CN112611789B - Biosensor based on lanthanum-doped zirconium-based MOF (Metal organic framework) derived composite material and preparation method thereof - Google Patents
Biosensor based on lanthanum-doped zirconium-based MOF (Metal organic framework) derived composite material and preparation method thereof Download PDFInfo
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Abstract
The invention provides a biosensor based on a lanthanum-doped zirconium-based MOF (metal organic framework) derivative composite material and a preparation method thereof 2 O 3 @ZrO 2 An IL layer, a glutaraldehyde layer, a neurofilament antibody layer, a bovine serum albumin layer and a neurofilament protein layer. The biosensor prepared by the method is based on the zirconium-based metal organic framework composite pyrolysis material. The biosensor prepared by the method has the characteristics of low detection limit, wide linear range and the like, and can be used for quickly detecting the neurofilament protein.
Description
Technical Field
The invention belongs to the field of electrochemistry, relates to a biosensor, and particularly relates to a biosensor based on a lanthanum-doped zirconium-based MOF (metal organic framework) derivative composite material and a preparation method thereof.
Background
Neurodegenerative diseases are caused by the loss of neurons or their myelin sheaths, which worsen over time and become dysfunctional. It can be divided into two symptoms of acute and chronic diseases, and the acute disease mainly includes Cerebral Ischemia (CI), brain Injury (BI), epilepsy, etc.; chronic disorders mainly include Alzheimer's Disease (AD), parkinson's Disease (PD), huntington's Disease (HD), amyotrophic Lateral Sclerosis (ALS), and the like. When neurodegenerative diseases occur in humans, the immune system carries out natural immune defenses against them, during which neurofilament proteins are released into the blood, so that immunoassays for neurofilament proteins in plasma can be used as indicators of axonal damage in neurological diseases, as markers for neurodegenerative diseases. The neurofilament NF-L is a useful marker for monitoring diseases such as amyotrophic lateral sclerosis, multiple sclerosis and Huntington's disease.
Zirconium-based MOF (Zr-MOF, uiO-66-NH) 2 ) Due to good chemical stability, high porosity, larger specific surface area, stronger thermal stability and good pH tolerance, the composite material has broad application prospect and is favored by researchers. More importantly, compared with other MOFs, the material is based on zirconium cluster and skeletonInteraction between the mesoamino ligands, zr-MOF due to-NH 2 The interaction of the groups results in selective adsorption of compounds containing the particular group. Thanks to the above excellent properties of Zr-MOFs, it has been widely used for catalysis, molecular adsorption and separation, drug delivery, and as a porous carrier. Zr-MOF usually exists in powder form, and is not easy to separate from a sample in practical use, so that the Zr-MOF is mostly fixed on various support materials for further application.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a biosensor based on a lanthanum-doped zirconium-based MOF (metal organic framework) derivative composite material and a preparation method thereof, and solve the technical problems that the detection limit of the biosensor is not low enough and the linear range is not wide enough in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
a biosensor based on a lanthanum-doped zirconium-based MOF (Metal organic framework) derived composite material comprises a carbon paste electrode and a modifier on the surface of the carbon paste electrode, wherein the modifier is La from inside to outside in sequence 2 O 3 @ZrO 2 a/IL layer, a glutaraldehyde layer, a neurofilament antibody layer, a bovine serum albumin layer and a neurofilament protein layer.
The invention also has the following technical characteristics:
specifically, the La 2 O 3 @ZrO 2 In the alloy, the mass percentage of the La element is 0.05-0.5%.
Preferably, said La 2 O 3 @ZrO 2 In the above formula, la is contained in an amount of 0.1% by mass.
The IL is ionic liquid [ BMIm]BF 4 。
Specifically, la 2 O 3 @ZrO 2 The preparation process comprises the following steps:
step 2.1, adding zirconium tetrachloride and 2-amino terephthalic acid into an acetic acid solution, adding a lanthanum nitrate solution, and putting the mixture into a 120 ℃ oven for reaction for 24 hours;
step 2.2, centrifugally separating the reaction product obtained in the step 2.1, washing for many times, and drying in vacuum;
step 2.3, putting the product obtained in the step 2.2 into a double-tube carbon determination furnace in N 2 Heating to 900 ℃ for 3h under the condition; when the temperature is cooled to room temperature, slight grinding is carried out to obtain powdered La 2 O 3 @ZrO 2 。
Preferably, in the step 2.1, 0.163g of 2-amino terephthalic acid is added for every 0.21g of zirconium tetrachloride, and 40mL of acetic acid solution is added for every 0.21g of zirconium tetrachloride;
2-20 mL of lanthanum nitrate solution is correspondingly added into every 0.21g of zirconium tetrachloride;
the acetic acid solution is an N, N-dimethylformamide solution containing acetic acid, and the volume concentration of the acetic acid is 0.12mL/mL;
the lanthanum nitrate solution is an N, N-dimethylformamide solution containing lanthanum nitrate, and the concentration of the lanthanum nitrate is 1mg/mL.
Preferably, in step 2.2, methanol and N, N-dimethylformamide are used in a volume ratio of 1:4, washing the mixture for multiple times; the temperature for vacuum drying was 80 ℃.
The invention also provides a preparation method of the biosensor based on the lanthanum-doped zirconium-based MOF derivative composite material, which comprises the following steps:
step one, la is added 2 O 3 @ZrO 2 Ultrasonically dispersing the powder in PBS (phosphate buffer solution) of ionic liquid to form mixture suspension, transferring, dripping on the surface of carbon paste electrode, and air drying at room temperature to obtain La 2 O 3 @ZrO 2 The carbon paste electrode modified by the/IL composite membrane is marked as La 2 O 3 @ZrO 2 /IL/CPE；
Step two, in La 2 O 3 @ZrO 2 Dripping glutaraldehyde on the carbon paste electrode modified by the/IL composite membrane, drying in the air, dripping 5 mu L of neurofilament protein antibody, placing in a refrigerator, and drying in the air to obtain a modified electrode which is marked as anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE；
Step three, continuously dripping bovine serum albumin solution with mass fraction on the surface of the modified electrode in the step two,covering the area which is not modified by the antibody on the electrode, placing the electrode in a refrigerator, airing the electrode, washing the electrode with deionized water to prepare the biosensor based on the lanthanum-doped zirconium-based MOF derivative composite material, and marking the biosensor as NF-L/BSA/anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE。
Preferably, the method is carried out according to the following steps:
step one, 5.0mgLa 2 O 3 @ZrO 2 Ultrasonically dispersing the powder in 1ml PBS solution containing 10 μ L of ionic liquid to form mixture suspension, transferring 10 μ L of the suspension to the surface of carbon paste electrode, and air drying at room temperature to obtain La 2 O 3 @ZrO 2 The carbon paste electrode modified by the/IL composite membrane is marked as La 2 O 3 @ZrO 2 /IL/CPE；
Step two, in La 2 O 3 @ZrO 2 Dripping 5 mu L of glutaraldehyde with the volume fraction of 2.5% on a carbon paste electrode modified by an IL composite membrane, drying the glutaraldehyde, dripping 5 mu L of neurofilament protein antibody with the concentration of 40 mu g/mL after drying the glutaraldehyde, placing the neurofilament protein antibody in a refrigerator, and drying the neurofilament protein antibody at 4 ℃ to obtain a modified electrode, which is marked as anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE；
And step three, continuously dropwise coating 5 mu L of bovine serum albumin solution with the mass fraction of 1% on the surface of the modified electrode in the step two to cover the area which is not modified by the antibody on the electrode, placing the electrode in a refrigerator, airing the electrode at 4 ℃, washing the electrode with deionized water for 2 to 3 times to prepare the biosensor based on the lanthanum-doped zirconium-based MOF derivative composite material, and recording the biosensor as NF-L/BSA/anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE。
Preferably, before the carbon paste electrode is used, the carbon paste electrode needs to be polished into a mirror surface on weighing paper, and then the mirror surface is placed in a phosphate buffer solution to be subjected to cyclic voltammetry scanning for 10 periods, so that the carbon paste electrode is subjected to electrochemical activation treatment.
Compared with the prior art, the invention has the following technical effects:
the biosensor prepared by the method is a biosensor based on a zirconium-based metal organic framework composite pyrolysis material. The biosensor prepared by the method has the characteristics of low detection limit, wide linear range and the like, and can be used for quickly detecting the neurofilament protein.
(II) La synthesized by the method of the invention 2 O 3 @ZrO 2 The material is used for preparing the neurofilament protein biosensor, the substrate electrode of the material is a carbon paste electrode, and modifiers on the surface of the carbon paste electrode are sequentially La from inside to outside 2 O 3 @ZrO 2 IL, glutaraldehyde, neurofilament antibodies, bovine serum albumin, and neurofilament proteins. The peak current and the mass concentration of the neural thread protein of the sensor present two-stage linear relationship, and are respectively 0.05-5 ng.mL -1 And 5-200 ng. ML -1 The linear regression equations are respectively I p =3.26c (ng/mL) -40.92 and I p =0.024c (ng/mL) -22.16, detection limit 0.012ng mL -1 With a lower detection limit and a wider linear range.
Drawings
FIG. 1 (A) is La 2 O 3 @ZrO 2 XRD spectrum of the material.
FIG. 1 (B) is La 2 O 3 @ZrO 2 XPS spectrum of C element in material.
FIG. 1 (C) is La 2 O 3 @ZrO 2 XPS spectrum of Zr element in the material.
FIG. 1 (D) is La 2 O 3 @ZrO 2 XPS spectrum of La element in the material.
FIG. 2 is La 2 O 3 @ZrO 2 The total elemental mapping (B) of region (A) shown in (A), and the individual mapping of (C) Zr and (D) La atoms.
FIG. 3 shows La with different compounding ratios 2 O 3 @ZrO 2 Scanning electron micrographs of the materials (A), (B), (C), (D) and (E) and the result (F) of detection of the sensor produced from them.
FIG. 4 (A) is a cyclic voltammogram at different stages of the modified electrode.
FIG. 4 (B) is an AC impedance diagram (a.La) of the modified electrode at different stages 2 O 3 @ZrO 2 /IL/CPE,b.anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE,c.NF-L/BSA/anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE)。
FIG. 4 (C) shows anti-NF-L/GA/La 2 O 3 @ZrO 2 Cyclic voltammograms of/IL/CPE at different scan rates (inset shows the relationship of oxidation peak ip, a and reduction peak ip, c with scan rate).
FIG. 5 shows the optimization of the mass concentration (A), incubation temperature (B) and time (C) of the antibody, and pH (D) of the working solution in the detection process of the biosensor prepared according to the present invention.
FIG. 6 is a differential pulse voltammogram (A) of a biosensor prepared according to the present invention after immersion in a solution of neural thread protein of various concentrations; FIGS. 6 (B) and (C) are graphs obtained by linear fitting the results of the experiment for the analysis of the relationship between the NF-L mass concentration and the oxidation peak current.
The present invention will be explained in further detail with reference to examples.
Detailed Description
CPE refers to carbon paste electrode; IL refers to ionic liquids; GA refers to glutaraldehyde; anti-NF-L refers to a neurofilament protein antibody; BSA refers to bovine serum albumin; NF-L refers to neurofilament protein.
Ionic liquids [ BMIm]BF 4 I.e. 1-butyl-3-methylimidazolium tetrafluoroborate, is a greenhouse ionic liquid with the molecular formula C 8 H 15 N 2 BF 4 。
The instruments and materials used in this example:
CHI660B electrochemical workstation (Shanghai Chenghua instruments Co.), electronic analytical balance (Shenyang Longteng electronic weighing instrument Co., ltd.), electric hot air drying oven (Beijing Kogyi Yongxing instruments Co., ltd.), ultrasonic cleaner (Zhejiang Ningbo instruments Co., ltd.), magnetic heating stirrer (Hezhou Guohua instruments Co., ltd.), X-ray diffractometer (Germany Bruker), scanning electron microscope (U.S. FEI), vacuum drying oven (Tianjin Tester instruments Co., ltd.), double tube fixed carbon furnace (Beijing Kogyi Yongxing instruments Co., ltd.), zirconium chloride (Guangdong Guanghua science and technology Co., ltd.), 2-amino terephthalic acid (Hangzhou Kommer chemical reagent Co., ltd.), lanthanum nitrate (Xian chemical reagent factory), N, N-dimethylformamide (Guangdong Guanghua science and technology Co., ltd.), graphite powder (national medicine group chemical reagent Co., ltd.), liquid paraffin (Tianjin chemical reagent Co., ltd.), potassium chloride (Sigan chemical reagent plant), potassium dihydrogen phosphate (Sigan chemical reagent plant), dipotassium hydrogen phosphate (Sigan chemical reagent plant) potassium ferricyanide (remote chemical Co., ltd., tianjin City), potassium ferrocyanide (remote chemical Co., ltd., tianjin City), hydrogen peroxide (Sigan chemical Co., ltd.), neurofilament protein antibody (Duty company, shanghai 'an research Biotechnology), neurofilament protein (Duty company, shanghai' an research Biotechnology Co., ltd.), potassium ferricyanide (remote chemical Co., ltd., tianjin City, west An chemical Co., ltd.), and potassium ferrocyanide (Sedan, biotechnology Co., ltd.), bovine Serum Albumin (BSA) >98% (beijing obozin biotechnology, llc).
The working base solution for impedance test contains 5.0mM K 3 Fe(CN) 6 /K 4 Fe(CN) 6 +0.1M KCl in 0.01M PBS.
Other measurements were carried out using 0.01M K in PBS buffer 2 HPO 4 And KH 2 PO 4 The stock solution is prepared, and the pH value of the stock solution is 0.01M H 3 PO 4 And NaOH solution. The water used is double distilled water.
The following embodiments of the present invention are provided, and it should be noted that the present invention is not limited to the following embodiments, and all equivalent changes based on the technical solutions of the present invention are within the protection scope of the present invention.
Example 1:
this example presents a biosensor based on a lanthanum-doped zirconium-based MOF-derived composite, which is essentially the same as example 2, except that La is the only difference 2 O 3 @ZrO 2 In the above formula, the content of La was 0.05% by mass.
The material La 2 O 3 @ZrO 2 Was prepared essentially as in example 2, except that in step 2.1, 2mL of lanthanum nitrate solution (La (NO) was added 3 ) 3 ，1mg/mL DMF)。
The preparation method of the biosensor based on the lanthanum-doped zirconium-based MOF derived composite material of this example was the same as that of example 2.
Example 2:
the embodiment provides a biosensor based on a lanthanum-doped zirconium-based MOF (metal organic framework) derivative composite material, which comprises a carbon paste electrode and modifiers on the surface of the carbon paste electrode, wherein the modifiers are La from inside to outside in sequence 2 O 3 @ZrO 2 a/IL layer, a glutaraldehyde layer, a neurofilament antibody layer, a bovine serum albumin layer and a neurofilament protein layer.
La 2 O 3 @ZrO 2 In the above formula, la is contained in an amount of 0.1% by mass.
IL is an ionic liquid [ BMIm]BF 4 。
The preparation process of the carbon paste electrode comprises the following steps: 3.4g of graphite and 0.6g of liquid paraffin were thoroughly mixed and stirred to form a uniform paste. Putting into PVP tube with diameter of 3mm, compacting, inserting copper wire with appropriate length as wire, and making into Carbon Paste Electrode (CPE)
Before the carbon paste electrode is used, the carbon paste electrode needs to be polished into a mirror surface on weighing paper, then the mirror surface is placed in a phosphate buffer solution, cyclic voltammetry scanning is carried out for 10 periods, and electrochemical activation treatment is carried out on the carbon paste electrode.
The material La 2 O 3 @ZrO 2 The preparation process comprises the following steps:
step 2.1, 0.21g of zirconium tetrachloride (ZrCl) 4 ) And 0.163g of 2-amino-p-benzoic acid (H) 2 BDC-NH 2 ) To 40mL of N, N-dimethylformamide containing 4.8mL of acetic acid was added, and 4mL of lanthanum nitrate solution (La (NO) 3 ) 3 1mg/mL DMF), and placing the mixture into a 120 ℃ oven for reaction for 24 hours;
step 2.2, the product was centrifuged, washed multiple times with a mixture of methanol and N, N-dimethylformamide (v/v = 1:4) and dried under vacuum at 80 ℃;
step 2.3, the product (La/UiO-66-NH) is reacted 2 ) Placing in a double-tube carbon determination furnace in N 2 Heating to 900 deg.C for 3h. When the temperature is cooled to room temperature, slight grinding is carried out to obtain powdered La 2 O 3 @ZrO 2 。
The biosensor based on the lanthanum-doped zirconium-based MOF-derived composite material of the present example was prepared according to the following steps:
step one, 5.0mgLa is added 2 O 3 @ZrO 2 Ultrasonically dispersing the powder in 1ml PBS solution containing 10 μ L of ionic liquid to form mixture suspension, transferring 10 μ L of the suspension to the surface of carbon paste electrode, and air drying at room temperature to obtain La 2 O 3 @ZrO 2 La as carbon paste electrode modified by/IL composite membrane 2 O 3 @ZrO 2 /IL/CPE；
Step two, in La 2 O 3 @ZrO 2 Dripping 5 mu L of glutaraldehyde with the volume fraction of 2.5% on a carbon paste electrode modified by an IL composite membrane, drying the glutaraldehyde, dripping 5 mu L of neurofilament protein antibody with the concentration of 40 mu g/mL after drying the glutaraldehyde, placing the neurofilament protein antibody in a refrigerator, and drying the neurofilament protein antibody at 4 ℃ to obtain a modified electrode, which is marked as anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE；
And step three, continuously dripping 5 mu L of bovine serum albumin solution with the mass fraction of 1% on the surface of the modified electrode obtained in the step two to cover the area which is not modified by the antibody on the electrode, placing the electrode in a refrigerator, airing the electrode at 4 ℃, washing the electrode with deionized water for 2 to 3 times to obtain the biosensor based on the lanthanum-doped zirconium-based MOF-derived composite material, and recording the biosensor as NF-L/BSA/anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE。
Example 3:
this example presents a biosensor based on a lanthanum-doped zirconium-based MOF-derived composite, which is essentially the same as example 2, except that La is the only difference 2 O 3 @ZrO 2 In the above formula, the content of La was 0.25% by mass.
The material La 2 O 3 @ZrO 2 Was prepared essentially as in example 2, except that, in step 2.1, 10mL of lanthanum nitrate solution (La (NO) was added 3 ) 3 ，1mg/mL DMF)。
The preparation method of the biosensor based on the lanthanum-doped zirconium-based MOF derived composite material of this example was the same as that of example 2.
Example 4:
this example presents a method for the preparation of a biosensor based on a lanthanum-doped zirconium-based MOF-derived composite, which is substantially identical to that of example 2, except that La is the only difference 2 O 3 @ZrO 2 In the above formula, the content of La was 0.5% by mass.
The material La 2 O 3 @ZrO 2 Was prepared essentially as in example 2, except that in step 2.1, 20mL of lanthanum nitrate solution (La (NO) was added 3 ) 3 ，1mg/mL DMF)。
The preparation method of the biosensor based on the lanthanum-doped zirconium-based MOF derived composite material of this example was the same as that of example 2.
La 2 O 3 @ZrO 2 Characterization of the materials:
FIG. 1 is a graph showing the microstructure of the prepared La 2 O 3 @ZrO 2 The material was characterized.
FIG. 1 (A) is La 2 O 3 @ZrO 2 The XRD pattern of (a) shows that the sample has a broad diffraction peak at about 24 °, which corresponds to the diffraction of the (002) crystal plane of graphitic carbon. It can also be seen that peaks appear in the vicinity of 30, 50 and 59, which correspond to ZrO respectively 2 The (111), (202) and (131) crystal planes of (a).
At the same time, for the prepared La 2 O 3 @ZrO 2 The material was XPS characterized to further elucidate the La synthesized 2 O 3 @ZrO 2 The state of the individual elements in the material. As a result, as shown in FIG. 1 (B), the spectrum of C1 s can be decomposed into three peaks at 284.8eV, 286.1eV, and 288.9eV, which correspond to the three states of C-C, C-O, or C-N and O-C ═ O for C. As can be seen from FIG. 1 (C), the spectrum of Zr 3d has two peaks, the peak at 182.5eV belongs to Zr 3d5/2 and the peak at 185.0eV belongs to Zr 3d3/2, and these two peaks indicate that Zr is Zr in the synthesized material 4+ The state of (2) exists. FIG. 1 (D) shows two doublets around 834.9eV and 853.8eV, which are the peaks for La 3D5/2 and La 3D3/2, indicating that La is La by La after calcination 2 O 3 Doped in the material.
The elemental surface distribution of the material was analyzed by an energy spectrometer in an SEM electron microscope, and the results are shown in fig. 2. The red-box region in FIG. 2 (A) was analyzed, wherein FIG. 2 (B) is the total mapping map, and (C) and (D) are the mapping of Zr and La elements, respectively. As can be seen from the figure, both Zr and La are uniformly distributed in the material, but the spectrum brightness is lower compared to Zr because of the smaller La content.
Influence of addition of La element on the material:
in order to study the effect of the addition of La on the material, SEM images of materials with different La composite ratios are compared. As shown in fig. 3 (a), the surface of the material exhibited a pillar-like, particle-close packed morphology prior to the un-recombination of La. From FIGS. 3 (B) and 3 (C), it can be seen that La (NO) is added 3 ) 3 The surface of the material is more uniform and appears more loose, showing a dendritic structure. The composition of the La element improves the specific surface area of the material and simultaneously improves the porosity of the material. However, after the compounding ratio is further increased, the material may be agglomerated to some extent, and the specific surface area and porosity of the material are rather decreased (fig. 3 (D, E)). Meanwhile, la with different composite ratios is compared 2 O 3 @ZrO 2 The sensor made from the material detected the same concentration of NF-L, as can be seen in FIG. 3 (F), 0.1% La in La composite ratio 2 O 3 @ZrO 2 With the best detection results. The appearance of the synthesized material is easy to adsorb under the composite proportion, which is beneficial to the loading of protein, increases the immobilized amount of enzyme on the surface of an electrode and improves the electrocatalytic efficiency of the material. Therefore, the composite proportion of 0.1 percent is selected as La 2 O 3 @ZrO 2 The optimal La composite ratio.
Electrochemical properties of electrodes in sensors at different modification stages:
the electrochemical properties of the electrodes in the sensor at different modification stages can be analyzed by CV. As shown in FIG. 4 (A), curve a is La 2 O 3 @ZrO 2 CV curve of/IL/CPE, la can be seen 2 O 3 @ZrO 2 /IThe L/CPE has a pair of reversible redox peaks, the peak shape is obvious and the peak current is large, which indicates that La 2 O 3 @ZrO 2 The material has good electron transfer capability. And further modifying the surface of the electrode with a cross-linking agent for fixing anti-NF-L, wherein the redox peak shape is unchanged but the peak current is reduced (curve b), and because the antibody is a biological macromolecule, the conductivity is poor, and when the antibody is modified on the surface of the electrode, the conductivity of the electrode is reduced. Finally, after the modified electrode is specifically and immunologically combined with NF-L, the biosensor based on the lanthanum-doped zirconium-based MOF derivative composite material NF-L/BSA/anti-NF-L/GA/La 2 O 3 @ZrO 2 There is a significant decrease in peak current of the CV curve for/IL/CPE (curve c). This is because the antigen-antibody binding covers the electrode surface, and the addition of BSA also blocks the regions of the modified electrode surface not covered by the antibody specific binding.
In addition to CV, EIS can also analyze the surface electron transport at different stages of electrode modification. As shown in FIG. 4 (B), it is evident that the radius of the CPE impedance curve is the largest, indicating that the bare electrode is not conductive, and the electrode surface is modified with La 2 O 3 @ZrO 2 after/IL, la 2 O 3 @ZrO 2 The radius of the impedance curve of/IL/CPE becomes minimal, indicating that La 2 O 3 @ZrO 2 The material can effectively improve the electron transfer capability on the surface of the electrode. Then, anti-NF-L is fixed on the surface of the electrode through a cross-linking agent, and BSA/anti-NF-L/GA/La is formed after the incubation in NF-L 2 O 3 @ZrO 2 /IL/CPE with impedance values compared to La 2 O 3 @ZrO 2 the/IL/CPE is obviously increased because the substances such as antigen antibody, BSA, etc. have poor conductivity, and after the substances are combined with each other on the surface of the electrode in an interaction way, the transfer of electrons on the surface of the electrode is hindered. And analyzing the electrode surface states of all stages in the sensor construction process by combining the obtained results analyzed by the CV, which indicates that the NF-L immunosensor is successfully prepared and can be used for detecting NF-L.
The electrochemistry of the immunosensor can be further analyzed through CV curves under different sweepsAnd (6) behaviors. FIG. 4 (C) shows NF-L/BSA/anti-NF-L/GA/La at different scanning speeds in the range of 0.05-0.5V/s 2 O 3 @ZrO 2 The CV curve of/IL/CPE is shown in the figure, when the scanning speed is lower, the oxidation reduction peak type of the curve is obvious, and the symmetrical peak heights of the peak shapes are equal, which indicates that the reversibility of the electrode reaction is good; when the scanning rate is increased, the peak current of the oxidation-reduction peak is increased, and at the same time, the oxidation peak potential is shifted positively, the reduction peak potential is shifted negatively, and the potential difference is increased accordingly. The calculation result shows that the current magnitude and the scanning rate are in a linear relation, and the linear equations are respectively oxidation peaks: ip, a = -472.55v1/2 (V · s-1) +16.95 (R = 0.999), reduction peak: ip, c =602.76v1/2 (V · s-1) -64.95 (R = 0.999), which indicates that the redox reaction process at the electrode surface is diffusion-controlled.
Selection of NF-L immunosensor experimental conditions:
FIG. 5 is a selection of NF-L immunosensor assay conditions. NF-L immunosensor is a neurofilament protein biosensor.
In the electrochemical experiment process, the experimental condition of the immune reaction has a great influence on the magnitude of the response signal. The following different experimental conditions were investigated herein for their effect on the current response of an electrochemical sensor. First, the influence of the amount of antibody immobilized on the electrode surface on the detection result. As shown in FIG. 5 (A), the sensor current response value gradually decreases when the antibody concentration gradually increases, and when the antibody mass concentration reaches 40. Mu.g/mL, the sensor response current does not change much with the increase of the antibody concentration, which indicates that the fixed amount of the antibody on the electrode surface is saturated. Therefore, the antibody concentration of 40. Mu.g/mL was used as the optimum experimental condition for detection. For the electrochemical immunosensor, not only the preparation condition of the sensor has an influence on the detection result, but also the detection condition has a certain influence on the result. Wherein the incubation conditions alter the current response of the sensor by affecting the binding of the antigen-antibody in an immune response. The temperature and time during the incubation process were optimized by first analyzing the effect of the incubation temperature on the assay results, testing the immunosensor obtained above for a 1ng/mL NF-L solution, setting the assay temperatures to 27, 32, 37, 42, and 47 ℃ in that order, and recording the response current of the sensor after 40min of incubation. As shown in fig. 5 (B), the peak current value of the sensor becomes small as the temperature becomes high, but the peak current value of the sensor becomes large conversely when the temperature is greater than 37 ℃. This is because the binding of antigen-antibody is accelerated by increasing the temperature below 37 ℃, but the detection effect is reduced by inactivating the antigen-antibody to some extent when the temperature is above 37 ℃. Therefore 37 ℃ was chosen as the optimal incubation temperature. FIG. 5 (C) is a graph showing the effect of incubation time on the test results, in which the immunosensor was immersed in NF-L solution at 1ng/mL and incubated at 37 ℃ for selected times, wherein the incubation times were 20, 30, 40, 50, and 60min, respectively. As the immune reaction proceeds, the antigen-antibody is combined on the surface of the electrode to block the electron transfer, so that the response current of the immunosensor is gradually reduced, and when the incubation time reaches 50min and the incubation is continued, the response current is not changed greatly, because the reaction of the antigen-antibody on the surface of the electrode reaches the dynamic equilibrium, and the reaction can not be promoted any more by the continued incubation. Therefore 50min was chosen as the optimal incubation time. Meanwhile, the pH value of the working base solution can influence the activities of the antigen and the antibody, and can also influence the stability and the affinity of the antigen-antibody compound on the surface of the electrode, thereby influencing the sensitivity of the immunosensor. The influence of different pH values on the test results was investigated here, and the immunosensor prepared was immersed in a 1ng/mL NF-L solution and incubated at 37 ℃ for 50min, with pH values of 5,6,7,8,9, respectively. As a result, as shown in fig. 5 (D), it can be seen that the value of the response peak current is the largest when the pH value is 7, and the detection effect of the sensor is reduced both above and below pH 7. Therefore, the working base solution with pH 7.0 was selected as the optimum experimental condition.
And (3) NF-L analysis and detection:
when NF-L analysis and detection are carried out, the optimum experimental conditions discussed above are selected, and NF-L/BSA/anti-NF-L/GA/La is carried out at 37 DEG C 2 O 3 @ZrO 2 the/IL/CPE was immersed in solutions to be tested at different NF-L concentrations at pH =7.0 and incubated for 50min before testing the immunosensor for DPV response. The results are shown in FIG. 6 (A), with NF-L concentratedThe increasing of the degree, the antigen antibody generates immunoreaction on the surface of the electrode to generate a complex, the formed complex is gathered on the surface of the electrode, the current response is blocked, and the peak current value of the complex is gradually reduced. In order to analyze the relationship between the mass concentration of NF-L and the oxidation peak current, the obtained experimental results are subjected to linear fitting. Two methods are respectively selected for fitting, one method is direct linear analysis of NF-L mass concentration and oxidation peak current, and the result shows two-section linear relation which is respectively 0.05-5 ng.mL -1 And 5-200 ng. ML -1 The linear regression equation is Ip =3.26c (ng · mL) respectively -1 ) -40.92 (R = 0.986) and Ip =0.024c (ng · mL) -1 ) 22.16 (R = 0.996), with a signal-to-noise ratio of 3 and a detection limit of 0.012ng · mL -1 . The other is to analyze the NF-L mass concentration and the oxidation peak current index, and the result shows that the concentration is between 0.1 and 100 ng.mL -1 In the range of (b), the linear regression equation is Ip =7.84log c (ng · mL) -1 ) 34.28 (R = 0.981), detection limit of 0.034ng · mL at signal-to-noise ratio of 3 -1 . Both of these indicate that the immunosensor has a lower detection limit and a wider linear range.
Claims (9)
1. The biosensor based on the lanthanum-doped zirconium-based MOF derivative composite material comprises a carbon paste electrode and is characterized by also comprising a modifier on the surface of the carbon paste electrode, wherein the modifier is La from inside to outside in sequence 2 O 3 @ZrO 2 An IL layer, a glutaraldehyde layer, a neurofilament antibody layer, a bovine serum albumin layer and a neurofilament protein layer;
the IL is ionic liquid [ BMIm]BF 4 。
2. The biosensor based on a lanthanum-doped zirconium-based MOF derivative composite material of claim 1, wherein the La 2 O 3 @ZrO 2 In the alloy, the mass percentage of the La element is 0.05-0.5%.
3. The biosensor based on a lanthanum-doped zirconium-based MOF derivative composite material of claim 2, wherein the La 2 O 3 @ZrO 2 In the above formula, la is contained in an amount of 0.1% by mass.
4. The biosensor of claim 1, wherein the La doped zirconium based MOF derivative composite material 2 O 3 @ZrO 2 The preparation process comprises the following steps:
step 2.1, adding zirconium tetrachloride and 2-amino terephthalic acid into an acetic acid solution, adding a lanthanum nitrate solution, and putting the mixture into a 120 ℃ oven for reaction for 24 hours;
step 2.2, centrifugally separating the reaction product obtained in the step 2.1, washing for many times, and drying in vacuum;
step 2.3, putting the product obtained in the step 2.2 into a double-tube carbon determination furnace in N 2 Heating to 900 ℃ for 3h under the condition; when the temperature is cooled to room temperature, slight grinding is carried out to obtain powdered La 2 O 3 @ZrO 2 。
5. The biosensor based on a lanthanum doped zirconium based MOF derivative composite material according to claim 4, wherein in step 2.1, for every 0.21g of zirconium tetrachloride, 0.163g of 2-amino terephthalic acid is added, for 40mL of acetic acid solution;
adding 2-20 zxft 8978 of lanthanum nitrate solution into every 0.21g of zirconium tetrachloride;
the acetic acid solution is an N, N-dimethylformamide solution containing acetic acid, and the volume concentration of the acetic acid is 0.12mL/mL;
the lanthanum nitrate solution is an N, N-dimethylformamide solution containing lanthanum nitrate, and the concentration of the lanthanum nitrate is 1mg/mL.
6. The biosensor based on a lanthanum-doped zirconium-based MOF derivative composite according to claim 4, wherein in step 2.2, methanol and N, N-dimethylformamide are used in a volume ratio of 1:4, washing the mixture for multiple times; the temperature for vacuum drying was 80 ℃.
7. A method for preparing a biosensor based on a lanthanum doped zirconium based MOF derivative composite material according to any of claims 1 to 6, wherein the method is performed according to the following steps:
step one, la is added 2 O 3 @ZrO 2 Ultrasonically dispersing the powder in PBS (phosphate buffer solution) of ionic liquid to form mixture suspension, transferring, dripping on the surface of carbon paste electrode, and air drying at room temperature to obtain La 2 O 3 @ZrO 2 The carbon paste electrode modified by the/IL composite membrane is marked as La 2 O 3 @ZrO 2 /IL/CPE；
Step two, in La 2 O 3 @ZrO 2 Dripping glutaraldehyde on the carbon paste electrode modified by the/IL composite membrane, drying in the air, dripping 5 mu L of neurofilament protein antibody, placing in a refrigerator, and drying in the air to obtain a modified electrode which is marked as anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE；
And step three, continuously dripping bovine serum albumin solution with the mass fraction on the surface of the modified electrode in the step two to cover the area which is not modified by the antibody on the electrode, placing the electrode in a refrigerator, airing the electrode, washing the electrode with deionized water to prepare the biosensor based on the lanthanum-doped zirconium-based MOF derivative composite material, and recording the biosensor as NF-L/BSA/anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE。
8. The method of making a lanthanum doped zirconium based MOF derivative composite based biosensor according to claim 7, wherein the method is performed according to the following steps:
step one, 5.0mgLa is added 2 O 3 @ZrO 2 Ultrasonically dispersing the powder in 1ml of LPBS solution containing 10 mu L of ionic liquid to form mixture suspension, transferring 10 mu L of the mixture suspension to the surface of a carbon paste electrode, and airing at room temperature to obtain La 2 O 3 @ZrO 2 The carbon paste electrode modified by the/IL composite membrane is marked as La 2 O 3 @ZrO 2 /IL/CPE；
Step two, in La 2 O 3 @ZrO 2 Dripping 5 mu L of glutaraldehyde with the volume fraction of 2.5% on a carbon paste electrode modified by an IL composite membrane, drying the glutaraldehyde, and dripping 5 mu L of concentrated solutionPlacing the neurofilament protein antibody with the degree of 40 mu g/mL in a refrigerator to be dried at 4 ℃ to obtain a modified electrode which is marked as anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE；
And step three, continuously dropwise coating 5 mu L of bovine serum albumin solution with the mass fraction of 1% on the surface of the modified electrode in the step two to cover the area which is not modified by the antibody on the electrode, placing the electrode in a refrigerator, airing the electrode at 4 ℃, washing the electrode with deionized water for 2 to 3 times to prepare the biosensor based on the lanthanum-doped zirconium-based MOF derivative composite material, and recording the biosensor as NF-L/BSA/anti-NF-L/GA/La 2 O 3 @ZrO 2 /IL/CPE。
9. The method of claim 7, wherein the carbon paste electrode is polished to a mirror surface on a weighing paper before use, and then placed in a phosphate buffer solution for cyclic voltammetric scanning for 10 periods to perform electrochemical activation treatment.
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