference. DELTA.I between the current signal at the corresponding concentration and the background signal) and the different concentrations of the normal human serum diluted 100-fold + CD123 protein (0.2, 0.5, 1, 2, 2.5. mu.g/mL for the CD123 standard, respectively).
FIG. 8 is a linear plot of the difference in current response (difference Δ I between current signal and background signal at corresponding concentrations) versus the different concentrations of undiluted serum + CD123 protein (0.01, 0.03, 0.04, 0.2, 0.5 μ g/mL for the CD123 standard, respectively).
FIG. 9 is a graph of the current response of the immunosensor electrical signal to different antigens.
FIG. 10 is a graph comparing the content of the CD123 standard substance detected by the electrochemical immunosensor with that of the known CD123 standard substance.
FIG. 11 is the comparison of the results of the electrochemical immunosensor and ELISA for detecting the serum CD123 of leukemia patients.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
As shown in figure 1, firstly, a nano gold electrode is prepared by an electrodeposition method, then ATA is polymerized by cyclic voltammetry, and then K is passed2S2O8And H2O2Activating the surface of the electrode to enhance electron transfer, and then coupling carboxyl with carboxyl through EDC and NHSAmino, closely connecting ATA and CD123 monoclonal antibody (primary antibody), assembling the primary monoclonal antibody to the surface of a gold electrode, using BSA to block nonspecific adsorption sites, combining CD123 antigen through specific reaction of the antigen-antibody, then sequentially assembling biotin-modified CD123 polyclonal antibody (secondary antibody) to an electrode interface, specifically combining avidin carried by HRP and biotin carried by the CD123 polyclonal antibody, assembling avidin-modified horseradish peroxidase (s-HRP) to the surface of the gold electrode, and finally forming a sandwich structure, wherein HRP is used for catalyzing H2O2TMB substrate reaction, thereby performing rapid and flexible electrochemical detection at an electrochemical workstation.
The embodiment of the invention is realized in such a way that a sandwich type electrochemical immunosensor of a multiple signal amplification technology is used for detecting a leukemia stem cell tumor marker, the marker to be detected is CD123, and the method comprises the following steps:
(1) pretreatment of the gold electrode, namely 1) pretreatment of the gold electrode: placing a gold electrode (AuE) at 0.3 μmAl2O3Particle size and 0.05 mu mAl of powder2O3Sequentially polishing chamois leather with powder particle size for 3min, and sequentially placing polished gold electrodes in deionized water and concentrated HNO3The volume ratio of the solution to the gold electrode is 1:1, absolute ethyl alcohol and deionized water are respectively subjected to ultrasonic treatment for 3min, and Al remained on the surface of the gold electrode is removed2O3And other impurities, washing the gold electrode with sufficient deionized water, then placing the gold electrode in 0.5mol/L dilute sulfuric acid solution to scan by cyclic voltammetry (CV method: scanning with potential between 0 and 1.6V), further cleaning and activating the electrode surface, washing with sufficient deionized water after the scanning signal is stable and presents the cyclic voltammetry characteristic peak of the corresponding gold, and finally washing with high-purity N2And drying the surface of the gold electrode for later use. 2) Preparing gold nano by adopting an electrodeposition method: dissolving 25 mu L of chloroauric acid with a mother solution of 2 wt% in 0.5mol/L dilute sulfuric acid to finally obtain a chloroauric acid deposition solution of about 0.1 wt%, and obtaining the electrode for depositing the nano-gold by using a time-lapse current method (IT) under the voltage of 0.2V, the scanning speed of 50mv/s and the scanning time of 100 s. Using 10mmol/L PBS solution with pH of 7.4 to lightenAnd (5) lightly washing and drying. 3) Electropolymerization of ATA: electrochemical copolymerization of Amino Terephthalic Acid (ATA) was performed using gold electrodes. 99mL of 0.1mol/L PBS (phosphate buffer solution) with pH of 7.4 is taken, 1mL of 1mol/L NaOH is added, 0.18115g of ATA is weighed and dissolved in the solution, ultrasonic cleaning is carried out for 30min until the ATA solution is uniformly dissolved, finally, the concentration of the ATA solution is 0.1mol/L, the potential of electropolymerized ATA is between 0.06V and 1.4V, the scanning speed is 50mv/s, and the scanning cycle is carried out for 4 periods. After the scanning, a gray film on the electrode surface can be observed by naked eyes. 4) And (3) oxidation: activating the surface of a gold electrode by adopting a reduction potential of a cyclic voltammetry method to further increase an electric signal on the surface of the gold electrode, firstly weighing 1.3516g of potassium persulfate to be dissolved in 50mL of 0.1mol/L PBS (phosphate buffer solution) with pH of 7.4, then carrying out ultrasonic treatment on the mixed solution for 10min until the mixed solution is completely dissolved, and then adding 300 mu L of 10mol/L H into the uniformly dissolved solution2O2And lightly shaking, and placing in a refrigerator at 4 deg.C for use. A three-electrode system is immersed into an oxidizing solution by adopting a cyclic voltammetry method under the potential of 0-1.6V, and a gold electrode is scanned for 6 periods through an electrochemical workstation at the scanning speed of 50 mv/s. The scanned electrode was then washed with 10mmol/L PBS pH7.4 and dried. Then all the electrodes are sequentially put into 0.5mol/L dilute sulphuric acid to carry out cyclic voltammetry scanning to remove impurities on the surfaces, then the electrodes are lightly washed by double distilled water, and finally high-purity N is used2And (5) drying for later use. 5) Coupling amino and carboxyl groups: weighing a certain amount of EDC and NHS, dissolving in the same sterile 0.1mol/L PBS solution with pH7.4 to ensure that the concentration of EDC and NHS is 100 mug/mL, immediately taking out 80 mug/L mixed solution, placing in a 2mL EP tube, immersing the electrode treated in the step 4) into the mixed solution, placing in a water bath kettle at 37 ℃, then taking out the electrode, and slowly washing with 10mmol/L PBS with pH7.4 for half an hour until the electrode is dried for later use.
(2) Ab1/AuE working droplets of 3 μ L of 50 μ g/mLCD123 monoclonal capture antibody were applied to the pretreated gold electrode AuE in (1), and the gold electrode was incubated at 4 ℃ for 11h overnight. Slowly washing off the antibody which is not stably bonded with the gold electrode and the antibody which is not bonded with the gold electrode by using 10mmol/L PBS (phosphate buffer solution) with the pH value of 7.4 in the morning next day, and naturally airing at room temperature to prepare an Ab1/AuE electrode;
(3) BSA/Ab1/AuE: the gold electrode Ab1/AuE modified with Ab1 was immersed in an EP tube containing 100 μ L of 1wt% BSA blocking solution, and the EP tube was placed in a 37 ℃ water bath for 1 h. After the reaction time reaches 1h, washing away the unbound BSA with 10mmol/L PBS (pH 7.4), and then placing at room temperature for air drying to obtain a BSA/Ab1/AuE electrode;
(4) CD123/BSA/Ab1/AuE: and dropping 3 mu L of CD123 protein solution with corresponding concentration (0.01-2.5 mu g/mL) on the surface of the electrode BSA/Ab1/AuE modified with the capture antibody, and incubating for 30min at 37 ℃ in a constant temperature and humidity box with humidity of 60%. Taking out, washing with 10mmol/L PBS (pH 7.4), and air drying at room temperature to obtain CD123/BSA/Ab1/AuE electrode;
(5) ab2/CD123/BSA/Ab1/AuE, namely, dripping 3 mu L of 2 mu g/mL of CD123 polyclonal detection antibody (bio-Ab2) with biotin modification on the surface of an electrode CD123/BSA/Ab1/AuE, continuing to react for 60min in a constant temperature and humidity box at 37 ℃ and 60 percent, taking out after the reaction time, washing with 10mmol/L PBS buffer solution of PH7.4, airing at room temperature, and preparing an Ab2/CD123/BSA/Ab1/AuE electrode;
(6) HRP/Ab2/CD123/BSA/Ab1/AuE mu.L of s-HRP was dropped on the surface of the above-mentioned electrode Ab2/CD123/BSA/Ab1/AuE, and the reaction was carried out at room temperature for 30min, followed by gently rinsing with 10mmol/L PBS buffer at pH 7.4. The liquid drops on the electrode surface were spun off to obtain an HRP/Ab2/CD123/BSA/Ab1/AuE electrode immediately containing H2O2Electrochemical detection was performed in the TMB probe solution.
In fig. 2, a is a blank using BSA blocking solution, B is a current signal respectively measured by selecting a CD123 standard of 2 μ g/mL, and it can be seen that there is an obvious difference between the blank value of the standard signal of 2 μ g/mL and the blank value of the background signal, which indicates that the construction of the immunosensor has higher sensitivity.
In FIG. 3, curve A is a bare electrode, curve B is deposited gold, curve C is ATA, and curve D is K2S2O8+H2O2And oxidizing, and modifying the cyclic voltammograms of the electrodes differently. From fig. 3, it can be concluded that bare AuE shows a distinct redox peak (curve-a) with a minimum redox current. In contrast to the electrode, AuNPs/AuE (Curve)The redox current of B) is increased because the electrode surface is modified with nano-gold, and the conductivity of gold is good, so that the diffusion of electrons to the electrode surface is increased, namely the nano-gold is successfully modified to the electrode surface, therefore, the curve of the modified electrode is larger and larger as ATA (curve-C) and potassium persulfate (curve-D) are sequentially oxidized due to the increase of the conductivity, which indicates that the method respectively modifies each compound to the electrode surface.
FIG. 4A is a blank representation, i.e., a scanning electron microscope image of a bare gold electrode without any modification, and FIG. 4B is a scanning electron microscope representation of a gold electrode modified with gold nanoparticles obtained by electrodeposition using the i-t method. As shown in the figure, it can be seen that the nano-gold electrode is significantly rougher than the bare electrode and appears with many flower-like structures, thus indicating that the gold nano-meter has been completely assembled on the gold electrode. Experimental results show that by electrodepositing the gold nanoparticles, a flower-like structure appears on the surface of the gold electrode, and the configuration increases the specific surface area of the surface of the gold electrode and the antibody loading capacity, thereby achieving the effect of amplifying signals.
As shown in fig. 5, it can be seen that the EDS plot shows different contents of Au, N, O elements, with no N, O elements at all in the original bare AuE electrode, and now shows N, O elements on the assembled electrode surface, indicating that ATA has been successfully assembled onto the gold electrode surface.
FIG. 6 shows that under the optimal experimental conditions, the immunosensor is used for detecting current response values of different CD123 protein concentrations by an i-t method. As shown in FIG. 6, when the concentration of CD123 is in the range of 0.02-2.5 mug/mL, a good linear relationship is shown between the response value I of the current signal and the concentration of CD 123. Linear equation is I =2.5363CCD123+0.6628, where R2Is 0.9971.
FIG. 7 shows that a simulated sample (healthy human serum plus CD123) is used as a detection target. As can be seen from FIG. 7, the measured difference Δ I between the electrochemical signal and the background signal and the CD123 (prepared by using 100-fold diluted normal serum) at different concentrations showed a good linear relationship within a certain range (the CD123 range is 0.2-2.5 μ g/mL), the linear equation is Δ I =1.2886CCD123+3.3073, and the correlation coefficient R is2Is 0.9902.
The difference Δ I between the electrochemical signal and the background signal measured in FIG. 8 and CD123 (prepared by undiluted normal human serum) with different concentrations showed a good linear relationship in a certain range (CD 123 range is 0.01-0.5 μ g/mL), the linear equation is Δ I =2.213CCD123+0.0599, and the correlation coefficient R is2Is 0.9907. The detection limit was 7 ng/mL. (SN =3, i.e., the concentration corresponding to a detection signal value greater than 3 times the blank standard deviation; n = 3).
FIG. 9 shows that the selectivity of this immunosensor was analyzed using LDL, TRF, ALB, TG and AFP as distinguishing proteins. Changes in current response were negligible when compared to 2 μ g/mL CD123 incubated immunosensors when incubated with LDL (1000 mg/mL), TRF (500 mg/mL), ALB (50 g/L), TG (10 μ g/mL) and AFP (50 ng/mL). And the current response value measured by the interference protein is very close to that of the blank, and the result shows that the immunosensor shows good specificity to the CD 123.
As shown in fig. 10, the measured value of the electrochemical immunosensor for detecting the CD123 standard is not much different from the known content of the CD123 standard, and the average deviation rate reaches 7.52%. Therefore, the electrochemical immunosensor can detect the CD123 with higher accuracy.
FIG. 11 shows that the two methods detect the CD123 content of the serum of the patient in the same order of magnitude and do not differ much, indicating that the immunosensor assay has good reliability.
Compared with the prior art: the electrochemical method established by the method can be well applied to the detection of CD123, provides a new detection platform for the diagnosis, curative effect judgment and prognosis evaluation of leukemia patients, is expected to be developed and applied to the detection of other membrane protein tumor markers, and provides possibility for clinical rapid diagnosis. The technology has the advantages of rapidness, simplicity, convenience, sensitivity, stability, reusability, high specificity and the like, and is favorable for popularization and use.
Claims (4)
1. A sandwich type electrochemical immunosensor of a multiple signal amplification technology is used for detecting a leukemia stem cell tumor marker CD123, and a sensitive, efficient and stable electrochemical immunosensor method for detecting the leukemia stem cell tumor marker CD123 is constructed by utilizing a nano-gold material, an amino terephthalic acid conductive polymer film and an enzyme catalysis multiple signal amplification technology and through CD123 detection research.
2. The sandwich-type electrochemical immunosensor according to claim 1, wherein the sandwich-type electrochemical immunosensor comprises a plurality of electrochemical amplification technologies, and the electrochemical immunosensor sequentially comprises: (1) depositing gold nanoparticles on the surface of the nano gold electrode by using an electrodeposition method; (2) polymerizing 2-amino terephthalic acid (2-Aminoterephthalic acid, ATA) by using a cyclic voltammetry method to form a layer of polymer film on the surface of the nano gold electrode prepared in the step (1); (3) activating the surface of the electrode by combining with a mixed solution of potassium persulfate and hydrogen peroxide to enhance electron transfer, and tightly connecting Amino Terephthalic Acid (ATA) and a CD123 monoclonal antibody by utilizing 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to couple carboxyl and amino, thereby uniformly and firmly assembling the monoclonal antibody on the surface of the electrode; (4) blocking nonspecific adsorption sites by using bovine serum albumin, combining a CD123 antigen through the specific reaction of the antigen antibody, and sequentially assembling the CD123 antigen and a biotin-modified CD123 polyclonal antibody on a nanogold electrode interface; (5) by means of specific combination of avidin carried by HRP and biotin carried by CD123 polyclonal antibody, avidin-modified horse radish peroxidase (s-HRP) is assembled on the surface of the nanogold electrode, and HRP is utilized to catalyze H2O2And (4) carrying out TMB substrate reaction to carry out rapid and flexible electrochemical detection on a leukemia stem cell tumor marker CD 123.
3. The method for detecting the leukemia stem cell tumor marker CD123 by the sandwich-type electrochemical immunosensor based on the multiple signal amplification technology as claimed in claim 2, wherein the nanogold electrode structure is combined with electropolymerization ATA, so that the specific surface area and the conductivity can be effectively increased, and the sensitivity of the sensing method is enhanced.
4. The method for detecting the leukemia stem cell tumor marker CD123 by using the sandwich-type electrochemical immunosensor based on the multiple signal amplification technology as claimed in claim 2, wherein potassium persulfate containing hydrogen peroxide is used for oxidation, so as to further enhance the electric signal, thereby realizing high-sensitivity detection of CD123 and further enhancing the sensitivity of the sensing method.
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