CN114354716A - Multilayer nano composite material, electrochemical immunosensor and preparation method thereof - Google Patents
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Abstract
The invention provides a CMK-3(Au/Fc @ MgAl-LDH) n multilayer nanocomposite, wherein n is more than or equal to 1, and the CMK-3 multilayer nanocomposite is composed of a CMK-3 and multilayer assembled AuNPs and Fc @ MgAl-LDH. The preparation method of the multilayer nanocomposite material comprises the following steps: preparing gold nanoparticles AuNPs; preparing Fc @ MgAl-LDH; preparing a CMK-3(Au/Fc @ MgAl-LDH) n multilayer nanocomposite material self-assembled layer by layer. The invention also provides an electrochemical immunosensor and a preparation method thereof, and the electrochemical immunosensor comprises a substrate electrode, a nano composite material modified on the substrate electrode and an antibody fixed on the nano composite material. The CMK-3(Au/Fc @ MgAl-LDH) n multilayer nano composite material prepared by the method has the excellent properties of strong adhesion, good film forming property, large specific surface area, strong loading capacity, strong conductivity, better electrochemical activity and the like.
Description
Technical Field
The invention belongs to the technical field of nanometer, particularly relates to a multilayer nanocomposite material, an electrochemical immunosensor and a preparation method thereof, and particularly relates to a CMK-3(Au/Fc @ MgAl-LDH) n multilayer nanocomposite material, an electrochemical immunosensor and a preparation method thereof.
Background
Ordered Mesoporous Carbon (OMCs) is a class of porous carbon nanomaterials with ordered structures. CMK-3 is an OMC nanomaterial with good conductivity, large specific surface area and high porosity.
Gold nanoparticles (AuNPs) are well known for their excellent conductivity properties, are often used to immobilize biomolecules, and can accelerate the electron transfer rate and thus enhance the conductivity of the system.
Ferrocenecarboxylic acid (Fc) has good electrochemical stability and higher redox signal, and is often used for electrochemical analysis.
Layered Double Hydroxides (LDHs) are two-dimensional planar nanomaterials formed by stacking hydroxides of two metals, and are mainly found as ionic clays. In recent years, LDHs are widely used for immobilization of nanomaterials, and LDH-functional nanomaterials can be synthesized by electrostatic adsorption. The LDH functional nano material has been widely applied to catalysis, optics and electrochemistry, and has high biocompatibility, a bidirectional structure, no toxicity and large specific surface area.
The double metal hydroxides are star products of the existing nano materials, and have the advantages of strong adhesion, good film forming property, strong stability, large specific surface area, functional modification and the like. In addition, they are also more readily available and less expensive; their disadvantages are however also very pronounced, for example: the double metal hydroxide has poor conductivity and smooth specific surface area, and is not easy to fix biological molecules.
The Fc @ MgAl-LDH nano material has the defects of poor conductivity and low Fc signal. In order to improve the disadvantages of the Fc @ MgAl-LDH nanomaterial, a new high-performance nanocomposite is needed, which has both the excellent properties of the double metal hydroxide and the excellent properties of other materials.
Disclosure of Invention
A first object of the present invention is to propose a multilayer nanocomposite.
The invention provides a multilayer nanocomposite CMK-3(Au/Fc @ MgAl-LDH)nWherein n is more than or equal to 1, and consists of AuNPs and Fc @ MgAl-LDH assembled by CMK-3 and n layers.
In the present invention, CMK-3(Au/Fc @ MgAl-LDH)nThe number of assembled layers of the multilayer nanocomposite was 4.
The second object of the present invention is to propose a process for the preparation of the above multilayer nanocomposite.
The preparation method is characterized in that magnesium-aluminum bimetal hydroxide (Fc @ MgAl-LDH) containing ferrocenecarboxylic acid, ordered mesoporous carbon (CMK-3) and nanogold (AuNPs) are used as materials, a layer-by-layer self-assembly technology is invented, and the positive-charge Fc @ MgAl-LDH, the positive-charge CMK-3 and the negative-charge AuNPs are subjected to layer-by-layer self-assembly through mutual attraction of positive charges and negative charges to form the CMK-3(Au/Fc @ MgAl-LDH) n multilayer nanocomposite. The method specifically comprises the following steps: firstly, dripping CMK-3 solution on a substrate electrode, then dripping AuNPs solution on the substrate electrode, dripping Fc @ MgAl-LDH solution on the substrate electrode, naturally drying, repeating the dripping operation of the AuNPs solution and the Fc @ MgAl-LDH solution, and superposing n layers to (Au/Fc @ MgAl-LDH)nTo obtain CMK-3(Au/Fc @ MgAl-LDH)nA nanocomposite assembled in multiple layers.
The preparation method of the AuNPs solution with negative charges comprises the following steps of dissolving chloroauric acid in boiling water solution, continuously stirring, dropwise adding citric acid into the solution under stirring and heating, changing the solution from light yellow to purple after fully stirring, cooling to room temperature, centrifuging for 30min at 10000r/min, fully washing precipitates with deionized water, and adding deionized water to obtain the AuNPs solution. The resulting solution was stored at 4 ℃ in the dark until use.
In the invention, the preparation method of the positively charged magnesium-aluminum bimetal hydroxide (Fc @ MgAl-LDH) solution containing ferrocenecarboxylic acid comprises the following steps of dissolving Fc into NaOH solution, and slowly dropwise adding Al (NO) under magnetic stirring3)3·9H2O and Mg (NO)3)2·6H2And adjusting the pH value of the solution O to 9-10, standing the obtained yellow precipitate for 24 hours, collecting the product, fully washing the product with deionized water, and drying the product in vacuum at 50 ℃, wherein the deionized water is introduced with nitrogen to remove oxygen.
The third purpose of the invention is to provide an electrochemical immunosensor. Comprises a substrate electrode, a nano composite material modified on the substrate electrode and an antibody fixed on the nano composite materialIs a multilayer nanocomposite CMK-3(Au/Fc @ MgAl-LDH) as claimed in claim 1nThe substrate electrode is a bare gold electrode or a glassy carbon electrode.
In the invention, the multilayer nanocomposite material is CMK-3(Au/Fc @ MgAl-LDH)4。
The fourth purpose of the present invention is to provide a method for preparing the electrochemical immunosensor, which comprises the following steps: dropping Glutaraldehyde (GA) on the surface of a substrate electrode modified by a CMK-3(Au/Fc @ MgAl-LDH) n multilayer nanocomposite material, incubating for 30 minutes at 37 ℃, then dropping an antibody on the surface of the substrate electrode for 12 hours at 4 ℃, washing with 0.01M PBS (phosphate buffered saline) solution with pH 7.0, then dropping 1% BSA (bovine serum albumin, M/V) on the substrate electrode, incubating for 30 minutes, washing with 0.01M PBS solution with pH 7.0, and incubating the substrate electrode for 40 minutes at 40 ℃ under different antigen concentrations to obtain the antibody.
The invention has the beneficial effects that:
the CMK-3(Au/Fc @ MgAl-LDH) n multilayer nanocomposite provided by the invention integrates the excellent performances of magnesium aluminum bimetal hydroxide containing ferrocenecarboxylic acid, ordered mesoporous carbon and nanogold. CMK-3(Au/Fc @ MgAl-LDH) of the present inventionnThe multilayer nanocomposite has a large number of carboxyl groups, and CMK-3(Au/Fc @ MgAl-LDH) is a hydrophilic groupnThe multi-layer nano composite material is more uniform in dispersion in aqueous solution and is not easy to agglomerate.
The preparation method of the nanocomposite provided by the invention constructs a CMK-3(Au/Fc @ MgAl-LDH) n multilayer nanocomposite by mutually attracting the CMK-3, AuNPs and Fc @ MgAl-LDH nanomaterials with excellent performance layer by layer through self-assembly of positive and negative electricity. The CMK-3(Au/Fc @ MgAl-LDH) n multilayer nanocomposite constructed by layer-by-layer self-assembly greatly improves the conductivity of the Fc @ MgAl-LDH nanocomposite. The method is low in cost, and the prepared CMK-3(Au/Fc @ MgAl-LDH) n multilayer nano composite material has the excellent properties of strong adhesion, good film forming property, large specific surface area, strong loading capacity, strong conductivity, good electrochemical activity and the like.
The invention carries out layer-by-layer assembly on the nano materials with opposite charges, thereby improving the defects of single nano material and amplifyingThe advantages of the nano material. The double metal hydroxide nano material is improved to prepare CMK-3(Au/Fc @ MgAl-LDH) with excellent performance of various nano materialsnA multilayer nanocomposite. The conductivity of the double-metal hydroxide is obviously improved, and the excellent properties of the CMK-3 and AuNPs nano materials are obtained. The unique multilayer structure of the MgAl-LDH composite material improves the load capacity, the adsorption capacity and the specific surface area of the MgAl-LDH. CMK-3(Au/Fc @ MgAl-LDH)nThe unique multilayer structure of the multilayer nano composite material endows the multilayer nano composite material with more binding sites, and is easy to modify and load functional groups and metal nano materials.
CMK-3(Au/Fc @ MgAl-LDH) of the inventionnThe biosensing system designed by the multilayer nano composite material has the advantages of simple steps, superior performance, practical value and easy batch production, integrates the excellent characteristics of various nano materials, and can obtain more excellent performance in the fields of biosensing, seawater desalination, biomedical treatment and the like.
Drawings
FIG. 1 is a diagram illustrating the design of an electrochemical immunosensor according to the present invention.
FIG. 2 SEM image of ferrocenecarboxylic acid-containing magnesium aluminum double hydroxide (Fc @ MgAl-LDH) prepared in example 1.
FIG. 3 is an SEM image of nanogold (AuNPs) prepared in example 1.
FIG. 4 FTIR plot of magnesium aluminum double hydroxide with ferrocenecarboxylic acid (Fc @ MgAl-LDH) prepared in example 1.
FIG. 5 XRD pattern of ferrocene carboxylic acid containing magnesium aluminum double hydroxide (Fc @ MgAl-LDH) prepared in example 1.
FIG. 6 is an XPS plot of the ferrocene carboxylic acid containing magnesium aluminum double hydroxide (Fc @ MgAl-LDH) prepared in example 1, wherein plot A is the XPS plot of Fc @ MgAl-LDH, plot B is the binding energy plot of Mg 1s, plot C is the binding energy plot of Al 2p, plot D is the binding energy plot of O1s, and plot E is the binding energy plot of Fe 2p 3/2, Fe 2p 1/2.
FIG. 7 Zeta potential diagrams for AuNPs, CMK-3 and Fc @ MgAl-LDH prepared in example 1, where Panel A is the Zeta potential diagram for AuNPs, Panel B is the Zeta potential diagram for CMK-3, and Panel C is the Zeta potential diagram for Fc @ MgAl-LDH.
FIG. 8 CMK-3(Au/Fc @ MgAl-LDH) in example 2nAnd optimizing the condition of the layer number (n) of the nano material.
FIG. 9 is an electrochemical impedance spectrum, a cyclic voltammogram and a differential pulse voltammogram of the electrochemical immunosensor in example 4, wherein panel A is an electrochemical impedance spectrum, panel B is a cyclic voltammogram, and panel C is a differential pulse voltammogram.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1 CMK-3(Au/Fc @ MgAl-LDH)nPreparation of multilayer nanocomposites
(1) Preparation of negatively charged gold nanoparticles (AuNPs): 20mg of chloroauric acid are dissolved in 100ml of boiling aqueous solution and stirred continuously, 4ml of 1% citric acid drops (M/V) are added to the solution with stirring and heating, and the solution changes from light yellow to purple with continuous stirring. And naturally cooling the reaction mixture to room temperature, centrifuging 5ml of nano gold solution at 10000r/min for 30min, washing the precipitate with deionized water for 2-3 times, and adding 5ml of deionized water to prepare the gold nano solution with negative charges. The resulting solution was stored at 4 ℃ in the dark until use.
(2) Preparing a positively charged magnesium aluminum double hydroxide (Fc @ MgAl-LDH) containing ferrocenecarboxylic acid: 1.09g Fc was weighed into 30mL 0.5M NaOH solution, and 15mL containing 0.127M Al (NO) was added under magnetic stirring3)3·9H2O and 0.317M Mg (NO)3)2·6H2And slowly dripping the solution of O into the solution, introducing nitrogen into deionized water required in the experimental process to remove oxygen, adjusting the pH to 9-10, standing the obtained yellow precipitate for 24 hours, collecting the product, fully washing the product with the deionized water for several times, and drying the product in vacuum at 50 ℃ to obtain the positively charged magnesium-aluminum bimetal hydroxide containing the ferrocenecarboxylic acid.
(3) Preparing a CMK-3(Au/Fc @ MgAl-LDH) n multilayer nanocomposite material with self-assembly layer by layer: firstly, 3 mu L CMK-3(1mg/ml) is dripped on a clean glassy carbon batteryAnd (3) coating 2.5 mu L of AuNPs obtained in the step (1) on the electrode in a dropwise manner, and coating 3 mu L of Fc @ MgAl-LDH (1mg/ml) obtained in the step (2) on the electrode in a dropwise manner, wherein the dropwise added materials are all naturally dried. The dropping operation of AuNPs and Fc @ MgAl-LDH was repeated 4 times, and 4 layers (Au/Fc @ MgAl-LDH) were superimposed to form CMK-3(Au/Fc @ MgAl-LDH)4An assembled nanocomposite.
FIG. 2 is an SEM image of ferrocene carboxylic acid containing magnesium aluminum double hydroxide (Fc @ MgAl-LDH), and it can be seen that the platelet-like structure is approximately 50nm in diameter size close to the scale.
Fig. 3 is an SEM image of nano gold (AuNPs), and it can be seen that the result is a spheroidal shape, which is a typical structure of nano gold.
FIG. 4 is an FTIR plot of ferrocene carboxylic acid containing magnesium aluminum double hydroxide (Fc @ MgAl-LDH) and the structural composition of the Fc @ MgAl-LDH material can be seen.
FIG. 5 is an XRD pattern of ferrocene carboxylic acid containing magnesium aluminum double hydroxide (Fc @ MgAl-LDH), and the crystal structure of this material can be seen.
FIG. 6 is an XPS plot of ferrocene carboxylic acid containing magnesium aluminum double hydroxide (Fc @ MgAl-LDH) and the elemental composition of the material can be seen.
FIG. 7 is a Zeta potential diagram of AuNPs, CMK-3 and Fc @ MgAl-LDH, which shows that AuNPs are negatively charged and CMK-3 and Fc @ MgAl-LDH are positively charged.
Example 2 CMK-3(Au/Fc @ MgAl-LDH)nOptimization experiment of multilayer nanocomposite n
Other conditions were identical to those of example 1, and only in step (3) was the dropping operations of AuNPs and Fc @ MgAl-LDH repeated 1, 2, 3, 5, 6, 7, 8 times, respectively, with 1, 2, 3, 5, 6, 7, 8 layers (Au/Fc @ MgAl-LDH) being superimposed to form CMK-3(Au/Fc @ MgAl-LDH)1、CMK-3(Au/Fc@MgAl-LDH)2、CMK-3(Au/Fc@MgAl-LDH)3、CMK-3(Au/Fc@MgAl-LDH)5、CMK-3(Au/Fc@MgAl-LDH)6、CMK-3(Au/Fc@MgAl-LDH)7、CMK-3(Au/Fc@MgAl-LDH)8Assembled nanocomposite, and CMK-3(Au/Fc @ MgAl-LDH) prepared in example 14The number of layers n is optimized by Differential Pulse Voltammetry (DPV) testing.
FIG. 8 is CMK-3(Au/Fc @ MgAl-LDH)nAnd (3) a condition optimization diagram of the number (n) of the layers of the nano materials. The 4-layer nanocomposite of example 1 was found to be optimal by Differential Pulse Voltammetry (DPV) testing.
Example 3 CMK-3(Au/Fc @ MgAl-LDH)nPreparation of multilayer nanocomposites
Other conditions were the same as in example 1, and only the electrode in step (3) was replaced with a bare gold electrode for a glassy carbon electrode.
Example 4 based on CMK-3(Au/Fc @ MgAl-LDH)nElectrochemical immunosensor of multi-layered nanocomposite for detection of cancer antigen 125(CA 125)
Preparing an electrochemical immunosensor: description of the design of the electrochemical immunosensor As shown in FIG. 1, first, 3. mu.L of 1% glutaraldehyde (V/V) was dropped to CMK-3(Au/Fc @ MgAl-LDH) in example 14Modifying the surface of the electrode by 4 layers of nano composite materials, incubating for 30min at 37 ℃, and connecting carboxyl (-COOH) of Fc and amino (-NH) of antibody2) Form GCE-CMK-3(Au/Fc @ MgAl-LDH)4. Then, 10. mu.L of CA 125 antibody (Ab) (1mg/ml) was dropped onto the surface of the modified electrode at 4 ℃ for 12 hours to immobilize the antibody molecule, and the unbound antibody was washed away with 0.01M PBS solution (pH 7.0) to form GCE-CMK-3(Au/Fc @ MgAl-LDH)4-Ab. Subsequently, 5. mu.L of 1% BSA (M/V) was dropped onto the modified electrode and incubated for 30min to block non-specific sites, and unbound BSA was washed with 0.01M PBS (pH 7.0) to form GCE-CMK-3(Au/Fc @ MgAl-LDH)4Ab-BSA. Finally, the modified electrode and CA 125 antigen (Ag) (the antigen concentration is in the range of 0.01-1000 U.ml)-1) Incubation at 40 ℃ for 40 min to form an unlabeled electrochemical immunosensor (GCE-CMK-3(Au/Fc @ MgAl-LDH)4-Ab-BSA-Ag)。
Preparation of electrodes according to the above procedure the electrodes were incubated in 100U/ml CA 125 antigen (Ag) at 40 ℃ for 40 minutes to form an unlabeled electrochemical immunosensor (GCE-CMK-3(Au/Fc @ MgAl-LDH)4Ab-BSA-Ag), as shown in FIG. 9, and FIG. 9 is an electrochemical impedance spectrum, a cyclic voltammogram and a differential pulse voltammogram of the electrochemical immunosensor, from which the assembly process of the electrochemical immunosensor and the principle design of the sensor can be seen. It is composed ofWherein, the graph A is an electrochemical impedance spectrogram, a represents GCE, and b represents GCE-CMK-3(Au/Fc @ MgAl-LDH)4And c represents GCE-CMK-3(Au/Fc @ MgAl-LDH)4Ab, d represents GCE-CMK-3(Au/Fc @ MgAl-LDH)4Ab-BSA, e represents GCE-CMK-3(Au/Fc @ MgAl-LDH)4-Ab-BSA-Ag; panel B is a cyclic voltammogram, a representing GCE, B representing GCE-CMK-3(Au/Fc @ MgAl-LDH)4And c represents GCE-CMK-3(Au/Fc @ MgAl-LDH)4Ab, d represents GCE-CMK-3(Au/Fc @ MgAl-LDH)4Ab-BSA, e represents GCE-CMK-3(Au/Fc @ MgAl-LDH)4-Ab-BSA-Ag; FIG. C is a differential pulse voltammogram, a representing GCE, b representing GCE-CMK-3(Au/Fc @ MgAl-LDH)4And c represents GCE-CMK-3(Au/Fc @ MgAl-LDH)4Ab-BSA-Ag. From fig. 9, it can be concluded that: electrochemical immunosensors have been successfully prepared.
Claims (9)
1. A multilayer nanocomposite, wherein the multilayer nanocomposite is CMK-3(Au/Fc @ MgAl-LDH)nWherein n is more than or equal to 1, and consists of AuNPs and Fc @ MgAl-LDH assembled by CMK-3 and n layers.
2. The multilayer nanocomposite of claim 1, wherein n is equal to 4.
3. A process for the preparation of the multilayer nanocomposite material according to claim 1, comprising the steps of: firstly, dripping CMK-3 solution on a substrate electrode, then dripping AuNPs solution on the substrate electrode, dripping Fc @ MgAl-LDH solution on the substrate electrode, naturally drying, repeating the dripping operation of the AuNPs solution and the Fc @ MgAl-LDH solution, and superposing n layers to (Au/Fc @ MgAl-LDH)nAnd (5) obtaining the product.
4. The method for preparing the multilayer nanocomposite as claimed in claim 3, wherein the AuNPs solution is prepared by dissolving chloroauric acid in boiling aqueous solution, continuously stirring, dropwise adding citric acid under stirring and heating, cooling to room temperature after sufficient stirring, centrifuging at 10000r/min for 30min, sufficiently washing precipitate with deionized water, and adding deionized water to obtain the AuNPs solution.
5. The method of claim 3, wherein the Fc @ MgAl-LDH solution is prepared by dissolving Fc in NaOH solution, and adding Al (NO) dropwise under magnetic stirring3)3·9H2O and Mg (NO)3)2·6H2And adjusting the pH value of the O solution to 9-10, standing for 24 hours, fully washing with deionized water, and drying in vacuum at 50 ℃, wherein nitrogen is introduced into the deionized water to remove oxygen.
6. An electrochemical immunosensor comprising a substrate electrode, a multi-layered nanocomposite decorated on the substrate electrode, and an antibody immobilized on the multi-layered nanocomposite, wherein the multi-layered nanocomposite is the multi-layered nanocomposite CMK-3(Au/Fc @ MgAl-LDH) of claim 1nThe substrate electrode is a bare gold electrode or a glassy carbon electrode.
7. The electrochemical immunosensor of claim 6, wherein the multi-layered nanocomposite material is CMK-3(Au/Fc @ MgAl-LDH)4。
8. The method for preparing an electrochemical immunosensor according to claim 6, comprising the steps of: glutaraldehyde was added dropwise to the solution in a solvent of CMK-3(Au/Fc @ MgAl-LDH)nAnd incubating the surface of the substrate electrode modified by the multilayer nano composite material at 37 ℃ for 30 minutes, dripping the antibody on the surface of the substrate electrode, standing for 12 hours at 4 ℃, washing with 0.01M PBS (phosphate buffer solution) with pH 7.0, dripping BSA (bovine serum albumin) with the mass volume ratio of 1% on the substrate electrode, incubating for 30 minutes, washing with 0.01M PBS with pH 7.0, and incubating the substrate electrode and the antigen at 40 ℃ for 40 minutes to obtain the electrochemical immunosensor.
9. The method for preparing an electrochemical immunosensor according to claim 8,wherein the antibody is CA 125 antibody, the antigen is CA 125 antigen, and the concentration range of the antigen is 0.01 U.ml-1～1000U·ml-1。
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| CN103018303A (en) * | 2012-12-29 | 2013-04-03 | 国家烟草质量监督检验中心 | Preparation method of nickel aluminum stratiform dual-metal hydroxide modified electrode |
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| CN112986359A (en) * | 2021-02-09 | 2021-06-18 | 天津大学 | Based on CuBTC @ MoS2-AuNPs modified electrode and CA125 detection method |
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| CN103018303A (en) * | 2012-12-29 | 2013-04-03 | 国家烟草质量监督检验中心 | Preparation method of nickel aluminum stratiform dual-metal hydroxide modified electrode |
| CN111458387A (en) * | 2020-05-27 | 2020-07-28 | 青岛科技大学 | Preparation method of GSH @ AgNCs/MgAl-E L DH modified electrode |
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