CN110702754B

CN110702754B - Method for measuring human chorionic gonadotropin

Info

Publication number: CN110702754B
Application number: CN201911087646.7A
Authority: CN
Inventors: 邓必阳; 覃东庙; 蒋小华; 莫桂春
Original assignee: Guangxi Normal University
Current assignee: Guangxi Normal University
Priority date: 2019-11-08
Filing date: 2019-11-08
Publication date: 2021-05-04
Anticipated expiration: 2039-11-08
Also published as: CN110702754A

Abstract

The invention discloses a method for measuring human chorionic gonadotropin. According to the method, waste biomass papaya peels are used as raw materials, a hydrothermal method is adopted to synthesize blue high-fluorescence carbon quantum dots in one step, and the blue high-fluorescence carbon quantum dots are combined with silver ions to form AgCQDs; meanwhile, through photolysis and coordination polymerization of ferrocene dicarboxylic acid molecules in methanol, PS is synthesized, AgCQDs with negative electricity and PS which is modified by PEI and becomes positive electricity are combined through electrostatic attraction, and the obtained composite material and the rGO @ Ag composite material are jointly prepared into a sandwich type immunosensor which is then used for HCG detection. The method provided by the invention has high sensitivity and good stability.

Description

Method for measuring human chorionic gonadotropin

Technical Field

The invention relates to a detection method of human chorionic gonadotropin, in particular to a method for detecting human chorionic gonadotropin.

Background

Human Chorionic Gonadotropin (HCG) is a glycoprotein hormone with a molecular weight of 37kDa, produced by trophoblasts of the gestational placenta and overexpressed by trophoblast tumors and various non-fibroblast tumors. In addition, HCG, as a biomarker, plays an autocrine role in human cancer biology, promoting tumor growth, invasion and development of malignant tumors. The sensitive and accurate detection of HCG in serum is of great significance for predicting pregnancy and can be used as a tumor marker for monitoring trophoblastic diseases. In reported work, HCG has been detected using various techniques, including electrochemical immunoassays (EC), fluorescence methods (FL), surface enhanced raman scattering spectroscopy (ERS), chromatographic test strips, chemiluminescent immunoassays (CE), and Photoluminescence (PL). Although some of these methods are sensitive, most require expensive laboratory equipment, time consuming procedures and complex procedures. Therefore, it remains a valuable research topic to develop a rapid, low-cost, sensitive and selective method for detecting HCG. Compared with the above technologies, Electrochemiluminescence (ECL) is widely used in bioanalysis and complex analysis in clinical fields because of its combination of advantages of electrochemical analysis and chemiluminescence, such as easy control, high sensitivity, low background signal, simplified optical equipment, low cost, good specificity, etc.

Carbon Quantum Dots (CQDs), as a novel carbon nanomaterial, have been widely used as ECL probes in biosensors due to their ease of preparation, good water solubility and biocompatibility, good optical properties, high electron conduction rate, low cellular toxicity, good biocompatibility, and ease of biofunctionalization. However, the electrochemical luminescence signal of water-soluble CQDs is low, and thus in order to fabricate an ultrasensitive ECL platform, the ECL intensity of CQDs must be increased. Nanomaterials, such as graphene, metal nanoparticles and Magnetic Beads (MB), have been widely used in biosensors due to their good biocompatibility and electrical conductivity, ability to accelerate signal transduction and amplify signals, in combination with signal amplification strategies. In this work, CQDs were used as support materials and reducing agents for reducing silver ions, synthesizing CQDs-supported silver nanoparticles (AgCQDs), and increasing the electrochemiluminescence signal. For electrochemical immunosensors, the ease and speed of electron transfer affects their sensitivity, and the electron mediator plays an important role in this process. Traditional electronic media such as thionine, methylene blue, alizarin red and ferrocene cannot be stably fixed on the electrode, so that the constructed immunosensor is not stable enough.

Disclosure of Invention

The invention aims to provide a method for measuring human chorionic gonadotropin with high sensitivity and good stability.

The invention relates to a method for measuring human chorionic gonadotropin, in particular to a method for measuring human chorionic gonadotropin by using a sandwich type immunosensor based on a nanometer compound of papaya peel synthetic carbon quantum dots, which comprises the following steps:

1) preparation of CQDs:

taking pawpaw peels as a carbon source, and synthesizing by adopting a hydrothermal method to obtain CQDs;

2) preparation of AgCQDs suspension:

CQDs is combined with Ag⁺Combining to obtain AgCQDs suspension;

3) preparing AgCQDs @ PS composite material:

modifying PS (electroactive polymer nanospheres) by using PEI (polyethyleneimine) to obtain PS with positive charge, then mixing the PS with positive charge with AgCQDs suspension and carrying out ultrasound to obtain AgCQDs @ PS composite material, wherein the AgCQDs @ PS composite material is stored in PBS buffer solution;

4) preparing an AgCQDs @ PS/HCG-Ab2 composite material:

mixing EDC solution and NHS solution, adding AgCQDs @ PS solution and HCG-Ab2 into the mixture, stirring for reaction, then adding BSA into the mixture to block non-specific binding sites of the obtained material, centrifuging, collecting precipitate to obtain AgCQDs @ PS/HCG-Ab2 composite material, and storing the material in PBS buffer solution; wherein before adding BSA, the concentration of EDC in the system is controlled to be 1.8-1.9 mol/mL^-1The concentration of NHS is 0.4-0.5 mol/mL^-1The concentration of AgCQDs @ PS is 1.8-1.9 mol/mL^-1The concentration of HCG-Ab2 is 0.9-1.0. mu.g/mL^-1；

5) Preparing an rGO @ Ag composite material;

6) preparing a sandwich type immunosensor:

dripping the rGO @ Ag solution on the surface of GCE (glass carbon electrode), and airing to obtain rGO @ Ag/GCE; then, dripping HCG-Ab1 (primary antibody corresponding to HCG antigen) on the surface of the obtained rGO @ Ag/GCE, incubating, washing to remove unbound antibody after incubation is finished, dripping BSA, incubating to block non-specific binding sites, and washing with PBS buffer solution to obtain BSA/HCG-Ab1/rGO @ Ag/GCE; then, dripping an HCG antigen solution on the surface of the obtained BSA/HCG-Ab1/rGO @ Ag/GCE and incubating to obtain HCG/BSA/HCG-Ab1/rGO @ Ag/GCE; dropwise adding AgCQDs @ PS/HCG-Ab2 solution on the surface of the obtained HCG/BSA/HCG-Ab1/rGO @ Ag/GCE and carrying out immunoreaction to obtain AgCQDs @ PS/HCG-Ab2/HCG/BSA/HCG-Ab1/rGO @ Ag/GCE, namely the sandwich type immunosensor;

7) and (3) detection:

the sandwich type immunosensor prepared by the method is used as a working electrode, an Ag/AgCl electrode and a platinum wire are respectively used as a reference electrode and an auxiliary electrode, and the working electrode and the auxiliary electrode are placed in PBS buffer solution containing a co-reactant for electrochemical luminescence detection;

the PBS buffer referred to in the above method is at a concentration of0.1～0.12mol·L^-1And a PBS buffer solution with pH of 7.3-7.5.

The PBS buffer solution used in the above method preferably has a concentration of 0.1 mol. L^-1PBS buffer at pH 7.4.

In step 1) of the method of the present invention, the method for preparing the carbon quantum dots specifically comprises: putting the papaya peel powder into water, carrying out ultrasonic dissolution, transferring into a hydrothermal reaction kettle for hydrothermal reaction (reaction is carried out for 10-12 h at the temperature of 80-200 ℃), cooling after the reaction is finished, centrifuging the reactant, filtering the supernatant through a microporous filter membrane (the aperture is 0.20-0.25 mu m), and dialyzing through a dialysis bag with the molecular weight of 800-1000 Da to obtain a pure carbon quantum dot solution. In this step, before adding BSA, it is preferable to control the EDC concentration in the system to 1.8 mol/mL^-1The concentration of NHS was 0.4 mol/mL^-1The concentration of AgCQDs @ PS is 1.8 mol/mL^-1The concentration of HCG-Ab2 was 0.9. mu.g.mL^-1。

In step 2) of the method of the invention, CQDs and Ag are mixed by the conventional method⁺And combining to obtain AgCQDs suspension. The preparation is preferably carried out as follows: combining the synthesized CQDs with AgNO₃And uniformly mixing the aqueous solution, adding NaOH solution (aiming at enabling silver nano particles to grow on the surface of carbon quantum dots in situ), then violently stirring and reacting for 24 hours at 37 ℃, wherein the color of the solution gradually changes from brown yellow to deep red in the reaction process, which indicates that a silver nano structure is formed on the surface of CQDs, and obtaining AgCQDs suspension (namely the AgCQDs composite material) after the reaction is finished. The obtained material is stored at low temperature and in dark.

In step 3) of the method, the PS solution is a solution prepared by dispersing PS in a PBS buffer solution, wherein the concentration of PS is 12-16 mg/mL^-1Wherein the PBS buffer solution has a concentration of 0.1-0.12 mol.L^-1And a PBS buffer solution with pH of 7.3-7.5. The PS can be prepared according to the conventional method, and preferably according to the following method: dissolving 1,1' -ferrocene dicarboxylic acid (Fc-COOH) in methanol by ultrasonic wave to obtain an orange solution; exposing the solution to sunlight until the solution turns grey brown in color; Fc-COOH was decomposed under natural light and air exposureInto deprotonated Fc-COO^-And Fe³⁺Simultaneously, in deprotonation of Fc-COO^-And Fe³⁺The polymerization reaction (generating precipitate) occurs, the obtained reactant is centrifuged, the precipitate is collected and washed by methanol, and the PS is obtained. The PS is stored in a PBS buffer solution (the concentration is 0.1-0.12 mol. L)^-1pH 7.3-7.5) and storing at low temperature.

In the step 3) of the method, the specific method for preparing the AgCQDs @ PS composite material comprises the following steps: adding a PEI solution into a PS solution, performing ultrasonic treatment, centrifuging, collecting supernatant, adding AgCQDs suspension into the supernatant, performing ultrasonic treatment, centrifuging, collecting precipitate, and washing with water to obtain an AgCQDs @ PS composite material; the PEI solution is a solution formed by dispersing PEI in water, wherein the concentration of PEI is 0.09-0.11 mg/mL^-1。

In the step 4) of the method, the concentration of the EDC solution is 100-110 mmol.L^-1The concentration of the NHS solution is 45-55 mmol.L^-1The concentration of HCG-Ab2 is 20 mu g/mL^-1. In the step, the AgCQDs @ PS solution is a solution obtained by dispersing an AgCQDs @ PS composite material solution in a PBS buffer solution, wherein the concentration of the AgCQDs @ PS composite material is 2.0 mg.mL^-1The PBS buffer solution is 0.1-0.12 mol/L^-1And a PBS buffer solution with pH of 7.3-7.5.

In step 5) of the method of the present invention, the rGO @ Ag composite material is prepared according to the conventional method, preferably according to the following method: ultrasonically dispersing GO (graphene oxide) in water, and sequentially adding AgNO at 80-100 DEG C₃And uniformly stirring and mixing the solution and the sodium citrate, then adding hydrazine hydrate into the solution, stirring for reaction, cooling after the reaction is finished, centrifuging the reactant, collecting precipitate, washing with ethanol and water respectively, and drying to obtain the rGO @ Ag composite material.

In step 6) of the method, the rGO @ Ag solution is a solution obtained by dispersing a rGO @ Ag composite material in water, wherein the concentration of the rGO @ Ag composite material is 1.0 mg/mL^-1(ii) a The concentration of HCG-Ab1 is 100 mu g/mL^-1(ii) a The HCG antigen solutionThe concentration of (b) is more than or equal to 10 mIU.mL^-1Preferably 10 mIU/mL^-1(ii) a The AgCQDs @ PS/HCG-Ab2 solution is a solution obtained by dispersing AgCQDs @ PS/HCG-Ab2 composite material in PBS buffer solution, wherein the concentration of the AgCQDs @ PS/HCG-Ab2 composite material is 2.0 mg/mL^-1. The concentration of BSA is preferably 1 wt%.

In the step 7) of the method, the concentration of the coreactant in the PBS buffer solution is 0.09-0.11 mol.L^-1Preferably 0.1 mol. L^-1The coreactant may be any conventional coreactant suitable for CQDs, such as potassium persulfate and the like. The conditions for performing the electrochemiluminescence detection are preferably: the high voltage of the photomultiplier is 800V, the scanning potential is 0 to-2.0V, and the scanning speed is 100mV^-1。

Compared with the prior art, the invention is characterized in that:

1. firstly, taking waste biomass pawpaw peels as a carbon source, and synthesizing by adopting a hydrothermal method in one step to obtain blue high-fluorescence carbon quantum dots;

2. through electrostatic attraction, AgCQDs with negative electricity and PS modified by PEI to be positively charged are combined, PEI not only connects the PS with the AgCQDs, but also forms a stable film on the surface of the PS to fix an electron mediator, so that the obtained AgCQDs @ PS shows good current signal amplification capacity;

3. the sandwich type immunosensor designed by the invention shows high sensitivity (the detection limit of HCG is 0.00033mIU & mL)^-1) And excellent stability (correlation coefficient of 0.9971), can be used for quantitatively detecting HCG in human serum.

Drawings

FIG. 1 is a morphology diagram of CQDs, AgCQDs, PS and AgCQDs @ PS prepared in example 1 of the present invention, wherein (a) is a TEM diagram of CQDs, (b) is a TEM diagram of AgCQDs, (c) is a TEM diagram of PS, (d) is a TEM diagram of AgCQDs @ PS, (e) is a Zeta potential diagram of PS (wherein curve 1 is an ultraviolet absorption diagram before PS exposure and curve 2 is an ultraviolet absorption diagram after PS exposure), (f) is a Zeta potential diagram of PS (bar diagram 1), modified PS (PS-PEI) (bar diagram 2), AgCQDs (bar diagram 3) and AgCQDs @ PS (bar diagram 4), (g) is a particle size distribution diagram of CQDs, and (h) is a particle size distribution diagram of AgCQDs.

FIG. 2 is an EDX map of AgCQDs and AgCQDs @ PS, wherein (a) is an EDX map of AgCQDs, and (b) is an EDX map of AgCQDs @ PS.

FIG. 3 is XRD spectra of CQDs, AgCQDs, GO, rGO @ Ag, wherein (a) is XRD images of CQDs and AgCQDs, and (b) is XRD image of GO, rGO @ Ag.

FIG. 4 is a FTIR spectrum and an XPS map of CQDs and AgCQDs, wherein (a) is a FTIR spectrum and (b) is a XPS map of CQDs; (c) XPS plots of C1s for CQDs; (d) XPS plots of N1s for CQDs; (e) XPS plots of O1s for CQDs, and (f) XPS plots for AgCQDs.

FIG. 5 is a UV-vis spectrum and a fluorescence spectrum of CQDs for CQDs and AgCQDs, wherein (a) is a UV-vis spectrum for CQDs and AgCQDs; (b) fluorescence emission spectra and fluorescence excitation spectra for CQDs; (c) fluorescence spectra of CQDs at different excitation wavelengths.

FIG. 6 shows CQDs and AgCQDs in 0.1 mol.L^-1Cyclic voltammogram of the modified electrode in PBS buffer at pH 7.4.

FIG. 7(a) and FIG. 7(b) are each 0.1 mol. L^-1No 0.1 mol. L in PBS buffer (pH 7.4)^- ¹K₂S₂O₈And has a molar ratio of 0.1 mol. L^-1K₂S₂O₈The ECL-potential curve and the CV curve of (1), wherein the curve is no 0.1 mol. multidot.L^-1K₂S₂O₈ECL-potential curve of CQDs/GCE under the condition that curve 2 is no 0.1 mol.L^-1K₂S₂O₈Under the condition of AgCQDs/GCE, the curve 3 is 0.1 mol.L^-1K₂S₂O₈The ECL-potential curve of CQDs/GCE under the conditions, curve 4, 0.1 mol.L^-1K₂S₂O₈ECL-potential curve of AgCQDs/GCE under the condition; scanning rate: 100 mV. s^-1。

FIG. 8 is a graph of ECL signals for CQDs/GCE, AgCQDs/GCE and AgCQDs @ PS/GCE; (b) ECL signal plots scanned 9 times repeatedly for CQDs/GCE (Curve 1), AgCQDs/GCE (Curve 2), and AgCQDs @ PS/GCE (Curve 3); detection conditions are as follows: 0.1 mol. L^-1PBS buffer (pH 7.4) contained 0.1 mol. L^-1K₂S₂O₈The potential scanning range is-2V to 0V, and the PMT voltage is set to 800V.

FIG. 9 shows a graph containing 0.1 mol. L^-1KCl solution 5.0 mmol. L^-1[Fe(CN)₆]^3-/4-And a corresponding EIS response plot, wherein: (a) is 0.1 mol.L^-1KCl solution 5.0 mmol. L^-1[Fe(CN)₆]^3-/4A CV curve of (b) is a curve containing 0.1 mol. L-¹KCl solution 5.0 mmol. L-¹[Fe(CN)₆]^3-/4-In FIGS. (a) and (b), curve 1 represents bare/GCE, curve 2 represents rGO @ Ag/GCE, curve 3 represents HCG-Ab1/rGO @ Ag/GCE, curve 4 represents BSA/HCG-Ab1/rGO @ Ag/GCE, curve 5 represents HCG/BSA/HCG-Ab1/rGO @ Ag/GCE, and curve 6 represents AgCQDs @ PS-Ab2-HCG/HCG/BSA/HCG-Ab1/rGO @ Ag/GCE; detection conditions are as follows: CV potential scan range: -0.2-0.6V, scan rate of 100mV · s^-1(ii) a EIS: the impedance frequency is 0.1Hz-10⁵Hz。

FIG. 10 is a graph of the optimization of the parameters in the experiment, where (a) is the ECL intensity profile of the immunosensor prepared in PBS at different pH values, (b) is the ECL intensity profile of the immunosensor in different BSA blocking volumes, (c) is the ECL intensity profile of the immunosensor at different concentrations of rGO @ Ag, (d) is the ECL intensity profile of the immunosensor at different concentrations of HCG-Ab1, (e) is the ECL intensity profile of the immunosensor at different concentrations of AgCQDs @ PS, (f) is the ECL intensity profile of the immunosensor at different volumes of HCG-Ab2, (g) is the ECL intensity profile of the immunosensor at different incubation times of HCG, (h) is the ECL intensity profile of HCG at different incubation temperatures; detection conditions are as follows: the immunosensor was mixed with 1 mIU. mL^-1Incubation with HCG antigen in a medium containing 0.1 mol.L^-1K₂S₂O₈0.1 mol. L of^-1ECL response was measured in PBS buffer, scan rate: 100 mV. s^-1The scanning potential is from-2-0V (n-4).

FIG. 11 is a graph showing the response of HCG at different concentrations to the electrochemiluminescence intensity and the curves of the electrochemiluminescence intensity versus the logarithm of the HCG concentration, in which (a) is the response of HCG at different concentrations to the electrochemiluminescence intensity, and (1) represents 0.001 mIU. mL^-1(ii) a (2) Represents 0.01 mIU/mL^-1(ii) a (3) Represents 0.1 mIU. mL^-1(ii) a (4) Represents 1 mIU. mL^-1(ii) a (5) Represents 10 mIU. mL^-1(ii) a (6) Represents 100 mIU. mL^-1(ii) a (7) Represents 200 mIU. mL^-1(ii) a (8) Represents 500 mIU. mL^-1(ii) a (b) The electrochemiluminescence intensity is plotted against the logarithm of the HCG concentration.

Fig. 12 is ECL stability results of the prepared immunosensor against HCG concentrations, wherein (a) is an ECL stability curve of the immunosensor against four HCG concentrations; (b) the ECL immunosensor prepared under the same condition is kept for 1 week, 2 weeks, 3 weeks and 4 weeks at 4 ℃ and then is continuously used for detecting HCG electrochemiluminescence intensity bar chart; (c) for the electrochemical luminescence intensity bar chart of the prepared immunosensor in the detection of different antigens or proteins: wherein Blank is Blank, Bovine Serum Albumin (BSA) (20 mg. mL)^-1)，CEA(10ng·mL^-1)，AFP(10ng·mL^-1)，CA15-3(10U·mL^-1)，HCG(10mIU·mL^-1) And mixtures (Misture); (d) the 4-branch immunosensor prepared under the same conditions is at 0.01 mIU.mL^-1(bar chart 1),1 mIU. mL^-1(bar graph 2),200 mIU. mL^-1(bar graph 3) bar graph of electrochemiluminescence intensity in the presence of HCG.

Detailed Description

The present invention will be better understood from the following detailed description of specific examples, which should not be construed as limiting the scope of the present invention.

Example 1

1 method of experiment

1.1 reagents and instruments

HCG antigen and HCG antibody (anti-HCG), antigen CEA, antigen AFP, antigen CA15-3 were purchased from Biocell Co. (zheng, china); graphene Oxide (GO) (nanjing piofeng nano technologies ltd, south kyo, china). Silver nitrate, sodium citrate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and potassium persulfate (Guangdong Shangshu chemical industry Co., Ltd., China); bovine Serum Albumin (BSA), N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), Polyethyleneimine (PEI), 1' -ferrocenedicarboxylic acid (Fc-COOH), methanol and sodium hydroxide (from Aladdin industries). The reagents used in the experiment are analytically pure, and the water is ultrapure water.

Recording ECL responses using an MPI-B ECL analyzer (sienna Remex electronics technologies ltd, west safety, china); the voltage of the photomultiplier tube (PMT) was set to 800V. Platinum wires, Ag/AgCl (saturated KCl) electrodes and modified GCE electrodes are respectively used as counter electrodes, reference electrodes and working electrodes. CHI660E a15312 electrochemical workstation (shanghai CH instruments ltd); (JEM-2100 Electron microscope JEOL Ltd., Japan); the IR spectra were recorded using a Fourier transform Infrared Spectrophotometer (FTIR, Perkin-Elmer, USA). The uv-vis absorption spectrum was obtained using a Cary 60 uv-vis spectrophotometer (Agilent Technologies, USA); FSCALAB250Xi X-ray photoelectron spectrometer (XPS, Thermo Fisher scientific, USA); rigaku D/max 2500/pc type X-ray powder diffractometer (XRD, Rigaku, Japan); model H1650-W high speed centrifuge (hunan instrument centrifuge, ltd.); dialysis bags (United states carbide, molecular weight cut-off 1000 Da).

1.2 Synthesis of carbon quantum dots

In the experiment, papaya peels are used as a carbon source, and the carbon quantum dots are synthesized by a hydrothermal method, which comprises the following specific steps: cleaning fresh papaya peel, air drying, oven drying at 80 deg.C for 10 hr, and grinding into powder. Accurately weighing 1g of papaya peel powder, adding 30mL of ultrapure water, ultrasonically dissolving for 10min to uniformly disperse, and transferring the solution to a 50mL polytetrafluoroethylene hydrothermal reaction kettle to heat at 200 ℃ for 12 h. After the reaction was completed, it was naturally cooled to room temperature. Then centrifuging the solution at 11000rpm for 30min, taking the supernatant, taking the microporous filter membrane, filtering with pore size of 0.22 μm, and removing large-particle impurities. Finally dialyzing for 24h by using a dialysis bag with the molecular weight of 1000Da to obtain a pure carbon quantum dot solution, and storing at 4 ℃ in a dark place.

1.3 preparation of AgCQDs composite Material

The pawpaw peel carbon quantum dot synthesized by a hydrothermal method contains rich carboxyl, and is suitable for metal ion Ag through ion exchange or coordination reaction⁺And (4) combining. The specific steps for synthesizing the AgCQDs composite material are as follows: the synthesized CQDs (10mL) and AgNO were mixed₃Aqueous solution(2.5mL，10mmol·L^-1) After mixing for 5 minutes, NaOH solution (50. mu.L, 1 mol. L) was added^-1) And then, the solution is stirred vigorously at 37 ℃ for reaction for 24 hours, the color of the solution gradually changes from brown yellow to deep red in the reaction process, which shows that a silver nano structure is formed on the surface of the CQDs, and after the reaction is finished, the obtained AgCQDs composite material is stored in a refrigerator at 4 ℃ in a dark place.

1.4 preparation of PS

PS is prepared by adopting an infinite coordination polymerization method. Accurately weighed 5.0mg of 1,1' -ferrocene dicarboxylic acid (Fc-COOH) was dissolved in 5.0mL of methanol and after sonication, an orange solution was obtained. The solution was then exposed to sunlight for 2 hours and the solution turned a grey brown color. Under natural light and air exposure, Fc-COOH is decomposed into deprotonated Fc-COO^-And Fe³⁺Simultaneously, in deprotonation of Fc-COO^-And Fe³⁺Polymerization occurs in between. Subsequently, the precipitate was centrifuged at 11000rpm for 10min and washed three times with methanol. Finally, the polymer was stored and dissolved in 2mL of 0.1 mol. L^-1PBS buffer pH 7.4 and stored in a refrigerator at 4 ℃ for further use.

1.5 preparation of AgCQDs @ PS

AgCQDs @ PS is prepared by a self-contained method. Modification of PS was performed using AgCQDs as an aid. Firstly, 1.0mL of PEI solution is added into 2.0mL of PS solution, the solution is subjected to ultrasonic treatment for 2 hours to synthesize PS with positive charges, then the PS solution is centrifuged for 10 minutes at the rotating speed of 11000rpm, then 1.0mL of AgCQDs suspension is added into the PS solution and subjected to ultrasonic treatment for 2 hours, and a stable structure of AgCQDs @ PS is formed through electrostatic attraction. Finally, the prepared AgCQDs @ PS was centrifuged at 11000rpm for 10min and washed three times with ultrapure water, and then the precipitate was redispersed in 2mL of PBS buffer (0.1 mol. L)^-1pH 7.4).

1.6 preparation of AgCQDs @ PS/HCG-Ab2

20 μ L of EDC solution and NHS solution were mixed at a mass ratio of 4:1 (volume ratio) and added to 2mL2.0 mg. multidot.mL^- ¹To the AgCQDs @ PS solution, stirring was carried out for 1h, and then 100. mu.L of Ab2 (20. mu.g.mL) was added^-1) Stirring was continued for 6 h. Followed by addition of 50. mu.L of 1% BSA overnight and centrifugation at 11000rpm for 10min, 15mL of 0.1 mol. L^-1Three washes with PBS buffer pH 7.4, and finally dissolving the mixture to 1mL of 0.1 mol.L^-1pH 7.4PBS buffer, then stored in a refrigerator at 4 ℃ for further use.

1.7 preparation of rGO @ Ag composite

25mg of GO is weighed and dispersed in 50mL of ultrapure water for 1.5h by ultrasonic treatment, and 10mg of AgNO is added in sequence at 90 DEG C₃And 300mg of sodium citrate and stirred for 1h, followed by addition of 30. mu.L of hydrazine hydrate to the mixture solution and stirring continued for 4 hours. And after the product is cooled to room temperature, centrifuging at the rotating speed of 11000rpm for 10min for separation, washing precipitates twice with ethanol and ultrapure water respectively, and then drying the precipitates in an oven at the temperature of 60 ℃ for 12 hours to obtain the rGO @ Ag composite material.

1.8 preparation of immunosensor

First, 0.3 μm and 0.05 μm Al were used₂O₃And (3) polishing the Glassy Carbon Electrode (GCE) into a mirror surface by powder polishing, ultrasonically cleaning the glassy carbon electrode by using ethanol and ultrapure water, and drying by using nitrogen. The rGO @ Ag composite (1.0 mg. mL) was then placed in a vacuum^-18.0. mu.L) was dropped on a clean GCE surface and air-dried at room temperature, followed by dropping 8.0. mu.L of HCG-Ab1 (100. mu.g.mL)^-1) Incubate at 4 ℃ for 12h on the surface of rGO @ Ag/GCE, then incubate with 0.1 mol. L^-1The unbound antibody was removed by washing with PBS buffer at pH 7.4. After washing, 4.0. mu.L of BSA solution (1.0 wt%) was dropped on the electrode surface and incubated for 40 minutes to block non-specific binding sites, followed by PBS buffer (0.1 mol. multidot.L)^-1pH 7.4) BSA/HCG-Ab1/rGO @ Ag/GCE and incubated with different concentrations of HCG (8.0. mu.L) at 37 ℃ for 1 hour to give HCG/BSA/HCG-Ab1/rGO @ Ag/GCE; finally, 8. mu.L of HCG-Ab2/AgCQDs @ PS solution was dropped onto HCG/BSA/HCG-Ab1/rGO @ Ag/GCE and immunoreaction was carried out at 37 ℃ for 1 hour to obtain AgCQDs @ PS/HCG-Ab2/HCG/BSA/HCG-Ab1/rGO @ Ag/GCE, i.e., a sandwich-type immunosensor (hereinafter also referred to as ECL immunosensor or immunosensor). Stored at 4 ℃ when not in use for further use.

1.9 detection method

In the presence of 0.1 mol.L^-1K₂S₂O₈1mL ofPBS buffer (0.1 mol. L)^-1pH 7.4) was performed in the test cell. In the ECL immunoassay, an Ag/AgCl electrode and a platinum wire were used as a reference electrode and an auxiliary electrode, respectively, and AgCQDs @ PS/HCG-Ab2/HCG/BSA/HCG-Ab1/rGO @ Ag/GCE was used as a working electrode. The detection conditions are as follows: the voltage of the photomultiplier tube (PMT) was maintained at 800V, the potential range was-2-0V, and the scanning rate was 100 mV. multidot.s^-1。

2 results and discussion

2.1 characterization of the synthetic materials

TEM was used to characterize the morphology of CQDs, AgCQDs, PS, AgCQDs @ PS nanocomposites (FIG. 1). As shown in FIG. 1(a), TEM images of CQDs show that CQDs are well-dispersed nanoparticles, and the particle size distribution of CQDs (as shown in FIG. 1 (g)) is mainly in the range of 2-10nm, with an average diameter of 6.5nm (images were selected for analysis in 100 individual particle regions). The carbon quantum surface is characterized by containing carboxyl through infrared, and silver ions are easily combined with the carboxyl part of the carbon quantum dot through ion exchange or coordination reaction. Fig. 1(b) is a TEM image of AgCQDs, fig. 1(h) is a particle size distribution diagram of AgCQDs, and it can be observed from fig. 1(h) that the prepared AgCQDs have a particle size of 15 to 22nm and an average diameter of about 19.5nm, and it can be clearly observed that the particle size is larger than that of carbon quantum dots, which may be caused by silver nano-particle growth on the surface of the AgCQDs. It is observed from the TEM image of FIG. 1(c) that PS are nanoscale spherical particles with an average diameter of about 110 nm. Furthermore, UV-vis absorption spectra were used to characterize the change before and after irradiation of Fc-COOH, as shown in FIG. 1(e), which shows absorption bands at 217,252,309 and 446nm (curve 1) before exposure (curve 1) due to aromatic rings (217 and 252nm) and metallocycle charge transfer (309 and 446nm) in Fc-COOH itself. After exposure to sunlight for 2 hours, three absorption peaks are observed at curve 2 of FIG. 1(e), two aromatic rings (215 and 272nm) and one metal ring charge transfer (353nm), respectively, indicating that the precipitate formed contains ferrocene units. Meanwhile, the absorption peak of the charge transfer (446nm) of the metal ring disappears due to photodecomposition of Fc-COOH. As can be observed from the TEM image of fig. 1(d), the PS is surrounded by AgCQDs, revealing the successful synthesis of AgCQDs @ PS composites. FIG. 1(f) is a Zeta potential value of AgCQDs @ PS from which it can be observed that the Zeta potential value of PS (curve 1) is-16.33 mV due to the electronegativity of its-COOH moiety, and that of PS + PEI (curve 2) is +1.48mV when the PS is modified with PEI, the change in the Zeta potential value indicates that PS successfully binds positively charged PEI due to the large number of positively charged amino groups on the PEI surface that can electrostatically adsorb negatively charged PS. It was also observed from the figure that the zeta potential value of the AgCQDs (curve 3) composite was-13.7 mV, and when PEI modified PS was bound to the AgCQDs composite, it was observed that the zeta potential became-10.64 mV, respectively. Thus, the success of the AgCQDs @ PS (curve 4) nanocomposite preparation was further demonstrated by Zeta potential measurements on several nanomaterials.

In order to analyze the element composition of AgCQDs and AgCQDs @ PS, EDS characterization was performed on the AgCQDs and AgCQDs @ PS, respectively, and the results are shown in FIG. 2(a) and FIG. 2(b), respectively. As shown in FIG. 2(a), AgCQDs comprise four elements of 5.25% C, 18.69% O, 13.17% Na and 62.89% Ag, and the successful synthesis of AgCQDs composite material is proved. FIG. 2(b) is an EDS characterization diagram of AgCQDs @ PS, from which it can be observed that AgCQDs @ PS comprises six elements of 8.61% of C, 4.26% of N, 29.49% of O, 24.64% of Na, 32.16% of Ag and 0.88% of Fe, which proves that the AgCQDs @ PS composite material is successfully synthesized.

FIG. 3 is an XRD spectrum of CQDs, AgCQDs, GO, rGO @ Ag. As can be observed from fig. 3(a), CQDs have a broad diffraction peak at 23.45 ° 2 θ, which corresponds to the amorphous carbon crystal plane of (002), indicating a lattice spacing of 0.38 nm. While the (002) peak of AgCQDs is difficult to observe on the surface of the CQDs when AgNPs grow, it is possible that AgNPs grow in the CQDs interlayer to separate, resulting in the increase of the interlayer spacing of the CQDs and the broadening of the (002) diffraction peak. (002) The peak shift to 22.1 ° also confirms the above conclusion, while also verifying that other nanoparticles are modified at the surface carbon points, whose typical peak will be diminished. In addition, the crystalline characteristics and diffraction peaks of characteristic bands of silver nanoparticles were observed at 2 θ of 38.06 ° (111),44.27 ° (200),64.38 ° (220) and 77.40 ° (311), respectively. This indicates that silver nanoparticles are grown on the surface of CQDs. From fig. 3(b) it can be observed that GO shows a graphite diffraction peak at 11.5 ° (fig. 3(b)), corresponding to an interlayer d-spacing of 0.73 nm. The diffraction peak of the reduced graphene oxide appeared at 25.1 °, corresponding to the (002) crystal plane of graphene (fig. 3 (b)). Once metal (oxide) nanoparticles are introduced to the surface of rGO, their corresponding peaks are evident. The silver nanoparticles above rGO @ Ag showed peaks at 38.15 °, 44.33 °, 64.32 ° and 77.32 °, which could be assigned to (111), (200), (220) and (311) crystal planes (matching JCPDS 01-1174) (fig. 3(b)), indicating that the silver nanoparticles were successfully modified at the surface of rGO. This further demonstrates the successful synthesis of CQDs, AgCQDs, rGO @ Ag.

To understand the surface group profiles of CQDs and AgCQDs, they were characterized herein using Fourier transform Infrared Spectroscopy (FT-IR). The FTIR spectrum of CQDs is shown in FIG. 4(a), and it can be seen from the data in the figure that it is located at 3427cm^-1The broad peak at the position corresponds to the stretching vibration peak of O-H and N-H, 1641cm^-1Peak at corresponds to-NH₂Characteristic peak of (1), 1127cm^-1The peak at (a) corresponds to the stretching vibration peak of C-O. The infrared result shows that the CQDs surface contains O-H, -NH₂and-COOH functional groups. The metal ion is most likely bound to the carboxyl moiety by ion exchange or coordination reactions. The carbon quantum dots with reducibility can easily convert Ag⁺Reducing the Ag NPs to the surface of the carbon quantum dots. Compared with carbon quantum dots, the O-H absorption peak of AgCQDs in Fourier transform infrared spectroscopy is 1127cm^-1Disappearance of-NH₂Has an absorption peak of 1641cm^-1And disappear. Furthermore, at 1596cm^-1And 1385cm^-1The intensity of the absorption peak of the carbonyl compound is obviously enhanced, and the absorption peak is at 3427cm^-1The intensity of the O-H absorption peak is obviously reduced. This indicates that the O-H functionality is converted to a carboxyl functionality upon oxidation. XPS characterization of CQDs as shown in fig. 4(b), it can be seen from XPS survey that the peaks of synthesized CQDs at 291.0eV, 400.0eV and 536.0eV correspond to the characteristic peaks of C1s, N1s and O1s, respectively, indicating that they are mainly composed of C, N, O three elements. Fig. 4(C) is a high resolution plot of a C1s spectrum, containing 4 characteristic peaks, 284.4eV (C-C/C ═ C), 286.1eV (C-O), 288.1eV (C ═ N/C ═ O), 285.1eV (C-OH/C-N), respectively; FIG. 4(d) is an XPS spectrum of N1s with a peak at 399.5eV of C-N-C and a peak at 400.0eV of N- (C)₃The peak at 401.4eV is N-H; FIG. 4(e) is a high resolution O1s spectrum showing two peaks, 531.6eV (C-O)H/C-O-C), 532.9eV (-COOH). Through the analysis of FTIR spectrum and XPS characterization chart, it is fully proved that the surface of the synthesized carbon quantum dot is rich in-OH, -COOH and-NH₂Hydrophilic groups such as groups and the like enable the carbon quantum dots to have good water solubility. Meanwhile, the strong electron donating groups enable the CQDs to have certain electron donating capability, and the fluorescence of the CQDs is improved to a great extent. In addition, fig. 4(f) is an XPS spectrum of AgCQDs, and it can be seen from the XPS survey that peaks of synthesized AgCQDs at 284.1eV, 368.2eV, 406.5eV and 532.1eV correspond to characteristic peaks of C1s, Ag 3d, N1s and O1s, respectively. It is shown that AgCQDs contain elements of C, Ag, N and O. FIG. 4(f) also shows Ag 3d_3/2And Ag 3d_5/2The binding energies of (A) and (B) are respectively 373.8eV and 367.9eV, and the distance between the two energies is 6.0eV, which is consistent with the characteristic spectrum peak of the simple substance silver. Indicating the successful preparation of AgCQDs.

2.2 optical Properties of CQDs

The synthesized CQDs and AgCQDs were characterized using ultraviolet-visible spectroscopy. As shown in fig. 5(a), the uv-vis spectrum of CQDs has 1 distinct characteristic peak at 283nm, which may be caused by ii → ii transition of the electrons from the C ═ C functional group in the CQDs. In the ultraviolet-visible spectrogram of AgCQDs, a new absorption peak appears at 409nm, which corresponds to a characteristic peak caused by surface plasmon resonance of silver nanoparticles. FIG. 5(b) is a fluorescence spectrum of CQDs, and as shown in the figure, the optimal excitation wavelength of the synthesized carbon quantum dots is 340nm, and the optimal emission wavelength is 428 nm. The fluorescence spectrum of CQDs under different excitation wavelengths is shown in fig. 5(c), according to the report, the position and intensity of the fluorescence emission peak of CQDs are related to the excitation wavelength, the excitation wavelength changes, the emission peak changes, and the intensity also changes, when the excitation wavelength is increased from 300nm to 400nm, the intensity of the corresponding emission peak increases and then gradually decreases, which indicates that the emission wavelength of the synthesized carbon quantum dot is red-shifted, and the reason for this phenomenon may be that different emission sites are generated due to different forms or energy traps on the surface of the material, thereby causing the wavelength dependence of CQDs. With quinine sulfate (54%, 0.1 mol. L)^-1H₂SO₄) As a reference substance, the fluorescence quantum yield of CQDs was measuredThe rate was 17.3%.

The synthesized AgCQDs can be further confirmed electrochemically. Compared with the CQDs modified electrode without oxidation-reduction current peak in CV (figure 6), the AgCQDs modified electrode is 0.1 mol.L^-1The CV in PBS buffer (pH 7.4) showed a pair of typical silver nanoparticle redox peaks in the potential range of-0.1-0.5V (fig. 6), indicating the presence of silver nanoparticles on AgCQDs complexes.

2.3 electrochemical behavior of CQDs and AgCQDs

The electrochemical behavior of CQDs/GCE and AgCQDs/GCE was studied by CV. FIG. 7(a) shows the signal at 100mV · s^-1At a scanning rate of not less than 0.1 mol. L, respectively^-1K₂S₂O₈And has a molar ratio of 0.1 mol. L^-1K₂S₂O₈0.1 mol. L of^-1ECL Strength vs. potential curves for CQDs/GCE and AgCQDs/GCE in PBS buffer (pH 7.4) solution. In the scan range of 0 to-2.0V, in the absence of K₂S₂O₈No significant ECL emission was observed for both CQDs/GCE (curve 1) and AgCQDs/GCE (curve 2) as co-reactants, with the addition of 0.1 mol. L^-1K₂S₂O₈Thereafter, ECL emission at a peak potential of-1.99V occurred at CQDs/GCE with an initial potential of-1.56V (FIG. 7(b), curve 3). In addition, stronger ECL emission was obtained on AgCQDs/GCE at-1.90V with an initial potential of-0.56V (FIG. 7(b), curve 4). Meanwhile, the ECL strength of AgCQDs/GCE is 6.2 times higher than that of CQDs/GCE, positive shift of the onset voltage and ECL peak potential due to better conductivity of silver nanoparticles and more compact film formed by cross-linking between silver nanoparticles and CQDs. It is reasonable that the silver nanoparticles can act as a conductive bridge between the carbon quantum dots and the electrodes to increase the conductivity, thereby effectively improving ECL strength. The ECL emission mechanism is based on anionic quantum dot radicals (R)^·-) With co-reactant (SO)₄ ^·-) Due to electron transfer annihilation between the oxidized species. Corresponding AgCQDs/S₂O₈ ^2-The ECL process is as follows:

AgCQDs+e→AgCQDs^-· (1)

S₂O₈ ^2-+e^-→SO₄ ^2-+SO₄ ^-· (2)

AgCQDs^-·+SO₄ ^-·→AgCQDs*+SO₄ ^2- (3)

AgCQDs*→AgCQDs+hv (4)

in the method, multiple signal amplification ECL measurement is further researched, and K is used in the cyclic voltammetry scanning process of-2.0-0V₂S₂O₈(0.1mol·L^-1) As a coreactant, the coreactant was dissolved in PBS buffer (0.1 mol. L)^-1pH 7.4) the ECL behavior of the CQDs, AgCQDs and AgCQDs @ PS composites was measured and the results are shown in FIG. 8. As shown in FIG. 8(a), where Δ I is the relative ECL signal intensity of CQDs, the ECL intensity of AgCQDs nanocomposites is 6.2 times higher than that of CQDs. In order to obtain high sensitivity, an amino-functionalized PS with large specific surface area and excellent adsorption capacity was applied to load AgCQDs nanocomposites, building AgCQDs @ PS composites that excellently enhance ECL signal (fig. 8 (a)). The ECL strength of the composite was 7.3 times higher than CQDs. The reasons can be attributed to the following: (a) the AgCQDs have excellent conductivity, can greatly accelerate electron transfer and enhance ECL signals; (b) the passivation of silver to carbon quantum dots greatly changes the surface state of the carbon quantum dots, so that the electrochemical luminescence property of the carbon quantum dots is improved; (c) PS can provide a solid state structure to carry large amounts of AgCQDs, resulting in higher ECL signals. FIG. 8(b) is a graph of ECL signals from CQDs/GCE, AgCQDs/GCE and AgCQDs @ PS/GCE scanned continuously for 9 cycles between 0 and-2.0V, with no apparent change observed, indicating excellent stability of the ECL probe. These results indicate that the ultra-sensitive AgCQDs nanocomposite has potential application value in tumor marker analysis.

2.4 characterization of immunosensors

To confirm the successful step-by-step manufacturing process of the immunosensor, it was characterized by Cyclic Voltammogram (CV), and the detection results of CV are shown in fig. 9 (a). The peak current increased (curve 2) after immobilization of the rGO @ Ag material on a glassy carbon electrode compared to a bare electrode (curve 1) because rGO @ Ag has a large specific surface area and excellent conductivity. When HCG-Ab1, BSA and the antigen HCG were immobilized consecutively on rGO @ Ag/GCE, the redox peak current decreased consecutively (curves 3, 4 and 5) because poorly conducting biological proteins could severely impede the electron transfer rate. Finally, due to the specific immunoreaction between the antibody and the antigen, the successfully synthesized nano-composite HCG-Ab2-AgCQDs @ PS (curve 6) is crosslinked with the modified electrode, and the current peak value is increased because the signal probe AgCQDs @ PS has excellent conductivity.

EIS is an effective technique for studying the surface characteristics of surface-modified electrodes during the stepwise modification process. The impedance plot consists of a semicircular portion, which corresponds to the higher frequency electron transfer process, and a linear portion, which corresponds to the lower frequency electron diffusion process. The semi-circle diameter is equal to the electron transfer resistance Ret. FIG. 9(b) shows an impedance spectrum during the stepwise construction of the immunosensor. It can be observed from the figure that for bare GCE, Fe (CN)₆ ³/Fe(CN)₆ ⁴The redox process of the probe showed a small Ret value (curve 1). After modification of the bare electrode with the rGO @ Ag composite, the resistance of the redox probe was significantly reduced (Curve 2), probably because the rGO @ Ag composite could produce a surface with increased conductivity, enhancing Fe (CN)₆ ³/Fe(CN)₆ ⁴The probe enters the modified rGO @ Ag layer, and the result proves that the immobilized silver nanoparticles really enhance the electron transfer on the rGO sheet. While continuing to incubate HCG-Ab1, BSA, HCG (10 mIU. mL)^-1) Thereafter, the impedance gradually increased significantly (curves 3-5) due to the production of a non-conductive layer of protein preventing the transport of electrons at the electrode interface. And after AgCQDs @ PS-HCG-Ab2 is dripped, impedance is reduced (curve 6), because the electroactive polymer nanosphere (PS) is synthesized by infinite coordination polymer of ferrocene dicarboxylic acid, PS not only improves the antibody loading capacity, but also is rich in ferrocene units to accelerate electron transfer at an electrode interface, and in addition, because silver nanoparticles have conductivity, Fe (CN) can be promoted₆ ³/Fe(CN)₆ ⁴The electron transfer on the surface of the electrode shows that AgCQDs @ PS has excellent electron transfer performance. Circulation ofThe consistent change of the voltammogram and the EIS chart indicates that the layer-by-layer self-assembly process of the immunosensor is successful.

2.5 optimization of the Experimental conditions (1 mIU. mL)^-1)

To obtain good assay performance, experimental parameters were optimized, including pH of the detection solution, volume of blocking reagent, rGO @ Ag concentration, HCG-Ab1 concentration, HCG-Ab2 volume, AgCQD @ PS concentration, incubation time between antibody and antigen, and incubation temperature.

Since the activity of biomolecules is pH-dependent, we investigated the effect of this factor on the immunosensor response. This experiment examined the dependence in the pH range of 5.5 to 8.5. As can be seen from fig. 10(a), the ECL intensity of the immunosensor increased from 5.5 to 7.4 with pH, and then decreased in the range of 7.4 to 8.5. This may be due to the fact that in an excessively acidic or basic environment, in particular in an alkaline environment, proteins denature and lose their effectiveness for immobilization on the nanomaterial. Therefore, the optimal pH of 7.4 was selected for further experiments.

Nonspecific adsorption interferes with the accuracy of the immune reaction, and typically Bovine Serum Albumin (BSA) is used to block the nonspecific adsorption sites prior to the immune reaction. 1% BSA was chosen as blocking reagent in this experiment. The effect of blocking the volume of BSA on nonspecific adsorption was studied (fig. 10 (b)). When the amount of BSA was insufficient to block non-specific adsorption of the AgCQDs @ PS-Ab2-HCG antibody conjugate, the ECL intensity increased with increasing BSA volume, and then remained constant when the BSA volume was 10 to 100 μ L. It was found that blocking could be accomplished at a volume of 50 μ L. Therefore 50 μ L BSA was chosen for use in subsequent experiments.

The effect of the concentration of rGO @ Ag of the immobilized primary antibody on the immunosensor is also important. FIG. 10(c) shows that the @ Ag concentration increases from 0.2 to 1.0 mg-mL with rGO @^-1The ECL strength increased and then began to remain unchanged, probably due to the saturation of rGO @ Ag on the electrode. Therefore, 1.0 mg/mL was used^-1The rGO @ Ag of (1) constructs an immunosensor.

The amount of HCG-Ab1 immobilized on the sensor platform had a large effect on ECL signal. Thus, study Ab 1. The results are shown in FIG. 10 (d). With antibody concentration from 20. mu.g.mL^-1Increased to 100. mu.g.mL^-1ECL intensity gradually increased when Ab1 concentration was greater than 100. mu.g.mL^-1In this case, since ECL reaction hardly changed, Ab1 was selected at an optimum culture concentration of 100. mu.g/mL^-1。

The concentration of AgCQDs @ PS is an important parameter for the performance of the immunosensor. Higher or lower concentrations of AgCQDs @ PS affect the electron transfer capacity and immobilization of the antibody. In order to obtain the best performance of the immunosensor, the effect of using a range of concentrations of AgCQDs @ PS on the immunosensor was investigated. As shown in FIG. 10(e), the concentration of AgCQDs @ PS was varied from 0.5 mg.mL^-1Increased to 2.0 mg. multidot.mL^-1The electrochemical response increased significantly, then with concentration from 2.0mg · mL^-1Increased to 3.0 mg. multidot.mL^-1And decreases. Therefore, the optimum AgCQDs @ PS concentration in this study was 2.0 mg/mL^-1。

For sandwich immunosensors, the amount of HCG-Ab2 on the label had a significant effect on ECL signaling. Therefore, a concentration of 100. mu.g/mL was examined^-1The effect of HCG-Ab2 volumes from 20 to 200. mu.L on ECL intensity. As shown in fig. 10(f), ECL intensity increased as the volume increased from 20 to 100 μ L. Above 100 μ L, the ECL response levels off due to saturation of HCG-Ab2 on the AgCQDs @ PS surface. Therefore, 100 μ L was selected as the optimal volume for HCG-Ab 2.

The effect of incubation temperature is very important for the activity of the antibody and antigen. In order to obtain the best ECL signal, the effect of incubation time on ECL was studied, and during the incubation, when the antigen reached Els electrode surface antibody, it took time for the contacting species to form immune complex, and as a result, as shown in fig. 10(g), the ECL signal gradually increased with increasing incubation time, and then tended to stabilize over 60 minutes, indicating that the binding capacity of the antigen on the sensor gradually tended to saturate. Therefore, an incubation time of 60 minutes was chosen for the subsequent experiments. In addition, the incubation temperature of HCG was studied, ranging from 10 ℃ to 60 ℃. It can be observed from fig. 10(h) that the electrochemiluminescence intensity gradually increases as the temperature increases from 10 ℃ to 37 ℃, and the luminescence intensity starts to decrease as the temperature exceeds 37 ℃. Therefore, the incubation temperature of the sandwich-type immune device was selected to be 37 ℃.

2.6 immunosensor detection of HCG

Under optimized experimental conditions, the ECL immunosensor was designed to detect the concentration of HCG. As shown in FIG. 11(a), the logarithmic value of the ECL intensity with HCG concentration was 0.001 mIU. multidot.mL^-1To 500.0 mIU.mL^-1The linear regression equation for y 9138+2436lg (C) increases linearly within the range as shown in fig. 11(b)_HCG) The correlation coefficient was 0.9971, and the detection limit of HCG was 0.00033 mIU. mL^-1(S/N-3). Results the proposed ECL immunosensor can be used for quantitative detection of HCG antigens. In addition, as shown in table 1, the sandwich type immunosensor of the present invention has lower detection limit and wider linear range than the reported method for detecting HCG.

Table 1 comparison of HCG measurements by different methods.

The individual documents in the table are illustrated below:

[1]Wen G,Liang X,Liu Q,Liang A,Jiang Z.A novel nanocatalytic SERS detection of trace human chorionic gonadotropin using labeled-free Vitoria blue 4R as molecular probe[J].Biosensors and Bioelectronics,2016,85,450-456.

[2]Xia N,Chen Z,Liu Y,Ren H,Liu L.Peptide aptamer-based biosensor for the detection of human chorionic gonadotropin by converting silver nanoparticles-based colorimetric assay into sensitive electrochemical analysis[J].Sensors and Actuators B:Chemical,2017,243,784-791.

[3]Roushani M,Valipour A.Voltammetric immunosensor for human chorionic gonadotropin using a glassy carbon electrode modified with silver nanoparticles and a nanocomposite composed of graphene,chitosan and ionic liquid,and using riboflavin as a redox probe[J].Microchimica Acta,2016,183(2),845-853.

[4]Lei J Q,Jing T,Zhou T T,Zhou Y S,Wu W,Mei S R,Zhou Y K.A simple and sensitive immunoassay for the determination of human chorionic gonadotropin by graphene-based chemiluminescence resonance energy transfer[J].Biosensors and Bioelectronics,2014,54,72-77.

[5]Tao M,Li XF,Wu ZS,Wang M,Mei H,Yang YH.The preparation of label-free electrochemical immunosensor based on the Pt–Au alloy nanotube array for detection of human chorionic gonadotrophin[J].Clinica Chimica Acta,2011,412(7-8),550-555.

2.7 stability, reproducibility and specificity Studies of immunosensors

In the presence of 0.1 mol.L^-1K₂S₂O₈0.1 mol. L of^-1The stability of the proposed biosensors with different concentrations was evaluated by continuous cycle scanning in PBS buffer (pH 7.4) solution. As shown in fig. 12(a), as the concentration of HCG increases, the ECL intensity increases and the scan curve is relatively stable at three consecutive cycles of each concentration. As shown in fig. 12(b), the ECL signal of the immunosensor for HCG detection decreased by 7.3% when the immunosensor was stored at 4 ℃ for 30 days. In summary, Els was constructed with acceptable stability and reproducibility.

A key characteristic parameter for assessing the performance of an immunosensor assay is selectivity. In order to investigate the selectivity of the proposed ECL sensor, some other types of tumor markers were also used as interfering proteins. As shown in FIG. 12(c), CEA (10 ng. mL) was included when each immunosensor was modified with a non-specific bioprotein^-1)，AFP(10ng·mL^-1)，BSA(20mg·mL^-1) And CA15-3 (10U. mL)^-1) There was no significant increase in ECL strength compared to the blank solution. However, the ECL signal obtained from the mixture-incubated immunosensor was only approximately from 10mIU · mL^-1The intensity of HCG collection proves that the constructed immunosensor has good selectivity and specificity for HCG detection.

As shown in fig. 12(d), the reproducibility of the ECL immunosensor was evaluated by measuring three different concentrations of HCG antigen using four electrodes under the same conditions. The RSD of the prepared ECL immunosensor is respectively 2.2%, 2.7% and 1.5%, which shows that the ECL immunosensor prepared by the invention has excellent reproducibility.

2.8 sample analysis

Under the optimal experimental conditions, the prepared immunosensor has good reliability in clinical tests. We determined HCG concentrations in serum samples collected from the fifth national hospital of Guilin. And (3) detecting the recovery rate of HCG in human serum by adopting a standard addition method on human serum samples. As shown in table 2, the relative standard deviation of HCG detection was between 1.3% and 2.6% and HCG recovery was between 97.2% and 102%.

Table 2 assay results and spiked recovery in human serum (n ═ 6)

3 conclusion

In this work, we ultrasensitively detected human chorionic gonadotropin by preparing AgCQDs composite materials for ECL sensor fabrication and a PS-assisted dual signal amplification strategy. The prepared rGO @ Ag not only has large surface area and excellent catalytic performance, but also can fix a large amount of primary antibodies. In addition, the finally synthesized AgCQDs @ PS-Ab2 can be captured by HCG antigen through a sandwich immune reaction between the antibody and the antigen, resulting in a large enhancement of ECL signal. Under the optimal experimental conditions, the constructed immunosensor can be 0.001-500 mIU.mL^-1The HCG antigen measured in the range has good stability, good reproducibility and good selectivity, and the detection limit is 0.00033 mIU.mL^-1The kit is successfully applied to the detection of human chorionic gonadotropin in human serum.

Claims

1. A method of assaying human chorionic gonadotropin comprising the steps of:

1) preparation of CQDs:

taking pawpaw peels as a carbon source, and synthesizing by adopting a hydrothermal method to obtain CQDs; the preparation method comprises the following steps:

placing papaya peel powder into water, carrying out ultrasonic dissolution, transferring into a hydrothermal reaction kettle for hydrothermal reaction, carrying out reaction for 10-12 h at 80-200 ℃, cooling after the reaction is finished, centrifuging a reactant, filtering supernate by using a microporous filter membrane, and dialyzing by using a dialysis bag with the molecular weight of 800-1000 Da to obtain a pure carbon quantum dot solution;

2) preparation of AgCQDs suspension:

CQDs is combined with Ag⁺Combining to obtain AgCQDs suspension;

3) preparing AgCQDs @ PS composite material:

4) preparing an AgCQDs @ PS/HCG-Ab2 composite material:

5) Preparing an rGO @ Ag composite material;

6) preparing a sandwich type immunosensor:

dripping the rGO @ Ag solution on the surface of the GCE, and airing to obtain rGO @ Ag/GCE; then, dripping HCG-Ab1 on the surface of the obtained rGO @ Ag/GCE, incubating, washing to remove unbound antibodies after incubation is finished, dripping BSA, incubating to block non-specific binding sites, and washing with PBS buffer solution to obtain BSA/HCG-Ab1/rGO @ Ag/GCE; then, dripping an HCG antigen solution on the surface of the obtained BSA/HCG-Ab1/rGO @ Ag/GCE and incubating to obtain HCG/BSA/HCG-Ab1/rGO @ Ag/GCE; dropwise adding AgCQDs @ PS/HCG-Ab2 solution on the surface of the obtained HCG/BSA/HCG-Ab1/rGO @ Ag/GCE and carrying out immunoreaction to obtain AgCQDs @ PS/HCG-Ab2/HCG/BSA/HCG-Ab1/rGO @ Ag/GCE, namely the sandwich type immunosensor;

7) and (3) detection:

the PBS buffer solution involved in the method is 0.1-0.12 mol.L^-1And a PBS buffer solution with pH of 7.3-7.5.

2. The method of assaying human chorionic gonadotropin according to claim 1 wherein: in the step 1), the preparation of the carbon quantum dots comprises the following steps: putting papaya peel powder into water, carrying out ultrasonic dissolution, transferring into a hydrothermal reaction kettle for hydrothermal reaction, cooling after the reaction is finished, centrifuging the reactant, filtering the supernatant through a microporous filter membrane, and dialyzing with a dialysis bag with the molecular weight of 800-1000 Da to obtain a pure carbon quantum dot solution.

3. The method of assaying human chorionic gonadotropin according to claim 1 wherein: in the step 3), the preparation of the AgCQDs @ PS composite material comprises the following steps: adding a PEI solution into a PS solution, performing ultrasonic treatment, centrifuging, collecting supernatant, adding AgCQDs suspension into the supernatant, performing ultrasonic treatment, centrifuging, collecting precipitate, and washing with water to obtain an AgCQDs @ PS composite material; wherein the content of the first and second substances,

the PS solution is formed by dispersing PS in a PBS buffer solution, wherein the concentration of the PS is 12-16 mg/mL^-1(ii) a The PBS buffer solution has a concentration of 0.1-0.12 mol.L^-1PBS buffer solution with pH value of 7.3-7.5;

the PEI solution is formed by dispersing PEI in water, wherein the concentration of PEI is 0.09-0.11 mg/mL^-1。

4. The method of assaying human chorionic gonadotropin according to claim 1 wherein: in the step 4), the concentration of the EDC solution is 100-110 mmol.L^-1。

5. The method of assaying human chorionic gonadotropin according to claim 1 wherein: in the step 4), the concentration of the NHS solution is 45-55 mmol.L^-1。

6. The method of assaying human chorionic gonadotropin according to claim 1 wherein: in the step 4), the AgCQDs @ PS solution is a solution obtained by dispersing the AgCQDs @ PS composite material solution in a PBS buffer solution, wherein the concentration of the AgCQDs @ PS composite material is 2.0 mg.mL^-1The PBS buffer solution is 0.1-0.12 mol/L^-1And a PBS buffer solution with pH of 7.3-7.5.

7. The method of assaying human chorionic gonadotropin according to claim 1 wherein: in the step 4), the concentration of HCG-Ab2 is 20 mu g/mL^-1。

8. The method of assaying human chorionic gonadotropin according to claim 1 wherein: in the step 6), the rGO @ Ag solution is a solution obtained by dispersing a rGO @ Ag composite material in water, wherein the concentration of the rGO @ Ag composite material is 1.0 mg/mL^-1。

9. The method of assaying human chorionic gonadotropin according to claim 1 wherein: in step 6), the concentration of HCG-Ab1 is 100. mu.g.mL^-1。

10. The method of assaying human chorionic gonadotropin according to claim 1 wherein: in the step 6), the concentration of the HCG antigen solution is more than or equal to 10 mIU.mL^-1(ii) a The AgCQDs @ PS/HCG-Ab2 solution is a solution obtained by dispersing AgCQDs @ PS/HCG-Ab2 composite material in PBS buffer solution, wherein the concentration of the AgCQDs @ PS/HCG-Ab2 composite material is 2.0 mg/mL^-1。