CN110146699B

CN110146699B - Bi-component ratio type electrochemical immunosensor and preparation method thereof

Info

Publication number: CN110146699B
Application number: CN201910473262.2A
Authority: CN
Inventors: 张宏芳; 韩秀娟
Original assignee: Northwestern University
Current assignee: Northwestern University
Priority date: 2019-05-31
Filing date: 2019-05-31
Publication date: 2020-09-15
Anticipated expiration: 2039-05-31
Also published as: CN110146699A

Abstract

The invention discloses a bi-component ratio type electrochemical immunosensor and a preparation method thereof, and the bi-component ratio type electrochemical immunosensor comprises the construction of a sensing interface and a soft core-hard shell type colloid body, wherein the sensing interface is prepared by modifying N, S co-doping graphene on the surface of a matrix electrode; the soft core-hard shell type colloid is an MB @ PbS colloid and an AQ @ CdS colloid; the two-component ratio type analysis method comprises the following steps: and (3) carrying out quantitative analysis on peak current obtained by scanning with differential pulse voltammetry by taking acetic acid-sodium acetate buffer solution as supporting electrolyte solution, wherein the ratio of oxidation current of MB and Pb is used for HE4 analysis, and the ratio of oxidation current of AQ and Cd is used for CA125 analysis. The bi-component ratio type electrochemical immunosensor can be used for simultaneously measuring the human epididymis protein 4 and the carbohydrate antigen 125.

Description

Bi-component ratio type electrochemical immunosensor and preparation method thereof

Technical Field

The invention belongs to the technical field of electrochemical analysis, relates to a bi-component ratio type electrochemical immunosensor and a preparation method thereof, and particularly relates to a bi-component ratio type electrochemical sensor based on metal sulfide colloid marking.

Background

The electrochemical immunosensing analysis combines the antigen-antibody specific reaction with the high sensitivity of the electrochemical analysis method, and is an important method for analyzing immune related substances of a complex sample system. In recent years, it has been widely used and studied in the fields of clinical diagnosis, food analysis, and the like. These applications often require the detection of the content of various components in a sample. In environmental monitoring, concentration indexes of various pesticides, herbicides and the like in a water body are often required to be monitored simultaneously so as to comprehensively evaluate the environmental pollution level. In clinical diagnosis, although the types of disease markers that have been found are numerous, there is little if any actual clinical diagnosis stage. The medical experts think that the combined detection of several tumor markers is of great help to improve the diagnosis accuracy in clinical tumor diagnosis and to popularize the application of the tumor markers in clinical diagnosis. For example, CA125 has been used for over 30 years in clinical diagnosis, treatment response assessment, prognosis evaluation, and recurrence monitoring of ovarian cancer, and is considered to be the "gold standard" for biomarker detection in ovarian cancer at present. However, CA125 has a low positive rate in stage I ovarian cancer, and has the disadvantages of poor tissue specificity and the like. HE4 is a newly discovered ovarian cancer marker, the specificity of which in ovarian cancer diagnosis is higher than that of CA125, but the application of the marker alone in ovarian cancer screening diagnosis still has great limitations such as false negative and the like. Thus, united states food and drug administration has approved 2011 to monitor disease progression and recurrence of ovarian cancer via combined CA125 and HE4 detection diagnostics. Enzyme-linked immunoassay generally used in clinic is mainly based on HRP labeling and enzyme-linked immunosorbent assay detection, and the sensitivity of the enzyme-linked immunosorbent assay is limited. The electrochemical immunosensor has the problems of large influence of a sample matrix and low accuracy although the detection sensitivity is relatively high. The patent utilizes two soft core-hard shell structure metal sulfide colloidal bodies of MB @ PbS and AQ @ CdS to construct a two-component ratio type electrochemical immunosensor for simultaneously detecting HE4 and CA 125.

Disclosure of Invention

The invention aims to solve the problem of high-sensitivity simultaneous detection of two disease markers, designs and prepares a dual-component ratio type electrochemical immunosensor marked by two soft core-hard shell structure metal sulfide colloids based on MB @ PbS and AQ @ CdS, and can be used for simultaneous determination of HE4 and CA 125.

In order to achieve the technical purpose, the invention is specifically realized by the following technical scheme:

the immunosensor comprises a sensing interface and an electrochemical marker, wherein the sensing interface is prepared by modifying N, S-rGO on the surface of a substrate electrode, and the electrochemical marker is a metal sulfide colloid.

The substrate electrode is a glassy carbon electrode, and the metal sulfide colloid body comprises MB @ PbS and AQ @ CdS colloid bodies with soft core-hard shell structures.

The soft core-hard shell colloid is prepared by wrapping MB and AQ in the cavity of PbS and CdS colloid by a reverse microemulsion method. Amino groups are introduced on the surfaces of the two colloids through hydrolysis of trimethoxy silane.

The immunosensor also comprises a reference electrode, a counter electrode and a supporting electrolyte solution, wherein the reference electrode is saturated calomel, the counter electrode is a platinum wire electrode, and the supporting electrolyte solution is an acetic acid-sodium acetate buffer solution.

In the immunosensor, the soft core-hard shell type colloid is used as a signal substance, and four electrochemical signals are utilized to realize the simultaneous and accurate determination of two antigens.

In another aspect of the present invention, a method for preparing the above two-component ratiometric electrochemical immunosensor is provided, in which N, S-doped graphene-chitosan dispersion is drop-coated on the surface of a polished substrate electrode, a first antibody of a first target analyte is cross-linked on the surface of the electrode to obtain a sensing interface, and an electrochemical marker is introduced into the sensing interface by using sandwich type immune complex formation.

The preparation method of the N, S-doped graphene-chitosan dispersion liquid is that N, S-doped graphene is ultrasonically dispersed in acetic acid solution of chitosan.

In another aspect of the invention, the electrochemical immunosensor takes acetic acid-sodium acetate buffer solution as supporting electrolyte solution, and peak current obtained by scanning through differential pulse stripping voltammetry is used for quantitative analysis.

Further, the oxidation current ratio of MB and Pb was used for the first component analysis, and the oxidation current ratio of AQ and Cd was used for the second component analysis; the first component is HE4 and the second component is CA 125.

The invention has the beneficial effects that:

the bi-component ratio type electrochemical immunosensor provided by the invention is constructed on the basis of two soft core-hard shell structure metal sulfide colloids MB @ PbS and AQ @ CdS. The core-shell structure colloid can simultaneously generate two electrochemical signal substances, the ratio of the two signals can be used as a quantitative signal, and the ratio type analysis can greatly reduce errors caused by uneven grain size of a nanometer material, passivation of the surface of an electrode, influence of a sensing interface on a sample matrix and the like. And the two colloids are combined to be used, so that a two-component ratio type electrochemical immunosensor for simultaneously detecting HE4 and CA125 can be constructed.

Drawings

FIG. 1 is a scanning electron microscope image of a PbS colloidal body according to the present invention;

FIG. 2 is a transmission electron micrograph of CdS NPs and CdS colloids according to the invention; wherein A is CdS NPs; b is CdS;

a in FIG. 3 is a scanning projection electron microscope image of the CdS colloid of the invention; b and C are respectively a surface scanning diagram of Cd element and S element;

FIG. 4 shows a working electrode of the present invention containing 10. mu. mol. L^-1MB, AQ and 1.0. mu. mol. L^-1Pb²⁺、Cd²⁺Differential pulse voltammogram in the supporting electrolyte;

FIG. 5 is a photograph of MB @ PbS colloids according to the present invention with the addition of ethanol at various times;

FIG. 6 is a graph of the UV-Vis spectra of AQ @ CdS colloids of the present invention with ethanol added over time;

FIG. 7 is a differential pulse voltammogram of the MB @ PbS and AQ @ CdS colloids of the present invention with respect to the controlled release of ethanol and nitric acid;

FIG. 8 is a differential pulse voltammogram of the electrochemical immunosensor of the present invention corresponding to the response of HE4 and CA 125.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The electrochemical immunosensor disclosed by the invention is prepared by the following method:

1) polishing the substrate electrode;

2) dropwise coating the N, S-rGO dispersion liquid on the surface of the polished substrate electrode;

3) crosslinking the antibody on the surface of the electrode to prepare a sensing interface;

4) forming a sandwich type immune complex on the surface of the electrode;

5) and (4) performing electrochemical detection.

Preferably, 0.05 μm Al is used in the step (1)₂O₃Polishing the substrate electrode with the powder, performing ultrasonic treatment in ultrapure water for 1min, and drying at room temperature.

Preferably, the preparation method of the N, S-rGO dispersion in the step (2) is:

1) synthesizing N, S-rGO in a graphene oxide aqueous dispersion by a one-pot hydrothermal method by using thiourea as a chemical doping agent;

2) 5.0mg of chitosan was dissolved in 1.0mL of 0.1 mol. L^-1Stirring in acetic acid solution at room temperature until a transparent solution is obtained;

3) and (3) adding the N, S-rGO (1.0mg) prepared in the step (1) into the transparent solution obtained in the step (2) and carrying out ultrasonic treatment until the solution is uniformly dispersed.

Preferably, the construction method of the immunosensing interface in the step (3) is as follows:

1) mu.L of 10. mu.g/mL^-1Ab_1-HE4And Ab_1-CA125The mixture was dropped onto the electrode and placed in a refrigerator at 4 ℃ overnight, and excess and weakly adsorbed antibodies were washed away with PBS and PBST;

2) mu.L of 1% BSA solution was dropped on the electrode surface of step (1) for reaction for 1h, and PBS and PBST were washed again and left at 4 ℃ until use.

Preferably, the step of forming the sandwich-type immune complex in the step (4) is:

1) incubation of different concentrations of HE4 and CA125 mixtures on immunosensory interfaces for 1h, PBS and PBST washes;

2) mu.L of MB @ PbS-Ab_2-HE4And AQ @ CdS-Ab_2-CA125And incubating the biological nano-composite and the obtained sensing interface for 45min at room temperature, washing by PBS, and carrying out electrochemical detection.

Preferably, the electrochemical detection in step (5) is:

1) immersing the electrode in 200 μ L ethanol for 10min, and then 200 μ L HNO₃Solution (1 mol. L)^-1) Neutralizing for 10min, mixing the obtained solution with HAc-NaAc buffer solution (0.1 mol. L)^-1pH5.0) are mixed;

2) and detecting by differential pulse anodic stripping voltammetry. The potential range is-1.2V to 0.2V, the deposition potential is-1.2V, the deposition time is 300s, the pulse amplitude is 50mV, the pulse width is 50ms, and the rest time is 2 s.

Example 1

1. Preparation of PbS and CdS nano-particles

The entire synthesis was carried out at room temperature and under argon atmosphere. Firstly, PbCl is added₂(0.28g) and 5.0mL Oleylamine (OLA) were added to a 50mL three-necked round bottom flask, the flask was sealed and degassed under vacuum for 5min, then heated to 90 ℃ with stirring and held at that temperature for 1h to form homogeneous PbCl₂-OLA dispersions. Meanwhile, sulfur (0.83mmol) was dissolved in 2.5mL OLA at room temperature, stirred for 30min, the resulting sulfur solution was added rapidly to the above dispersion, and the mixture was heated to 220 ℃ for 1h to obtain a black colloidal solution, which was PbS NPs. After cooling to room temperature, the PbS NPs was washed by adding ethanol (100mL), and centrifuged to obtain a solid product which was well dispersed in polar solvents such as heptane, toluene, etc.

By adopting the same preparation method, a reaction mixture with the molar ratio of Cd to S being 2:1 is used as a raw material to synthesize spherical CdS NPs. Specifically, an OLA dispersion containing 0.75mmol of sulfur was rapidly added to a solution containing 1.5mmol of CdCl at 160 deg.C₂-OLA dispersion and maintained at 160 ℃ for 1h, adding ethanol and centrifuging to obtain CdS NPs.

2. Preparation of MB @ PbS, AQ @ CdS

10mg of PbS NPs were added to 2mL of toluene and sonicated for 10 min. Subsequently, 400. mu.L of 10 mg. multidot.mL containing 4. mu.L of aqueous ammonia^-1MB, added to the PbS NPs dispersion (R)_o/w5) and then carrying out ultrasonic treatment for 1h to form Pickering emulsion, then standing for 1h, then adding 30 mu L of 3-aminopropyltrimethoxysilane into the emulsion, carrying out ultrasonic treatment for 1h, then carrying out standing reaction for 20h, centrifuging and washing with distilled water for three times to obtain the aqueous phase dispersion liquid of MB @ PbS. AQ @ CdS was also prepared in the same manner.

3. Preparation of biological nanocomposites

mu.L of MB @ PbS and 100. mu.L of AQ @ CdS were mixed with 100. mu.L of PBS buffer solution at pH7.4, respectively, and 4. mu.L of 1 mg/mL was added^-1Ab₂-HE4 and 4. mu.L 1 mg. multidot.mL^-1Ab₂-CA125, overnight at 4 ℃, followed by 20. mu.L of 1% BSA solution, shaking and standing for 1h, followed by centrifugation and washing with PBS buffer pH7.4 to give MB @ PbS-Ab₂-HE4 and AQ @ CdS-Ab₂CA125 biological nanocomplexes and disperse them in 0.1mL PBS buffer ph7.4 for further use.

4. Construction of immunosensing interface

First, 0.3 μm and 0.05 μm of Al were used for a glassy carbon electrode in this order₂O₃Polishing the surface with the powder, and adding anhydrous ethanol and distilled waterAnd (5) performing sound for 1min to remove substances adsorbed on the surface of the electrode. Subsequently, 6. mu.L of 1 mg. multidot.mL^-1The N, S-rGO dispersion was dropped onto the electrode and dried at room temperature. After the modified electrode was washed with water, 6. mu.L of 5% glutaraldehyde was added dropwise, reacted at 4 ℃ for 2.5 hours and washed with water three times. Then, 6. mu.L of 10. mu.g.mL^-1Ab₁-HE4 and Ab₁the-CA 125 mixture was dropped onto the electrode and placed in a refrigerator at 4 ℃ overnight, after which excess and weakly adsorbed antibody was washed away with PBS and PBST. Finally, 6 μ L of 1% BSA solution was dropped on the electrode surface for reaction for 1h to block non-specific sites, and PBS and PBST were washed again and left at 4 ℃ until use.

Example 2 characterization of metal sulfide nanoparticles and metal sulfide colloids

Fig. 1 is a scanning electron microscope image of PbS colloids, and it can be observed that PbS colloids are spherical nanoparticles having a size of about several tens to several hundreds nanometers. FIG. 2 is a transmission electron microscope image of the prepared CdS NPs and CdS colloids, and it can be seen from A in FIG. 2 that the CdS NPs prepared by the present invention have uniform particle size, are spherical with uniform particle size, and have an average particle size of about 10 nm. From B in fig. 2, it can be observed that CdS colloids are spherical nanoparticles with a size of about 100 nm. Amplifying a CdS colloid, it can be seen that it is a three-dimensional spherical structure assembled by single nanoparticles, which indicates that the colloid is assembled by a large number of nanoparticles. Fig. 3 a is a scanning transmission electron microscope image of CdS colloid, and fig. 3B and C are respectively a surface scanning image of Cd element and S element, and it can be seen that the two elements are uniformly distributed in the spherical structure of CdS colloid, indicating that the basic chemical composition of the colloid contains CdS.

Example 3

FIG. 4 shows the working electrode containing 10. mu. mol. L^-1MB, AQ and 1.0. mu. mol. L^-1Pb²⁺、Cd²⁺In a supporting electrolyte, in particular: the N, S-rGO/GCE modified electrode is subjected to differential pulse voltammetry on 10 mu mol.L in a pH4.5 HAc-NaAc buffer solution^-1MB, AQ and 1. mu. mol. L^-1Pb²⁺、Cd²⁺Simultaneous measurements were performed. MB, AQ, Pb²⁺And Cd²⁺Peaks of four substancesRespectively at-0.17V, -0.38V, -0.56V and-0.79V. The peaks of the four substances are not overlapped and the peak distance is proper, which preliminarily shows that the selected four signal substances can be used for the two-component ratio type electrochemical immunosensing analysis.

Example 4

The MB @ PbS and AQ @ CdS colloidal body provided by the invention takes a silicon film formed by a sol-gel method as a closed shell layer. The silica shell formed by the method contains a large number of small pores, and small molecules such as dye and the like are limited to a certain extent when passing through the small pores. The research finds that: ethanol can enhance the release of dye molecules in the silicon-coated colloid. This is probably because ethanol can improve the wettability of the pores of the silica gel to water. Therefore, in order to control the release of the electrochemical signaling substance of the prepared colloid, ethanol is first added to the colloid dispersion.

FIGS. 5 and 6 are photographs and UV-Vis spectra of the colloid MB @ PbS and AQ @ CdS, respectively, taken at different times with the addition of ethanol, as can be seen from FIG. 5, the colloid MB @ PbS is nearly colorless when ethanol is not added, indicating that MB is difficult to permeate from the cavity of the colloid MB @ PbS when ethanol is not added; after 10min of ethanol addition, the color of the solution turned blue, which is caused by MB released from the colloid; when ethanol was added for 30min, the blue color was slightly darker. Because the AQ color is not obvious, the ultraviolet-visible spectrum is adopted to research the controlled release of the ethanol to the AQ @ CdS colloid. As shown in FIG. 6, the intensity of the absorption peak of AQ at 256nm was significantly increased at 10min with the addition of ethanol, indicating that AQ was released from the colloid of AQ @ CdS, and the intensity of the absorption peak was not significantly increased at 30min, indicating that the dye molecules inside the colloid can be controlled to be substantially completely released within 10min by the addition of ethanol.

FIG. 7 is a differential pulse voltammogram of MB @ PbS and AQ @ CdS colloids versus ethanol and nitric acid controlled release. We examined the DPASV response of solutions obtained by destroying MB @ PbS and AQ @ CdS colloids with ethanol and nitric acid, and mixing them with HAc-NaAc buffer solution after 10 min. As shown in FIG. 7, when neither ethanol nor nitric acid was added, very weak Cd was observed on the voltammogram²⁺The dissolution peak and more obvious Pb²⁺May be due to colloidsCdS NPs and PbS NPs of the shell layer are slightly dissolved in a bottom solution HAc-NaAc buffer solution. When ethanol was added alone, significant oxidation peaks for MB and AQ were observed; when only nitric acid was added, significant Pb was observed²⁺And Cd²⁺The dissolution peak of (1). When ethanol and nitric acid are added in sequence, four signal substance responses appear on the obtained dissolution voltammogram at the same time, which shows that electrochemical signal substances of MB @ PbS and AQ @ CdS colloids can be released in a controlled manner through the combination of the ethanol and the nitric acid.

Example 5

FIG. 8 is a differential pulse voltammogram of an electrochemical immunosensor corresponding to the response of HE4 and CA 125. The DPASV response of the immunosensor with N, S-rGO/GCE as the matrix electrode and MB @ PbS, AQ @ CdS as the labels to the substances is examined. As shown in fig. 8, the response of the blank solution (curve 1) shows a weak Pb peak, which should be due to weak non-specific adsorption of MB @ PbS on the electrode surface. 1 U.mL^-1CA125 (Curve 2), it can be seen that in addition to the signal for Pb due to non-specific adsorption, significant AQ and Cd were produced²⁺Is generated by the release of the signal substance from AQ @ CdS labeled on the second antibody of CA 125. For 1 ng. mL^-1For HE4, the Pb signal increased significantly, while the oxidation peak of MB was generated (curve 3), indicating that the MB @ PbS colloids had been bound to the sensing interface via the formation of sandwich immunocomplexes. When the solution to be tested contains 1 ng.mL^-1HE4 and 1 U.mL^-1In CA125, it was observed that MB, AQ and Pb appeared simultaneously on the voltammetric response curve (curve 4) of the sensor²⁺And Cd²⁺Peaks for four signal species. When the amount is 100 ng.mL^-1HE4 and 100 U.mL^-1The peak currents for the four species increased significantly when CA125 (curve 5) was measured simultaneously, demonstrating that the peak currents for the four species correlate with the levels of HE4 and CA 125. Therefore, the two-component ratio type electrochemical immunosensing analysis can be carried out based on the colloidal bodies MB @ PbS and AQ @ CdS.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. The bi-component ratio type electrochemical immunosensor is characterized by comprising a sensing interface and an electrochemical marker, wherein the sensing interface is prepared by modifying N and S doped graphene on the surface of a substrate electrode, and the electrochemical marker is a metal sulfide colloid;

2. The two-component ratio-type electrochemical immunosensor according to claim 1, wherein the soft core-hard shell type colloid is prepared by encapsulating MB and AQ in the cavity of the PbS or CdS colloid by a reverse microemulsion method, and the surface of the colloid is introduced with amino groups by hydrolysis of trimethoxy silane.

3. The two-component ratiometric electrochemical immunosensor of claim 1, wherein the immunosensor further comprises a reference electrode, a counter electrode, and a supporting electrolyte solution, the reference electrode is saturated calomel, the counter electrode is a platinum wire electrode, and the supporting electrolyte solution is an acetate-sodium acetate buffer solution.

4. The method for preparing the electrochemical immunosensor according to claim 1, wherein the immunosensor comprises a sensing interface and an electrochemical marker, the sensing interface is prepared by modifying N and S-doped graphene on the surface of a substrate electrode, and the electrochemical marker is a metal sulfide colloid; the substrate electrode is a glassy carbon electrode, and the metal sulfide colloid body comprises MB @ PbS and AQ @ CdS colloid bodies with soft core-hard shell structures;

and dropwise coating the N, S-doped graphene-chitosan dispersion liquid on the surface of the polished substrate electrode, crosslinking a first antibody of a first target analyte on the surface of the electrode to obtain a sensing interface, and introducing an electrochemical marker into the sensing interface to form a sandwich type immune complex.

5. The preparation method according to claim 4, wherein the N, S-doped graphene-chitosan dispersion liquid is prepared by ultrasonically dispersing N, S-doped graphene in an acetic acid solution of chitosan.

6. An analytical method using a two-component rate type electrochemical immunosensor according to claim 1, wherein the two-component rate type electrochemical immunosensor is used for quantitative analysis by using an acetic acid-sodium acetate buffer solution as a supporting electrolyte solution and scanning a peak current obtained by differential pulse stripping voltammetry.

7. The analytical method according to claim 6, wherein the oxidation current ratio of MB to Pb is used for the first component analysis, and the oxidation current ratio of AQ to Cd is used for the second component analysis.

8. The assay of claim 7, wherein said first component is HE4 and said second component is CA 125.