CN112986359B - Based on CuBTC @ MoS 2 -AuNPs modified electrode and CA125 detection method - Google Patents
Based on CuBTC @ MoS 2 -AuNPs modified electrode and CA125 detection method Download PDFInfo
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Abstract
The invention relates to a MoS based on CuBTC @ MoS 2 -AuNPs modified electrodes and CA125 detection method; the CuBTC is composed of metal Cu as a node and an organic ligand as a connector and is a metal organic framework porous material; cuBTC @ MoS 2 Mixing CuBTC, mo source and S source by hydrothermal method to prepare electrode modification material CuBTC @ MoS 2 (ii) a Fixing it on working electrode by physical adsorption to form CuBTC @ MoS 2 An electrode; using NaAuCl 4 ·2H 2 And reducing the O solution on the electrode, and then depositing the gold nanoparticles on the working electrode to fix the gold nanoparticles on the working electrode through Au-S bonds. The electrode is placed at room temperature to physically adsorb the CA125 antibody, and the antibody is continuously modified on the electrode; preparation of CuBTC @ MoS 2 AuNPs/CA125Ab electrode. The requirement of accurate detection of CA125 is met.
Description
Technical Field
The invention belongs to the field of sensors, and particularly relates to a novel material CuBTC @ MoS 2 Preparation of (copper metal organic framework @ molybdenum disulfide) and application of CuBTC @ MoS 2 -AuNPs (copper metal organic framework @ molybdenum disulfide-gold nanoparticles) modified working electrode and a method of using the modified electrode to detect the cancer marker CA 125.
Background
CA125 is an important serum tumor marker for detecting ovarian cancer [1] . Common detection methods include fluorescence spectroscopy, electrochemiluminescence, surface plasmon resonance, fluorescence resonance energy transfer, and the like [2-5] . These methods tend to be expensive, require professional operation, and are time-consuming and labor-intensive, and electrochemical immunosensors with high affinity antibody-antigen binding are considered to be detection methods with fast response, low cost, high sensitivity, and good selectivity. In order to reduce the detection down-line and improve the signal-to-noise ratio of electrochemical biosensors, various advanced nanomaterials are used for the functionalization of biosensors [6] . In particular, metal Organic Frameworks (MOFs), as novel crystalline materials with periodic network structures, are considered as promising candidates due to their large pore volume, high specific surface area and tailorable chemistry [7] . However, MOFs typically have disordered orientation and low conductivity, which is detrimental to electron transfer [8] . And MoS 2 Coated MoS with embedded morphology, high electrochemical activity and excellent chemical stability 2 The catalyst is a polar substance and a catalyst, and provides more active sites for the nano composite material. In addition, some electroactive MOFs, such as CuBTC, have a three-dimensional porous cellular network, consisting of empty metal sites to receive adsorbed molecules, exhibit excellent catalytic properties and excellent electrical conductivity [9] . Compared to most MOFs, the heat resistance and relatively convenient precursor synthesis of CuBTC can meet the demand for rapid detection.
Disclosure of Invention
The invention designs CuBTC @ MoS 2 AuNPs modifies the working electrode in a three-electrode system consisting of the working electrode, the counter electrode and the reference electrode to be used for rapid, reliable and accurate trace analysis of CA 125.
The CuBTC is a metal organic framework porous material, is composed of metal Cu as a node and an organic ligand as a connector, is a good dielectric material, and can increase reaction sites of a working electrode due to the large specific surface area of the material. Compared with CuBTC powder prepared by an acid-base neutralization method, the anaerobic activation method provided by the invention has better load capacity.
The invention provides a water heaterThe method mixes CuBTC, mo source and S source to prepare a new electrode modification material CuBTC @ MoS 2 。
CuBTC @ MoS synthesized by hydrothermal method 2 The structure has an amplification effect on electrode detection current, can well increase the reaction surface area, and has certain guiding significance on modification of a working electrode. The gold nanoparticles can be connected on the working electrode through Au-S bonds, the current response signals of the electrode can be further amplified due to the good conductive characteristic of the gold nanoparticles, and the modification method of the gold nanoparticles mainly utilizes NaAuCl 4 ·2H 2 The O solution is reduced on the electrode and then deposited on the working electrode, and the electrochemical scanning method enables the modification to be more uniform and the working electrode to be more flat.
And the CA125Ab is used as a working electrode recognition sensitive layer, and is used for detecting CA125 by using an antigen-antibody specific binding method, so that the electrode is placed at room temperature to physically adsorb the CA125 antibody, and the antibody is continuously fixed on the working electrode. Therefore, the invention prepares CuBTC @ MoS 2 AuNPs/CA125Ab electrode. Prepared CuBTC @ MoS 2 The AuNPs/CA125 antibody electrode can realize the detection of the wide linear range of 0.0005U/mL-500U/mL of CA125, because of CuBTC @ MoS 2 The AuNPs substance is added, the electrode has extremely low detection lower limit, and contains 35U/mL of clinical detection index, so that the electrode has great research and application value for clinic, and rapid detection of CA125 can be realized by adopting an electrochemical method.
The purpose of the invention is realized by the following technical scheme:
CuBTC @ MoS 2 -AuNPs modified electrodes; the CuBTC is characterized in that CuBTC is composed of metal Cu as a node and an organic ligand as a connector and is a metal organic framework porous material; cuBTC @ MoS 2 CuBTC, mo source and S source are mixed by hydrothermal method to prepare electrode modification material CuBTC @ MoS 2 (ii) a Fixing it on working electrode by physical adsorption to form CuBTC @ MoS 2 An electrode; next, naAuCl was used 4 ·2H 2 And reducing the O solution on the electrode, and then depositing the gold nanoparticles on the working electrode to fix the gold nanoparticles on the working electrode through Au-S bonds.
CuBTC @ MoS based on the invention 2 The method for detecting the CA125 by the AuNPs modified electrode is characterized in that the electrode is placed at room temperature to physically adsorb a CA125 antibody, and the antibody is continuously fixed on the electrode; preparing CuBTC @ MoS 2 AuNPs/CA125Ab electrode.
CuBTC @ MoS of the present invention 2 -a method for preparing an AuNPs modified electrode, comprising the steps of:
1) Using the volume ratio of DMF: etOH: ultrapure water =1:1.33:1 to prepare a solvent to prepare trimesic acid (C) in a concentration ratio 9 H 6 O 6 )：Cu(NO 3 ) 2 ·3H 2 Placing the solution of O =1:2 in a clean glass bottle, performing ultrasonic treatment until the turbid solution in the glass bottle becomes transparent, transferring the solution into an autoclave with a polytetrafluoroethylene lining, placing the autoclave in an oven, and reacting for 24 hours at 85 ℃ to obtain a CuBTC solution;
2) Centrifuging the CuBTC solution obtained in the step 1) to obtain CuBTC particles, washing the CuBTC particles for 3-5 times by using ethanol, drying the CuBTC particles at 30-50 ℃ in vacuum for 6-10 h each time to obtain light blue particle substances, finally activating the obtained light blue particles in a nitrogen purging atmosphere, and keeping the light blue particles at 120-180 ℃ until the light blue particles gradually become dark blue, which indicates that the activation process is finished, and synthesizing CuBTC powder;
3) According to the mass fraction ratio of CuBTC: na (Na) 2 MoO 4 ·2H 2 O：CH 4 N 2 S =1 2 MoO 4 ·2H 2 O and CH 4 N 2 S, pouring the mixed aqueous solution into a polytetrafluoroethylene lining high-pressure kettle, and placing the kettle in an oven at 180-220 ℃ for 720min;
4) Washing, ultrasonically treating and drying the substance obtained in the step 3) to obtain solid powder, weighing, dissolving in ultrapure water again, and preparing into 10mg/mL CuBTC @ MoS 2 Standing the solution; cuBTC @ MoS modified and synthesized on printing electrode by physical adsorption method 2 A complex;
5) Preparation of NaAuCl with a mass fraction of 1% 4 ·2H 2 O solution and Na with concentration of 0.5M 2 SO 4 Nano goldThe reducing source solution is mixed and dripped into CuBTC @ MoS according to the volume ratio of 1:1 2 On the electrode, auNPs are modified on the surface of the electrode by utilizing a linear cyclic voltammetry electrochemical reduction method to form CuBTC @ MoS 2 -AuNPs modified electrodes.
CuBTC @ MoS based on the invention 2 -method for detecting CA125 by AuNPs modified electrode, comprising the steps of:
1) In the preparation of CuBTC @ MoS 2 Fixing a CA125 antibody on a working electrode of the AuNPs modified electrode through electrostatic adsorption, and performing electrochemical detection on CA125 with different concentrations and a blank control PBS solution by using a differential pulse voltammetry method;
2) Establishing a standard curve, drawing a fitting curve according to the absolute value of the difference between the differential pulse current peak value detected by the CA125 standard solution with different detected concentrations and the current peak value detected under the PBS solution and the logarithm of the concentration of the CA125 standard solution to obtain a current correlation equation:
△I＝-50.29766/[1+(ln[CA125]/1.68886E9)^0.15001]+50.92737
coefficient of correlation R 2 0.99801, lower limit of detection 0.5mU/mL;
3) Mixing the prepared CuBTC @ MoS 2 AuNPs/CA125 antibody electrodes were used for sample analysis.
The concrete description is as follows:
based on CuBTC @ MoS 2 -a method for CA125 detection of AuNPs modified electrodes comprising the steps of:
1. preparation of CuBTC @ MoS 2 -AuNPs modified electrode
(1) Synthesis of CuBTC: compared with CuBTC powder prepared by an acid-base neutralization method, the anaerobic activation method provided by the invention has better load capacity.
(2) Synthesis of CuBTC @ MoS 2 The compound is as follows: ready for working electrode modification.
(3) Preparation of CuBTC @ MoS 2 Electrode:
method for utilizing physical adsorption on printing electrode (only a certain amount of CuBTC @ MoS is dripped on working electrode (covering working electrode) 2 Solution, drying in an oven) modifying the CuBTC @ MoS synthesized in the step (2) 2 CompoundingThe compound provides chemical bond support for subsequent AuNPs modification.
(4) Preparation of CuBTC @ MoS 2 -AuNPs electrodes
CuBTC @ MoS modified in step (3) 2 A certain amount of gold nano reducing solution (covering the whole electrode) is dripped on the electrode, and AuNPs (AuNPs) are modified on the surface of the working electrode by utilizing a linear cyclic voltammetry electrochemical reduction method (wherein the scanning voltage is-1.0V-0.2V, the scanning rate is 100mV/s, and the number of scanning cycles is 5).
2. Based on CuBTC @ MoS 2 CA125 detection of AuNPs modified electrodes
(1) CuBTC @ MoS prepared in step 1 2 Fixing the CA125 antibody on the working electrode through electrostatic adsorption on the AuNPs modified electrode, and dropwise adding a certain amount of CA125 antibody (only the working electrode is covered) on the working electrode.
(2) Electrochemical detection is carried out on the CA125 solution with different concentrations and a blank control PBS solution by using differential pulse voltammetry (wherein the scanning voltage is-0.4V, the pulse amplitude is 50mV, the pulse width is 50ms, and the detection range is 0.0005U/mL-500U/mL).
(3) Establishing a standard curve, namely drawing a fitting curve according to absolute values (delta I) of differences between differential pulse current peak values detected by the CA125 standard solutions with different concentrations detected in the step (2) and current peak value differences detected under a PBS solution and logarithmic concentration (ln [ CA125 ]) points of the CA125 standard solutions to obtain a current correlation equation, and establishing the standard curve of the electrode for later clinical application:
△I＝-50.29766/[1+(ln[CA125]/1.68886×10 9 )^0.15001]+50.92737
coefficient of correlation R 2 0.99801, with a lower detection limit of 0.5mU/mL.
(4) The prepared CuBTC @ MoS is verified by detecting CA125, bovine Serum Albumin (BSA), human epididymis protein 4 (HE 4) and carcinoembryonic antigen (CEA) by using differential pulse voltammetry 2 Specificity of AuNPs/CA125 antibody electrode, to demonstrate the clinical feasibility of this electrode:
diluting CA125, BSA, HE4 and CEA standard substances which are common interference substances in serum when detecting CA125 by using a PBS solution, and preparing by selecting clinical detection concentrations to obtain 35U/mL CA125, 10ng/mL BSA, 5ng/mL CEA and 140pM HE4;
the control was performed by detecting the peak value (Ip) of the differential pulse voltammetry current in the PBS solution system, and then the peak values of the current in the four different solution systems were measured, and the absolute value of the difference from Ip was calculated.
(5) The prepared CuBTC @ MoS 2 The AuNPs/CA125 antibody electrode Is applied to the analysis of actual patient samples, wherein the content of CA125 in serum Is detected by a hospital gold standard Roche instrument as a reference value, the differential pulse voltammetry current peak value (Is) Is detected in an artificial serum system for comparison, the current peak value in the patient serum sample Is respectively detected, the absolute value of the difference between the current peak value and the Is calculated, and the clinical feasibility of the sensor Is evaluated by the judgment of a clinical 35U/mL cut-off point.
The beneficial effects of the invention are:
1. the invention firstly proposes CuBTC @ MoS 2 The material modified working electrode is applied to the field of electrochemical detection, the contact area of electrochemical reaction can be greatly increased due to the good characteristic of applying a metal organic framework, the detection sensitivity is improved, and MoS is utilized 2 The transition metal sulfide has good electrical properties and catalytic properties, and the combination of the electrical properties and the catalytic properties greatly improves the electrochemical performance of the electrode, as shown in fig. 3 in the specific embodiment.
2. The invention uses electrochemical reduction deposition method to make the gold nano-particles able to react with CuBTC @ MoS 2 The continuous and flat modification is carried out on the surface of the working electrode, the stability of the working electrode is improved due to the introduction of AuNPs, the interlayer capacitance of the working electrode is greatly reduced, and the electrochemical characteristics of the electrode are further improved, as shown in figure 3 in the specific embodiment.
3. When the cancer marker CA125 is detected, the nano-composite modified working electrode plays a role in front-end signal amplification, and can directly utilize specific binding of an antigen and an antibody without introducing a secondary antibody, so that the biological fixation step in the preparation process of the specific sensing electrode is reduced, a current signal is amplified, the detection precision is increased, the detection range (0.0005U/mL-500U/mL) is expanded, and the requirement of clinical accurate detection on trace CA125 can be met, as shown in fig. 7 and 8 in a specific embodiment.
Drawings
FIG. 1 is CuBTC @ MoS 2 -schematic representation of AuNPs electrode modification;
FIG. 2 is a graph of a process for modifying gold nanoparticles on a working electrode using linear cyclic voltammetry (LSV) scanning electrodeposition reduction;
FIG. 3 is a graph showing the results of step-by-step modification of cyclic voltammetry (Bare electrode, cuBTC @ MoS) 2 Electrode and CuBTC @ MoS 2 -AuNPs electrodes);
FIG. 4 is a scanning electron microscope image of a blank electrode;
FIG. 5 is a modified CuBTC @ MoS 2 Scanning electron microscope images of the electrodes;
FIG. 6 is a modified CuBTC @ MoS 2 -scanning electron microscopy images of AuNPs electrodes;
FIG. 7 is a graph using CuBTC @ MoS 2 AuNPs/CA125 antibody electrode detection of different concentrations of cancer antigen 125 (CA 125) differential pulse voltammetry detection curves (0.0005, 0.005, 0.05, 0.5, 5, 50, 500U/mL);
FIG. 8 is a graph of applying CuBTC @ MoS 2 A standard curve graph of the logarithmic fit of the absolute value of the difference between the current peak value of the differential pulse voltammetry detected by the AuNPs/CA125 antibody electrode detection of CA125 standard solutions with different concentrations and the current peak value detected in the PBS solution and the concentration logarithm of the CA125 standard solution;
FIG. 9 is a graph using CuBTC @ MoS 2 Specific histogram is drawn of the absolute value of the difference between the current peak detected by AuNPs/CA125 antibody electrode at 35U/mL CA125, 10ng/mL BSA, 5ng/mL CEA and 140pM HE4 solution and the current peak detected by PBS solution.
FIG. 10 is a graph of applying CuBTC @ MoS 2 The AuNPs/CA125 antibody electrode detects the actual serum sample of the ovarian cancer patient, and adds the absolute value of the difference between the current peak value detected in the actual serum sample and the current peak value detected under the artificial serum to the previous electrode response curve of FIG. 8.
In the figure, working electrode (1) of printing electrode, cuBTC @ MoS 2 Complex (2), auNPs (3), CA125 antibody (4), and target substance CA125 (5).
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention is not limited thereto.
The invention provides a method based on CuBTC @ MoS 2 The CA125 detection method of the AuNPs modified electrode can realize a wider linear range and a lower detection limit, and the preparation method comprises the following steps:
as shown in figure 1, the prepared CuBTC @ MoS is modified for 10min at 80 ℃ in an oven on the surface of a working electrode (1) of a printing electrode 2 A complex (2); and then scanned with NaAuCl using linear cyclic voltammetry 4 ·2H 2 O and Na 2 SO 4 The formed nano-gold reducing solution deposits gold nano-particles (3), the gold nano-particles (3) and CuBTC @ MoS 2 (2) S of (a) are connected through an Au-S chemical bond; thirdly, dripping the CA125 antibody (4) at room temperature, standing for 30min, and connecting the gold nanoparticles (3) with the cancer marker CA125 antibody (4) through amino; finally, at room temperature, left to stand for 5min to specifically capture the cancer marker CA125 (5).
The specific implementation process is as follows:
(1) Synthesis of CuBTC:
(1.1) using the volume ratio of DMF: etOH: ultrapure water =1:1.33:1 ratio preparation solvent to prepare 8.4mg/mL trimesic acid (C) 9 H 6 O 6 ) And 16.8mg/mL Cu (NO) 3 ) 2 ·3H 2 Placing the solution of O in a clean glass bottle, performing ultrasonic treatment for 60min to ensure that the solution in the glass bottle is changed from turbid to transparent, transferring the solution into a high-pressure autoclave with a polytetrafluoroethylene lining, and placing the high-pressure autoclave in a drying oven to react for 24h at 85 ℃ to obtain a CuBTC solution;
(1.2) centrifuging the CuBTC solution obtained in the step (1.1) to obtain CuBTC particles, washing the CuBTC particles with ethanol for 3 times, each time for 6 hours, placing the CuBTC particles at 40 ℃ for vacuum drying for 24 hours to obtain light blue particle substances, finally activating the obtained light blue particles in a nitrogen purging atmosphere, keeping the temperature at 160 ℃ for 12 hours, and gradually changing the light blue particles into dark blue, which indicates that the activation process is finished and CuBTC powder is synthesized.
(2) Synthesis of CuBTC @ MoS 2 The compound is as follows:
(2.1) mixing CuBTC: na (Na) 2 MoO 4 ·2H 2 O：CH 4 N 2 S =1 2 MoO 4 ·2H 2 O and CH 4 N 2 S, pouring the solution into a polytetrafluoroethylene lining high-pressure kettle, and placing the kettle in an oven at 200 ℃ for 720min;
(2.2) washing, ultrasonic treating and drying the substance obtained in the step (2.1) to obtain solid powder, weighing, dissolving in ultrapure water again, and preparing into 10mg/mL CuBTC @ MoS 2 The solution was allowed to stand for modification of the working electrode.
(3) 4 mu L of CuBTC @ MoS synthesized in the step (2) is dripped on a working electrode on a printing electrode 2 And (3) placing the compound in an oven at 80 ℃ for 10min for physical adsorption, and providing chemical bond support for modification of AuNPs after the compound is adsorbed on the working electrode, thereby greatly increasing the surface specific area of the working electrode.
(4) Preparation of NaAuCl with mass fraction of 1% 4 ·2H 2 O solution and Na with concentration of 0.5M 2 SO 4 Adding 70 μ L of nano gold reducing solution into the modified CuBTC @ MoS obtained in step (3) according to the volume ratio of 1:1 2 On the electrode, as shown in fig. 2, auNPs are continuously modified on the surface of the working electrode by using a linear cyclic voltammetry electrochemical reduction method, a peak of gold nanoparticle deposition can be seen when the voltage is-0.7V during the first scanning, the working electrode plate becomes golden yellow, and then the scanning is continuously carried out for four times to ensure that the gold particles are completely deposited (wherein the scanning voltage is-1.0V-0.2V, the scanning rate is 100mV/s, and the number of scanning cycles is 5). The CV curve of fig. 3 shows: the reduction peak of the bare electrode occurs at a voltage of 0.04V and a current of 60.3 muA; cuBTC @ MoS 2 The reduction peak of the electrode appeared at a voltage of 0.02V and a current of 81.4. Mu.A, and the comparison of the curves shows that CuBTC @ MoS 2 The electrochemical characteristics of the electrode are well increased, and as a dielectric material, the CV curve enclosed area is remarkably increased, which indicates that the capacitance of the electrode is also remarkably enhanced; cuBTC @ MoS 2 The oxygen reduction peak of the AuNPs electrode appears at the voltage of 0.08V and the current of 98.6 muA, the addition of AuNPs leads the current signal to be further amplified and modified layer by layerThe electrochemical performance of the electrode is obviously improved. Then, the prepared electrode is characterized by a scanning electron microscope, namely a bare electrode, cuBTC @ MoS 2 Electrode and CuBTC @ MoS 2 Scanning electron micrographs of the AuNPs electrode at a magnification of 3000 are shown in fig. 4, 5 and 6, respectively. It can be seen from the figure that the electrode surface layer material is more uniformly distributed and has better uniformity along with the self-assembly of the nanometer material layer by layer.
(5) CuBTC @ MoS prepared in step (4) 2 Fixing CA125 antibody on AuNPs modified electrode by electrostatic adsorption, dripping 4 μ L of 1mg/mL CA125 antibody on the surface of working electrode, connecting by electrostatic adsorption, standing at room temperature for 30min, washing excessive unbound CA125 antibody with pure water, and forming CuBTC @ MoS for specific detection of CA125 2 AuNPs/CA125 antibody electrodes.
(6) And (3) preparing an electrode to detect CA125 by using the step (5), sequentially dripping 70 mu L of PBS solution and 70 mu L of CA125 solution with different concentrations (the concentration is detected from low to high) on the electrode, standing for 10min at room temperature, and detecting current by using a differential pulse voltammetry electrochemical method after complete combination. CA125 solution: according to the method of ten-fold dilution of the concentration gradient, the CA125 standard substance concentration of 500U/mL-0.0005U/mL is prepared, and the total concentration is seven. The control group was 0.01m, ph =7.4 in PBS; and (4) detecting the prepared CA125 standard solutions with different concentrations by using the electrode prepared in the step (5), and detecting by using a differential pulse voltammetry method, wherein the scanning pulse period is 500ms, the pulse width is 50ms, the pulse amplitude is 50mV, and the voltage range is-0.4V. As shown in FIG. 7, the first curve measures 0.0005U/mL CA125, 70. Mu.L of 0.0005U/mL CA125 is dropped on the electrode, and the peak current is 83.1. Mu.A when the voltage is-0.04V; measuring 0.005U/mL CA125 on the second curve, dripping 70 μ L of 0.005U/mL CA125 on the electrode, and generating peak current of 82.9 μ A when the voltage is-0.03V; measuring 0.05U/mL CA125 by the third curve, dripping 70 mu L of 0.05U/mL CA125 on the electrode, and generating peak current of 82.5 mu A when the voltage is-0.03V; the fourth curve measures 0.5U/mL CA125, 70 mu L of 0.5U/mL CA125 is dripped on the electrode, and the peak current is 81.9 mu A when the voltage is-0.03V; measuring 5U/mL CA125 by a fifth curve, dripping 70 mu L of 5U/mL CA125 on an electrode, and generating peak current of 81.6A when the voltage is-0.03V; measuring 50U/mL CA125 by using a sixth curve, dripping 70 mu L of 50U/mL CA125 on an electrode, and leading the peak current to be 80.2 mu A when the voltage is minus 0.03V; the seventh curve measures 500U/mL CA125, 70 μ L of 500U/mL CA125 is dripped on the electrode, the peak current is 78.4 μ A when the voltage is-0.03V, and the current peak value of the DPV curve is gradually reduced along with the higher concentration of the CA 125.
(7) Repeating the experimental method in the step (6) for three times, establishing a standard curve according to three results, drawing a fitting curve of absolute values (delta I) of the difference between the current peak values of the differential pulse voltammetry detected by the CA125 standard solutions with different concentrations and the current peak value difference detected by the PBS solution and the logarithm of the concentration of the CA125 standard solution (ln [ CA125 ]) points, and obtaining a current correlation equation:
△I＝-50.29766/[1+(ln[CA125]/1.68886×10 9 )^0.15001]+50.92737
the detection range is 0.0005-500U/mL, and the correlation coefficient R 2 Is 0.99801, the lower detection limit can reach 0.5mU/mL, and the fitting curve is shown in FIG. 8.
(8) The prepared CuBTC @ MoS is verified by detecting CA125, bovine Serum Albumin (BSA), human epididymis protein 4 (HE 4) and carcinoembryonic antigen (CEA) by using differential pulse voltammetry 2 Specificity of AuNPs/CA125 antibody electrode:
(8.1) diluting CA125, BSA, HE4 and CEA standard substances which are common interference substances in serum when the CA125 is detected by using a PBS solution, and preparing the interference substances by selecting clinical detection concentrations to obtain 35U/mL CA125, 10ng/mL BSA, 5ng/mL CEA and 140pM HE4;
(8.2) to detect the peak value Ip of the differential pulse voltammetry current in the PBS solution system, then to measure the current peak in these four different solution systems, calculate the absolute value (DeltaI) of the difference from Ip to draw a specific histogram, as shown in FIG. 9, the DeltaI of 35U/mL CA125 is about 3 muA, the DeltaI of other substances 10ng/mL BSA, 5ng/mL CEA and 140pM HE4 are all within 1 muA, the changes brought by these interfering substances are very weak, the weak changes are due to that they are macromolecular proteins which cause a little current change in the solution, and only CA125 is bound to the working electrode which can cause a much larger range of current change.
(9) Mixing the prepared CuBTC @ MoS 2 The AuNPs/CA125 antibody electrode is applied to the analysis of actual patient samples, and the clinical feasibility of the electrode is verified:
(9.1) five samples of the serum of ovarian cancer patients are provided by Tianjin tumor Hospital, and the content of CA125 in the serum is detected by a Hospital gold standard Roche instrument: sample 1 (73.1U/mL), sample 2 (19.06U/mL), sample 3 (109U/mL), sample 4 (594U/mL), and sample 5 (36.3U/mL).
(9.2) comparing the detected differential pulse voltammetry current peak value Is in an artificial serum system, then measuring the current peak values in the serum samples of the five patients, and calculating the absolute value of the difference between the current peak values and the Is, as shown in figure 10, 5 groups of samples can give accurate judgment on the judgment of a clinical 35U/mL cut-off point, so that the electrode designed by the invention has good clinical feasibility.
Compared with other CA125 sensors, the sensor designed by the invention has wider detection range and good linearity compared with the related CA125 detection sensors published in years; has lower detection lower limit and high sensitivity. And the detection range comprises 35U/mL of detection index commonly used in clinical medicine, and certain optimization is also carried out in the upper detection limit according to the CA125 value in the actual human body, so that 500U/mL can be detected, and the application value is higher. ( SWV square wave voltammetry; DPV, differential pulse voltammetry; CHA-time current method )
Primary references
[1]Pakchin,P.S.,Fathi,M.,Ghanbari,H.,Saber,R.,Omidi,Y.,2020.A novel electrochemical immunosensor for ultrasensitive detection of CA125 in ovarian cancer.Biosensors&Bioelectronics 153.
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While the methods and techniques of the present invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications of the methods and techniques described herein may be practiced without departing from the spirit and scope of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and content of the invention.
Claims (4)
1. CuBTC @ MoS 2 -AuNPs repairDecorating the electrode; the CuBTC is characterized in that CuBTC is composed of metal Cu as a node and an organic ligand as a connector and is a metal organic framework porous material; cuBTC @ MoS 2 Mixing CuBTC, mo source and S source by hydrothermal method to prepare electrode modification material CuBTC @ MoS 2 (ii) a Fixing it on working electrode by physical adsorption to form CuBTC @ MoS 2 An electrode; next, naAuCl was used 4 ·2H 2 And reducing the O solution on the electrode, and then depositing the gold nanoparticles on the working electrode to fix the gold nanoparticles on the working electrode through Au-S bonds.
2. CuBTC @ MoS according to claim 1 2 The method for detecting the CA125 by the AuNPs modified electrode is characterized in that the electrode is placed at room temperature to physically adsorb the CA125 antibody, and the antibody is continuously fixed on the working electrode; preparing CuBTC @ MoS 2 AuNPs/CA125Ab electrode.
3. The CuBTC @ MoS of claim 1 2 -a method for preparing AuNPs modified electrode, which is characterized by comprising the following steps:
1) Using the volume ratio of DMF: etOH: ultrapure water =1:1.33:1 to prepare a solvent to prepare trimesic acid in a concentration ratio: cu (NO) 3 ) 2 ·3H 2 Placing the solution of O =1:2 in a clean glass bottle, performing ultrasonic treatment until the turbid solution in the glass bottle becomes transparent, transferring the solution into an autoclave with a polytetrafluoroethylene lining, placing the autoclave in an oven, and reacting for 24 hours at 85 ℃ to obtain a CuBTC solution;
2) Centrifuging the CuBTC solution obtained in the step 1) to obtain CuBTC particles, washing the CuBTC particles for 3-5 times by using ethanol, drying the CuBTC particles at 30-50 ℃ in vacuum for 6-10 h each time to obtain light blue particle substances, finally activating the obtained light blue particles in a nitrogen purging atmosphere, and keeping the light blue particles at 120-180 ℃ until the light blue particles gradually become dark blue, which indicates that the activation process is finished, and synthesizing CuBTC powder;
3) According to the mass fraction ratio of CuBTC: na (Na) 2 MoO 4 ·2H 2 O：CH 4 N 2 S =1 2 MoO 4 ·2H 2 O and CH 4 N 2 S, pouring the mixed aqueous solution into a polytetrafluoroethylene lining high-pressure kettle, and placing the kettle in an oven at 180-220 ℃ for 720min;
4) Washing, ultrasonically treating and drying the substance obtained in the step 3) to obtain solid powder, weighing, dissolving in ultrapure water again, and preparing into 10mg/mL CuBTC @ MoS 2 Standing the solution; cuBTC @ MoS modified and synthesized on printing electrode by physical adsorption method 2 A complex;
5) Preparation of NaAuCl with a mass fraction of 1% 4 ·2H 2 O solution and Na with concentration of 0.5M 2 SO 4 The nano gold reducing source solution is mixed and dripped into CuBTC @ MoS according to the volume ratio of 1:1 2 On the electrode, auNPs are modified on the surface of the electrode by utilizing a linear cyclic voltammetry electrochemical reduction method to form CuBTC @ MoS 2 -AuNPs modified electrodes.
4. CuBTC @ MoS according to claim 2 2 -method for detecting CA125 by AuNPs modified electrode, which is characterized by comprising the following steps:
1) In the preparation of CuBTC @ MoS 2 Fixing a CA125 antibody on a working electrode of the AuNPs modified electrode through electrostatic adsorption, and performing electrochemical detection on CA125 with different concentrations and a blank control PBS solution by using a differential pulse voltammetry method;
2) Establishing a standard curve, establishing a fitting curve according to the absolute value delta I of the difference between the differential pulse current peak value detected by the CA125 standard solution with different detected concentrations and the current peak value detected under the PBS solution, wherein the unit is mu A, and the logarithm ln [ CA125] point of the concentration of the CA125 standard solution, wherein the unit of the concentration of the CA125 is U/mL, and obtaining a current correlation equation:
△I＝-50.29766/[1+(ln[CA125]/1.68886×10 9 )^0.15001]+50.92737
coefficient of correlation R 2 0.99801, with a lower detection limit of 0.5mU/mL;
3) The prepared CuBTC @ MoS 2 AuNPs/CA125 antibody electrodes were used for sample analysis.
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