CN113155930B - Electrochemical immunosensor method for detecting leukemia stem cell tumor marker CD123 by multiple signal amplification technology - Google Patents
Electrochemical immunosensor method for detecting leukemia stem cell tumor marker CD123 by multiple signal amplification technology Download PDFInfo
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Abstract
The invention discloses an electrochemical immunosensor method for detecting leukemia stem cell tumor marker CD123 by a multiple signal amplification technology. The 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 researching CD123 detection. Experimental results show that under the optimal experimental conditions, the current signal of the immunosensor and the concentration of CD123 show good linear relation within the range of the concentration of CD123 of 0.02-2.5 mug/mL, and the detection limit is as low as 7ng/mL. The built electrochemical immunosensor is stable and reliable, is economical and convenient, has the advantages of accuracy, high sensitivity and high specificity, can be well applied to detection of CD123, provides a new detection platform for 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 leukemia stem cell tumor marker CD123 by a multiple signal amplification technology.
Background
The nano electrochemical immunosensor is an emerging biosensor, combines the nano technology, the electrochemical analysis method and the immunological technology, has simple operation, low cost, high analysis speed, high sensitivity and good selectivity, and is the first choice of a plurality of detection technologies. In recent years, electrochemical immunosensors have evolved rapidly, such as: electrochemical immunosensor studies for detection of markers such as carcinoembryonic antigen (CEA), alpha Fetoprotein (AFP), prostate Specific Antigen (PSA), CA125 have been reported in many documents. The nano electrochemical immunosensor takes tumor cells and related markers as research objects, and has been widely applied in clinical medicine, basic medicine, chemistry, environment, biology, agriculture and other aspects.
Leukemia (leukemia) is a clonal heterogeneous disease of malignant proliferation of cells at some stage during hematopoietic stem cell development. Among them, acute leukemia (Acute leukemia, AL) is the most common. The leukemia patient has partial micro cell population which has unlimited proliferation and self-renewal capacity, is a starting cell for worsening leukemia and is in a stationary phase, and the cell population is generally insensitive to chemotherapeutics and can escape from the attack of the chemotherapeutics, thus being a root cause of difficult treatment and relapse of leukemia. This group of leukemia cells is called leukemia stem cells (leukemia stem cells, LSCs), and the interleukin-3 receptor alpha chain (Cluster of Differentiation, CD 123) is a currently accepted marker that can recognize LSCs from normal hematopoietic stem cells. CD123 is therefore a sensitive and specific tumor marker associated with leukemia. CD123 is reported to be highly expressed in the bone marrow blood of patients with acute myelogenous leukemia and not expressed in the blood of normal people. The sensitive detection of CD123 has important significance for diagnosis, treatment and prognosis judgment of leukemia.
The existing detection methods of leukemia stem cell markers such as CD123 mainly comprise flow cytometry, immunohistochemical technology, enzyme-linked immunosorbent assay, microarray technology, gene amplification technology and the like, however, the technologies need large-scale precise instruments or have the defects of high cost, time and labor waste and certain limitation. Therefore, a simple, reliable and low-cost detection technology is developed to be hopeful to replace the traditional method, and the method has wide application prospect. Electrochemical immunosensors have attracted considerable attention in immunoassays due to their simple sample pretreatment, common instrumentation, rapid test times, and high sensitivity. At present, the electrochemical immunosensor technology is applied to research on detection of leukemia stem cell tumor markers, and has not been reported yet. Therefore, the research uses CD123 as a detection target, combines an immunological technique, a nano technique and an electrochemical sensing technique, explores and researches from the aspects of functional preparation of nano materials, construction of a novel sensing interface and the like, is intended to construct a nano electrochemical immunosensor for rapid, economical, simple and convenient to operate and sensitive detection of CD123, and establishes a novel method for detecting the leukemia stem cell marker CD123 sensitively, rapidly and economically.
The experiment described below combines nanotechnology, immunological technology and electrochemical sensing analysis technology, uses electrodeposition method to deposit chloroauric acid on gold electrode surface, uses electrochemical method to deposit gold nano-particle on gold electrode surface, then uses cyclic voltammetry to polymerize 2-amino terephthalic acid (2-Aminoterephthalic acid, ATA) to make gold electrode surface form a layer of polymer film, uses mixed solution of potassium persulfate and hydrogen peroxide to activate electrode surface to enhance electron transfer, then uses 1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide [ (1-Ethyl-3- (3-dimethyl amine) carbodiimide hydrochloride, EDC]Coupling carboxyl and amino with N-Hydroxysuccinimide (NHS), tightly connecting amino terephthalic acid (2-Aminoterephthalic aci, ATA) with CD123 monoclonal antibody, uniformly and firmly assembling the monoclonal antibody on the surface of gold electrode, blocking nonspecific adsorption site with bovine serum albumin, combining CD123 antigen by antigen-antibody specific reaction, sequentially assembling CD123 antigen and biotin-modified CD123 polyclonal antibody on the electrode interface, and passing avidin-modified horseradish through by means of specific binding of avidin carried by HRP and biotin carried by CD123 polyclonal antibodyThe oxidase (s-HRP) is assembled on the surface of the gold electrode to finally form a sandwich structure, namely the catalyst can be used for catalyzing H by the HRP 2 O 2 In the TMB substrate reaction, a rapid, flexible electrochemical detection is thereby performed. Experimental results show that under the optimal experimental conditions, the current signal of the immunosensor and the concentration of CD123 show good linear relation in the concentration range of CD123 of 0.02-2.5 mug/mL, and the detection limit is as low as 7ng/mL. Immunosensor detection exhibits excellent stability, reproducibility, and specificity. The detection result of the CD123 standard substance is basically consistent with the actual concentration thereof. The built electrochemical immunosensor is stable and reliable, is economical and convenient, has the advantages of accuracy, high sensitivity and high specificity, can be well applied to detection of CD123, provides a new detection platform for diagnosis, curative effect judgment and prognosis evaluation of leukemia patients, is hopefully expanded to be applied to detection of other membrane protein tumor markers, and has excellent potential clinical application value.
Disclosure of Invention
1. The invention aims to construct a sensitive, rapid and economical sandwich type electrochemical immunosensor based on a multiple signal amplification technology for detecting a leukemia stem cell tumor marker CD 123.
2. The preparation method of the nano electrochemical immunosensor for detecting CD123 provided by the invention comprises the following steps in sequence:
the invention utilizes a nano gold material, an amino terephthalic acid conductive polymer film and an enzyme catalysis multiple signal amplification technology, and constructs a sensitive, efficient and stable electrochemical immunosensor method for detecting leukemia stem cell tumor marker CD123 through detecting and researching CD 123.
The sandwich type electrochemical immunosensor of the multiple signal amplification technology is used for detecting the leukemia stem cell tumor marker CD123, and is characterized by sequentially comprising the following steps: (1) Depositing gold nanoparticles on the surface of a nano gold electrode by using an electrodeposition method; (2) Polymerizing 2-amino terephthalic acid (2-Aminoterephthalic acid, ATA) by cyclic voltammetry to form the nano gold electrode surface prepared in the step (1)A layer of polymer film; (3) Then combining the mixed solution of potassium persulfate and hydrogen peroxide to activate the electrode surface, enhancing electron transfer, coupling carboxyl and amino by using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and tightly connecting Amino Terephthalic Acid (ATA) and CD123 monoclonal antibodies, thereby uniformly and firmly assembling the monoclonal antibodies on the electrode surface; (4) Blocking a nonspecific adsorption site by using bovine serum albumin, combining a CD123 antigen through the specific reaction of an antigen antibody, and sequentially assembling the CD123 antigen and a biotin-modified CD123 polyclonal antibody on a nano-gold electrode interface; (5) Assembling avidin-modified horseradish peroxidase (s-HRP) on the surface of a nano-gold electrode by virtue of specific binding of avidin carried by HRP and biotin carried by a CD123 polyclonal antibody, and catalyzing H by using HRP 2 O 2 -TMB substrate reaction, and rapid and flexible electrochemical detection of leukemia stem cell tumor marker CD123 is carried out.
The sandwich type electrochemical immunosensor based on the multiple signal amplification technology is used for detecting the leukemia stem cell tumor marker CD123, and is characterized in that the specific surface area and the conductivity can be effectively increased by combining a nano gold electrode structure and electropolymerization ATA, and the sensitivity of the sensing method is enhanced.
The sandwich type electrochemical immunosensor based on the multiple signal amplification technology is used for detecting the 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 the leukemia stem cell tumor marker CD123, and comprises the following steps: (1) Ab1/AuE, namely, 3 [ mu ] L of 50 [ mu ] g/mL of CD123 monoclonal capture antibody is dripped on a pretreated gold electrode, and the gold electrode is placed at 4 ℃ and incubated for 11 hours overnight. Washing off the antibodies which are not bound with the gold electrode stably and the unbound antibodies in the morning, and naturally airing at room temperature;
(2) BSA/Ab1/AuE: immersing 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 mu L of CD123 protein solution with corresponding concentration on the surface of the electrode modified with the capture antibody in the step (2), and incubating for 30min at constant temperature. Taking out and washing, and airing at room temperature;
(4) Dripping 3 [ mu ] L2 [ mu ] g/mL of CD123 polyclonal detection antibody (bio-Ab 2) with biotin modification onto the surface of the electrode treated in the step (3), taking out and flushing after 60% constant temperature and humidity reaction for 60min at 37 ℃, 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 after 30min of reaction at room temperature, the electrode was gently rinsed. Throwing off the liquid drop on the electrode surface, and immediately forming the electrode on the electrode containing H 2 O 2 Electrochemical detection is performed in the TMB probe solution of (c).
Compared with the prior art, the invention has the characteristics and advantages that: immunosensor detection exhibits excellent stability, reproducibility, and specificity. The detection result of the CD123 standard substance is basically consistent with the actual concentration thereof. The built electrochemical immunosensor is stable and 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 a signal amplification strategy for a sandwich-type electrochemical sensor according to the present invention.
FIG. 2 shows the detection result of the electrochemical immunosensor i-T method of the present invention.
FIG. 3 is a cyclic voltammogram of a different modified electrode of the present invention.
FIG. 4A is a scanning electron microscope image of a blank representation, i.e., a bare gold electrode without any modification.
FIG. 4B is a diagram showing the characteristics of a gold electrode scanning electron microscope modified by nano gold obtained by electrodeposition by the i-t method.
Fig. 5 is an EDS diagram of an ATA electrode.
FIG. 6 is a graph of the current response (I) versus concentration of CD123 protein (CD 123 standard concentration 0.02, 0.05, 0.1, 1, 2, 2.5. Mu.g/mL, respectively).
FIG. 7 is a graph showing the linear relationship between the difference in current response (DeltaI, corresponding to the difference in concentration of current signal versus background signal) and the concentration of 100-fold diluted +CD123 protein in normal human serum (CD 123 standard was 0.2, 0.5, 1, 2, 2.5. Mu.g/mL, respectively).
FIG. 8 is a graph of the current response difference (ΔI corresponding to the difference in concentration current signal from background signal) versus the concentration of undiluted serum +CD123 protein (CD 123 standard 0.01, 0.03, 0.04, 0.2, 0.5 μg/mL, respectively).
FIG. 9 is a graph showing the galvanic response of immunosensor electrical signals to different antigens.
FIG. 10 is a graph comparing the results of electrochemical immunosensor detection of CD123 standard with known CD123 standard content.
FIG. 11 is a comparison of electrochemical immunosensor and ELISA for detecting CD123 serum from leukemia patients.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
As shown in FIG. 1, the gold nanoparticle electrode is prepared by electrodeposition, and then ATA is polymerized by cyclic voltammetry, and then K is used for preparing the gold nanoparticle electrode 2 S 2 O 8 And H 2 O 2 Activating electrode surface, enhancing electron transfer, coupling carboxyl and amino via EDC and NHS, tightly connecting ATA and CD123 monoclonal antibody (primary antibody), assembling monoclonal primary antibody to gold electrode surface, blocking nonspecific adsorption site with BSA, combining CD123 antigen via antigen-antibody specific reaction, assembling biotin-modified CD123 polyclonal antibody (secondary antibody) on electrode interface sequentially, and combining avidin with CD via HRPThe 123 polyclonal antibody carries biotin specific binding, and the avidin modified horseradish peroxidase (s-HRP) is assembled on the surface of a gold electrode to finally form a sandwich structure, and H is catalyzed by HRP 2 O 2 TMB substrate reaction, allowing for rapid and flexible electrochemical detection at the electrochemical workstation.
The embodiment of the invention is realized in such a way that the sandwich type electrochemical immunosensor with the multiple signal amplification technology is used for detecting leukemia stem cell tumor markers, wherein the markers to be detected are CD123, and the method comprises the following steps:
(1) Pretreatment of gold electrode 1) pretreatment of gold electrode: placing a gold electrode (AuE) at 0.3 mu mAl 2 O 3 Powder particle size and 0.05 mu mAl 2 O 3 Sequentially polishing the chamois leather with the powder particle size for 3min, and sequentially placing the polished gold electrode in deionized water and concentrated HNO 3 Respectively carrying out ultrasonic treatment on the solution, the absolute ethyl alcohol and the deionized water with the volume ratio of 1:1 for 3min to remove Al remained on the surface of the gold electrode 2 O 3 And other impurities, washing the gold electrode with enough deionized water, then placing in 0.5mol/L dilute sulfuric acid solution for cyclic voltammetry (CV method: potential is scanned between 0 and 1.6V), further cleaning and activating the electrode surface, after the scanning signal is stable and the cyclic voltammetry characteristic peak of the corresponding gold appears, washing with enough deionized water, finally using high-purity N 2 And drying the surface of the gold electrode for later use. 2) Preparing gold nanometer by adopting an electrodeposition gold method: dissolving 25 mu L of chloroauric acid with 2wt% of mother solution in 0.5mol/L of dilute sulfuric acid to finally obtain about 0.1wt% of chloroauric acid deposition solution, and obtaining the electrode for depositing nano gold by using a time-of-flight amperometry (IT) under the voltage of 0.2V, wherein the scanning speed is 50mv/s and the scanning time is 100 s. Gently rinsed with 10mmol/L PBS at pH7.4 and dried. 3) Electropolymerization ATA: the electrochemical copolymerization of Amino Terephthalic Acid (ATA) was carried out using gold electrodes. 99mL of PBS solution with the pH of 0.1mol/L and 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, the final concentration of the ATA solution is 0.1mol/L, and the electropolymerized ATA potential is 0.06-Between 1.4V, the scanning speed is 50mv/s, and the scanning turns scan 4 cycles. After the scanning is finished, a gray film can be observed on the surface of the electrode by naked eyes. 4) Oxidizing: activating the gold electrode surface by adopting cyclic voltammetry reduction potential to further increase the electric signal on the surface, firstly weighing 1.3516g of potassium persulfate dissolved in 50mL of PBS (phosphate buffer solution) with the pH of 0.1mol/L and 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 solution 2 O 2 And gently shaking, and placing in a refrigerator at 4 ℃ for standby. And immersing the three-electrode system into an oxidizing solution by adopting a cyclic voltammetry at a potential of 0-1.6V, and scanning the gold electrode for 6 cycles by an electrochemical workstation at a scanning speed of 50mv/s. The scanned electrode was then rinsed with 10mmol/L PBS pH7.4 and dried. Then all electrodes are sequentially put into 0.5mol/L dilute sulfuric acid for cyclic voltammetric scanning to remove impurities on the surface, then the electrodes are lightly washed by double distilled water, and finally the electrodes are lightly washed by high-purity N 2 Blow-drying for later use. 5) Coupling amino and carboxyl: weighing a certain amount of EDC and NHS, dissolving in the same sterile PBS solution with the concentration of 0.1mol/L and the pH of 7.4, taking out 80 mu L of mixed solution immediately after the EDC and NHS are 100 mu g/mL, placing the mixed solution in an EP tube with the concentration of 2mL, immersing the electrode treated by the step 4) in the mixed solution, placing the mixed solution in a water bath kettle with the temperature of 37 ℃ for half an hour, and then taking out the electrode, slowly flushing the electrode with 10mmol/L PBS with the pH of 7.4 until the electrode is dried for later use.
(2) Ab1/AuE working solution of 3 [ mu ] L of 50 [ mu ] g/mLCD123 monoclonal capture antibody is dripped on the pretreated gold electrode AuE in (1), and then the gold electrode is placed at 4 ℃ for incubation for 11h overnight. Slowly washing off an antibody which is not combined with a gold electrode stably and an antibody which is not combined with the gold electrode by using 10mmol/L PBS with the PH of 7.4 in the morning, and naturally airing at room temperature to prepare an Ab1/AuE electrode;
(3) BSA/Ab1/AuE: gold electrode Ab1/AuE modified with Ab1 was immersed in an EP tube containing 100. Mu.L of 1wt% BSA blocking solution, and the EP tube was placed in a water bath at 37℃for 1h. After the reaction time reaches 1h, washing the unbound BSA by using 10mmol/L PBS with the pH of 7.4, and then airing the BSA/Ab1/AuE electrode at room temperature;
(4) CD123/BSA/Ab1/AuE: and 3 mu L of CD123 protein solution with corresponding concentration (0.01-2.5 mu g/mL) is dripped on the surface of the electrode BSA/Ab1/AuE modified with the capture antibody, and the mixture is incubated for 30min at the temperature of 37 ℃ in a constant temperature and humidity box with the humidity of 60%. Taking out, washing with 10mmol/L PBS with pH of 7.4, and then airing at room temperature to obtain the CD123/BSA/Ab1/AuE electrode;
(5) Ab2/CD123/BSA/Ab1/AuE, 3 [ mu ] L2 [ mu ] g/mL of CD123 polyclonal detection antibody (bio-Ab 2) with biotin modification is immediately dripped on the surface of an electrode CD123/BSA/Ab1/AuE, the reaction is continued for 60min at 37 ℃ in a 60% constant temperature and humidity box, after the reaction time is reached, the electrode is taken out, and is washed by 10mmol/L PBS buffer solution with pH of 7.4, and the electrode is dried at room temperature to prepare the Ab2/CD123/BSA/Ab 1/AuE;
(6) HRP/Ab2/CD123/BSA/Ab 1/AuE. Mu.L of s-HRP was dropped on the surface of the above electrode Ab2/CD123/BSA/Ab1/AuE, reacted at room temperature for 30min, and then gently rinsed with 10mmol/L PBS buffer solution at pH 7.4. Throwing off the liquid drop on the electrode surface to obtain HRP/Ab2/CD123/BSA/Ab1/AuE electrode, immediately adding H 2 O 2 Electrochemical detection is performed in the TMB probe solution of (c).
In fig. 2, a is a blank of BSA blocking solution, and B is a current signal measured by selecting 2 mug/mL of CD123 standard substance, and it can be seen that a clear difference exists between the standard substance signal of 2 mug/mL and the background signal blank value, which indicates that the immunosensor is constructed with higher sensitivity.
In FIG. 3, curve A is a bare electrode, curve B is deposited gold, curve C is ATA, and curve D is K 2 S 2 O 8 +H 2 O 2 And (3) oxidizing, namely, cyclic voltammograms of different modified electrodes. From fig. 3, it can be seen that bare AuE exhibited a distinct redox peak (curve-a), with the redox current being at a minimum. Compared with the electrode, the AuNPs/AuE (curve-B) has increased redox current, because the electrode surface is modified with nano gold, and the gold has good conductivity, and the diffusion of electrons to the electrode surface is increased, namely, the nano gold is successfully modified to the electrode surface, so that the curve of the modified electrode is bigger and bigger along with ATA (curve-C) and potassium persulfate oxidation (curve-D) sequentially due to the increase of conductivity,such methods are described for modifying each of the complexes separately to the electrode surface.
FIG. 4A is a scanning electron microscope image of a blank characterization, namely a bare gold electrode without any modification, and FIG. 4B is a scanning electron microscope characterization image of gold nanoparticle modification obtained by electrodeposition using the i-t method. As shown, it can be seen that the nano-gold electrode is significantly rougher than the bare electrode and many flower-like structures appear, thus indicating that the gold nano-particles have been completely assembled on the gold electrode. Experimental results show that flower-like structures can appear on the surface of the gold electrode by electrodepositing gold nanoparticles, and the configuration increases the specific surface area of the gold electrode surface and increases the antibody load, so that the effect of amplifying signals is achieved.
As shown in fig. 5, it can be seen that EDS shows different contents of Au, N, and O elements, no N, O element is present in the original bare AuE electrode, and N, O element is present on the surface of the assembled electrode, which indicates that ATA has been successfully assembled to the surface of the gold electrode.
FIG. 6 shows the current response values of different CD123 protein concentrations using the immunosensor using the i-t method under optimal experimental conditions. As shown in fig. 6, the current signal response value I and the CD123 concentration show good linear relationship when the CD123 concentration ranges from 0.02 to 2.5 μg/mL. The linear equation is i=2.5363ccd123+0.6628, where R 2 0.9971.
FIG. 7 shows a simulated sample (serum-supplemented with CD123 from healthy persons) as the detection target. As can be seen from fig. 7, the difference Δi between the electrochemical signal and the background signal measured shows a good linear relationship with different concentrations of CD123 (formulated with normal serum diluted 100 times) in a certain range (CD 123 ranges from 0.2 to 2.5 μg/mL), the linear equation Δi=1.2886ccd123+3.3073, the correlation coefficient R 2 0.9902.
The difference DeltaI between the electrochemical signal and the background signal, which is measured in FIG. 8, shows good linear relationship with different concentrations of CD123 (formulated with undiluted normal human serum) in a certain range (CD 123 range is 0.01-0.5 mug/mL), the linear equation is DeltaI=2.213CCD123+0.0599, the correlation coefficient R 2 0.9907. The detection limit was 7ng/mL. (sn=3, i.e. detection signal valueA concentration corresponding to greater than 3 standard deviations of the blank; n=3).
FIG. 9 shows the selectivity of the immunosensor using LDL, TRF, ALB, TG and AFP as the differentiation proteins. The change in current response was negligible when the immunosensor was incubated with LDL (1000 mg/mL), TRF (500 mg/mL), ALB (50 g/L), TG (10. Mu.g/mL) and AFP (50 ng/mL) compared to the immunosensor incubated with 2. Mu.g/mL CD 123. 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 CD 123.
As shown in fig. 10, the measured value of the CD123 standard detected by the electrochemical immunosensor is not much different from the known content of the CD123 standard, and the average deviation rate reaches 7.52%. Thus, the electrochemical immunosensor has higher accuracy in detecting CD 123.
FIG. 11 shows that the two methods detect that the patient serum CD123 content values are on the same order of magnitude and are not very different, 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 hopeful to expand 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 (1)
1. The preparation method of the sandwich type electrochemical immunosensor of the multiple signal amplification technology is characterized by comprising the following steps of: (1) Depositing gold nanoparticles on the surface of a nano gold electrode by using an electrodeposition method; (2) Polymerizing 2-amino terephthalic acid ATA by cyclic voltammetry to form a layer of polymer film on the surface of the nano gold electrode prepared in the step (1); (3) Then combining the mixed solution of potassium persulfate and hydrogen peroxide to activate the electrode surface, enhancing electron transfer, and utilizing 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide EDC and N-hydroxyCoupling carboxyl and amino by using the NHS, and tightly connecting the amino terephthalic acid ATA and the CD123 monoclonal antibody, so that the monoclonal antibody is uniformly and firmly assembled on the surface of the electrode; (4) Blocking a nonspecific adsorption site by using bovine serum albumin, combining a CD123 antigen through the specific reaction of an antigen antibody, and sequentially assembling the CD123 antigen and a biotin-modified CD123 polyclonal antibody on a nano-gold electrode interface; (5) Assembling avidin-modified horse radish peroxidase s-HRP on the surface of a nano gold electrode by means of specific combination of avidin carried by HRP and biotin carried by a CD123 polyclonal antibody, and catalyzing H by using HRP 2 O 2 -TMB substrate reaction, and rapid and flexible electrochemical detection of leukemia stem cell tumor marker CD123 is carried out.
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