CN114230804A - Copper peroxide/hydrogen bond organic framework nano material, probe thereof and kit for detecting lipocalin-2 - Google Patents
Copper peroxide/hydrogen bond organic framework nano material, probe thereof and kit for detecting lipocalin-2 Download PDFInfo
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Abstract
The invention provides a copper peroxide/hydrogen bond organic framework nano material, a probe thereof and a kit for detecting lipocalin-2, belonging to the technical field of biosensing. According to the invention, HOF is used as a nano enzyme carrier, and the obtained copper peroxide/hydrogen bond organic framework nano material has good biological stability and chemical stability; meanwhile, HOF has large specific surface area and good adsorbability, and can increase HOF-CuO2The surrounding substrate concentration has enrichment effect on signal molecules, thereby improving HOF-CuO2Peroxidase activity and laccase activity. Further, HOF-CuO2Detection of lipocalin by ELISAAt 2 o, CuO2The nanoparticles can provide a powerful binding site for detecting antibodies or affinity peptides.
Description
Technical Field
The invention relates to the technical field of biosensing, in particular to a copper peroxide/hydrogen bond organic framework nano material, a probe thereof and a kit for detecting lipocalin-2.
Background
Acute Kidney Injury (AKI), previously known as acute renal failure, is a severe syndrome characterized by rapid loss of kidney function within hours to days. Although AKI is reversible in most cases, it still has a high morbidity and mortality rate if not treated in time.
Over the past decades, many potential biomarkers for early monitoring of AKI have been studied. Among these biomarkers, neutrophil gelatinase-associated lipocalin, also known as lipocalin-2 (NGAL), renal injury molecule-1 (KIM-1), cystatin c (cysc), hepatic fatty acid binding protein (L-FABP), and interleukin-18 (IL-18) were initially detected in AKI patients and associated with loss of renal function. According to clinical tests, the performance of the lipocalin-2 content in urine as an AKI early prediction index is found to be superior to other indexes. When AKI occurs, higher lipocalin-2 levels can be detected in the urine within only a few hours, which will effectively delay the progress of AKI.
The current methods for detecting lipocalin-2, such as western blotting and immunoblotting, usually require large laboratories equipped with precise analytical instruments, require complex operations and a large amount of waiting time, and are not favorable for clinical applications. Enzyme-linked immunosorbent assay (ELISA) has the advantages of high specificity, rapidness, simplicity and convenience, and is widely used. For traditional enzyme-linked immunosorbent assays, the detection antibody is usually labeled with a natural enzyme, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP), which have high efficiency and commercial availability. However, the above natural enzymes are unstable and easily denatured and inactivated under non-physiological conditions, which makes ELISA detection of lipocalin-2 difficult.
Disclosure of Invention
In view of the above, the present invention aims to provide a copper peroxide/hydrogen bond organic framework nanomaterial, a probe thereof, and a kit for detecting lipocalin-2. The copper peroxide/hydrogen bond organic framework nano material and the probe thereof provided by the invention have high stability and good peroxidase activity and laccase activity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a copper peroxide/hydrogen bond organic framework nano material and a probe thereof, which comprise a hydrogen bond organic framework material and CuO growing on the surface of the hydrogen bond organic framework material in situ2Nanoparticles; the organic ligand of the hydrogen bond organic framework material is 1,3,6, 8-tetra (4-carboxyphenyl) pyrene.
Preferably, the CuO2The mass ratio of the nano particles to the hydrogen bond organic framework material is 5 (0.5-10).
The invention provides a preparation method of the copper peroxide/hydrogen bond organic framework nano material, which comprises the following steps:
dissolving 1,3,6, 8-tetra (4-carboxyphenyl) pyrene in an alcohol-containing organic solvent for self-assembly to obtain a hydrogen bond organic framework material;
dissolving soluble cupric salt aqueous solution, dispersant and H2O2And mixing inorganic strong base with the hydrogen bond organic framework material to perform coordination reaction to obtain the copper peroxide/hydrogen bond organic framework nano material.
Preferably, the alcohol-containing organic solvent is a mixed solvent of N, N-dimethylformamide and methanol.
The invention provides a probe for detecting lipocalin-2, which comprises lipocalin-2 specific antigen affinity peptide and a copper peroxide/hydrogen bond organic framework nano material in coordination combination with the lipocalin-2 specific antigen affinity peptide.
Preferably, the amino acid sequence of the lipocalin-2 specific antigen affinity peptide is shown in SEQ ID No. 1.
Preferably, the mass ratio of the lipocalin-2 specific antigen affinity peptide to the copper peroxide/hydrogen bond organic framework nano material is 1: (1-20).
The invention provides application of a probe for detecting lipocalin-2 in preparing a kit for detecting lipocalin-2.
The invention provides a kit for detecting lipocalin-2, which comprises the probe for detecting lipocalin-2, a first antibody of lipocalin-2, a non-specific protein and a chromogenic substrate.
Preferably, the chromogenic substrate is 3,3',5,5' -tetramethylbenzidine;
or 4-AP and 2, 4-DP.
The invention provides a copper peroxide/hydrogen bond organic framework nano material, which comprises a hydrogen bond organic framework material and CuO growing on the surface of the hydrogen bond organic framework material in situ2Nanoparticles; the organic ligand of the hydrogen bond organic framework material is 1,3,6, 8-tetra (4-carboxyphenyl) pyrene. Copper peroxide nanoparticles (CuO)2) Has peroxidase activity and laccase activity, compared with natural enzyme, CuO2The nano enzyme has the advantages of low cost, good stability, easy preparation and the like. The hydrogen bond organic framework material (HOF) has the advantages of high biocompatibility, low toxicity and the like, and the invention takes the HOF as a nano enzyme carrier to obtain the copper peroxide/hydrogen bond organic framework nano material (HOF-CuO)2) Has good biological stability and chemical stability; meanwhile, HOF has large specific surface area and good adsorbability, and can increase HOF-CuO2The surrounding substrate concentration has enrichment effect on signal molecules, thereby improving HOF-CuO2The detection sensitivity of (3). Further, HOF-CuO2CuO for detection of lipocalin-2 by ELISA2The nanoparticles can provide a powerful binding site for detecting antibodies or affinity peptides.
The invention provides a lipocalin-2 detection probe, which comprises lipocalin-2 specific antigen affinity peptide and HOF-CuO coordinately bound with the lipocalin-2 specific antigen affinity peptide2. The invention uses affinity peptide of lipocalin-2 (NGAL) as recognition molecule of lipocalin-2, can specifically recognize lipocalin-2, and HOF-CuO combined with affinity peptide2Has good peroxidase activity and laccase activity, and can catalyze the color development of a color development substrate. Meanwhile, the present invention uses the affinity peptide of lipocalin-2 instead of the second antibody of lipocalin-2, which has a shorter production cycle and lower cost.
The invention provides a kit for detecting lipocalin-2, which comprises a lipocalin-2 detection probe, a first lipocalin-2 antibody, a non-specific protein and a chromogenic substrate. In the invention, the lipocalin-2 first antibody, the lipocalin-2 to be detected and the lipocalin-2 detection probe can form an antibody-antigen-antibody sandwich type enzyme-linked immunosorbent assay biosensor for detecting the specific antigen of the kidney injury marker, so that the aim of accurately and quantitatively detecting the specific antigen of the kidney injury marker is fulfilled, and the enzyme-linked immunosorbent assay biosensor has excellent stability and good reproducibility. The lipocalin-2 detection probe can specifically identify lipocalin-2, has good enzymatic activity, can catalyze chromogenic substrate to develop color, and realizes high-sensitivity and rapid detection of lipocalin-2. Meanwhile, the kit provided by the invention only uses one antibody, so that the kit has lower cost and better stability.
The lipocalin-2 detection probe is used for specifically identifying lipocalin-2 and catalyzing color development of a color development substrate, and the absorbance detected by ultraviolet analysis is enhanced along with the increase of the concentration of the lipocalin-2 and is 0.0001-316 ngmL-1Has a good linear relationship in the range of lipocalin-2 concentration, and the lowest detection limit is 33 fg/mL. Furthermore, the detection method provided by the invention has the characteristic of double-signal colorimetry, namely TMB color development characteristic and laccase colorimetric characteristic.
Drawings
FIG. 1 shows HOF and CuO obtained in example 12And HOF-CuO2X-ray diffraction patterns of (a);
FIG. 2 shows HOF and CuO obtained in example 12And HOF-CuO2An infrared spectrum of (1);
FIG. 3 shows HOF and CuO obtained in example 12And HOF-CuO2Transmission electron microscopy images of;
FIG. 4 is CuO obtained in example 12The particle size distribution map of (a);
FIG. 5 is a graph of the UV absorption spectrum of lipocalin-2 at various concentrations and a standard curve in example 2;
FIG. 6 is the UV absorption spectrum and standard curve of lipocalin-2 at different concentrations in example 3;
FIG. 7 shows the anti-interference test results of the immunobiosensor in example 4.
Detailed Description
The invention provides a copper peroxide/hydrogen bond organic framework nano material, which comprises a hydrogen bond organic framework material and CuO growing on the surface of the hydrogen bond organic framework material in situ2Nanoparticles; the organic ligand of the hydrogen bond organic framework material is 1,3,6, 8-tetra (4-carboxyphenyl) pyrene.
In the present invention, the CuO2The mass ratio of the nanoparticles to the hydrogen bond organic framework material is preferably 5: (0.5 to 10), more preferably 5: (2-6).
In the present invention, the CuO2The particle size of the nanoparticles is preferably 2.95-9.12 nm, and more preferably 5.2 nm.
The invention provides a preparation method of the copper peroxide/hydrogen bond organic framework nano material, which comprises the following steps:
1,3,6, 8-tetra (4-carboxyphenyl) pyrene solution is mixed with an alcohol-containing organic solvent for self-assembly to obtain a hydrogen bond organic framework material;
and mixing a soluble divalent copper salt aqueous solution, a dispersing agent, an oxidant and inorganic strong base with the hydrogen bond organic framework material, and carrying out coordination reaction to obtain the copper peroxide/hydrogen bond organic framework nano material.
In the invention, the alcohol-containing organic solvent is preferably a mixed solvent of N, N-dimethylformamide and methanol, and the volume ratio of the N, N-dimethylformamide to the methanol is preferably 5-40: 90, and more preferably 10-30: 90. In the present invention, the methanol can activate the pore channels of 1,3,6, 8-tetrakis (4-carboxyphenyl) pyrene.
In the invention, the self-assembly temperature is preferably room temperature, and the self-assembly time is preferably 6-36 h, and more preferably 12-24 h.
After the self-assembly reaction, the product is preferably washed and purified by using ethanol and acetone.
After the hydrogen bond organic framework material is obtained, the invention uses soluble cupric salt aqueous solution, dispersant and H2O2Inorganic strong base and the hydrogen bond organic boneMixing the frame materials, and carrying out coordination reaction to obtain HOF-CuO2A composite material. In the invention, the soluble cupric salt is preferably cupric chloride, and the concentration of the soluble cupric salt aqueous solution is preferably 0.01-0.1 mol/L, and more preferably 0.05 mol/L.
In the present invention, the dispersant is preferably polyvinylpyrrolidone; the strong inorganic base is preferably NaOH.
In the invention, the mass ratio of the soluble divalent copper salt to the hydrogen bond organic framework material is preferably 8.5: 0.5-10, and more preferably 8.5: 1; the mass ratio of the soluble divalent copper salt to the dispersing agent is preferably 8.5: 50-2000, and more preferably 8.5: 500.
In the present invention, the ratio of the mass of the soluble divalent copper salt to the molar weight of the inorganic strong base is preferably 8.5 g: 0.05 to 0.25mmol, more preferably 8.5 g: 0.02 mmol. In the present invention, OH in the strong inorganic base-Has the effect of deprotonating to promote H2O2And Cu2+Coordination of (3).
In the present invention, the mass of the soluble divalent copper salt and H2O2Preferably 8.5 g: 0.1 to 1mmol, more preferably 8.5 g: 0.5 mmol.
In the invention, the temperature of the coordination reaction is preferably room temperature, and the time is preferably 15-120 min.
After the coordination reaction, the obtained coordination reactant is preferably washed, and the washing detergent is preferably water, ethanol and acetone in sequence.
The invention provides a lipocalin-2 detection probe, which comprises lipocalin-2 specific antigen affinity peptide and the copper peroxide/hydrogen bond organic framework nano material in coordination combination with the lipocalin-2 specific antigen affinity peptide.
In the invention, the lipocalin-2 specific antigen affinity peptide contains sulfydryl and can be combined with CuO in a copper peroxide/hydrogen bond organic framework nano material2And (4) coordinating and combining the nanoparticles.
In the invention, the mass ratio of the lipocalin-2 specific antigen affinity peptide to the copper peroxide/hydrogen bond organic framework nano material is 1: (1-20), more preferably 1:1, 1:5, 1:10, 1:15 or 1: 20.
In the present invention, the method for preparing the lipocalin-2 detection probe preferably comprises the following steps:
and mixing the lipocalin-2 specific antigen affinity peptide with the copper peroxide/hydrogen bond organic framework nano material, and performing coordination reaction to obtain the lipocalin-2 detection probe.
In the invention, the amino acid sequence of the lipocalin-2 specific antigen affinity peptide is shown as SEQ ID NO.1, and specifically comprises: DRWVARDPASIFGGGGSC are provided. As a specific example of the present invention, the lipocalin-2 specific antigen affinity peptide is synthesized by Gill Biochemical Co., Ltd (Shanghai, China).
In the present invention, the coordination reaction is preferably carried out in a phosphate buffer solution, and the pH of the phosphate buffer solution is preferably 7.2.
In the present invention, the temperature of the coordination reaction is preferably 4 ℃ and the time is preferably 12 hours.
After the coordination reaction, the invention preferably centrifuges and washes the obtained coordination reaction liquid to obtain the lipocalin-2 detection probe solid.
In the present invention, the rate of the centrifugation is preferably 8000rpm, and the time is preferably 15 min.
In the present invention, the detergent for washing is preferably a phosphoric acid buffer solution having a pH of 7.2.
The invention provides a kit for detecting lipocalin-2, which comprises a lipocalin-2 detection probe, a first lipocalin-2 antibody, a non-specific protein and a chromogenic substrate.
In the present invention, the kit for detecting lipocalin-2 also preferably comprises a buffer and a washing solution.
In the present invention, the chromogenic substrate is 3,3',5,5' -tetramethylbenzidine. When the chromogenic substrate is 3,3',5,5' -tetramethylbenzidine, the buffer is preferably an acetate buffer solution, and the pH value of the acetate buffer solution is preferably 4.
Alternatively, the chromogenic substrate is preferably 4-AP or 2, 4-DP. In the invention, the 4-AP is 4-aminoantipyrine, and the 2,4-DP is 2, 4-dichlorophenol.
In the invention, the structural formula of the 4-AP is shown as a formula 1, and the structural formula of the 2,4-DP is shown as a formula 2.
In the present invention, the mass ratio of 4-AP to 2,4-DP is preferably 1: 1. In the present invention, when the chromogenic substrate is 4-AP or 2,4-DP, the buffer is preferably a MES buffer, and the pH of the MES buffer is preferably 6.8.
In the present invention, the nonspecific protein is preferably bovine serum albumin.
In the invention, the washing solution is preferably a phosphate buffer solution containing tween-20, the content of tween-20 in the phosphate buffer solution containing tween-20 is preferably 0.05%, and the pH value of the phosphate buffer solution is preferably 7.2.
The invention provides a method for detecting lipocalin-2 for non-diagnostic purposes, which comprises the following steps:
performing a first incubation on a lipocalin-2 first antibody to obtain a first incubation product;
adding non-specific protein into the first incubation product, performing second incubation, and removing unconjugated substances to obtain a second incubation product;
adding a sample to be tested into the second incubation product, performing third incubation, and removing unconjugated substances to obtain a third incubation product;
adding a lipocalin-2 detection probe into the third incubation product, performing fourth incubation, and removing unconjugated substances to obtain a fourth incubation product;
adding a chromogenic substrate into the fourth incubation product, carrying out chromogenic reaction, and testing the absorbance peak value of the obtained chromogenic solution;
obtaining the concentration of the lipocalin-2 in the sample to be detected according to a preset standard curve and the absorbance peak value; the standard curve is a linear relationship curve of the logarithm of the lipocalin-2 concentration to the peak of the absorbance.
The invention performs a first incubation of a lipocalin-2 first antibody to obtain a first incubation product. The present invention does not require any kind of the lipocalin-2 first antibody, and any commercially available lipocalin-2 first antibody conventionally used in the art may be used.
In the present invention, the first incubation is preferably performed in a 96-well microplate. In the invention, the temperature of the first incubation is preferably 4 ℃, and the time is preferably 8-12 h.
After the first incubation product is obtained, the invention adds non-specific protein into the first incubation product, carries out second incubation, and removes unconjugated substance to obtain a second incubation product. In the present invention, the nonspecific protein is preferably bovine serum albumin. In the present invention, the non-specific protein functions to block site-bound lipocalin-2 primary antibodies. In the present invention, the concentration of the nonspecific protein is preferably 1 wt%.
In the invention, the temperature of the second incubation is preferably room temperature, and the time is preferably 15-120 min, and more preferably 60 min.
In the present invention, the mode of removing the unconjugated substance is preferably washing with a phosphate buffer containing tween-20.
After the second incubation product is obtained, the sample to be tested is added into the second incubation product, the third incubation is carried out, and the unconjugated substance is removed, so that a third incubation product is obtained. In the present invention, the sample to be tested is preferably a serum sample. In the invention, the temperature of the third incubation is preferably room temperature, and the time is preferably 15-120 min, and more preferably 60 min.
In the present invention, the mode of removing the unconjugated substance is preferably washing with a phosphate buffer containing tween-20.
After the third incubation product is obtained, the lipocalin-2 detection probe is added into the third incubation product, fourth incubation is carried out, and unconjugated substances are removed to obtain a fourth incubation product. In the present invention, the temperature of the fourth incubation is preferably room temperature, and the time is preferably 15 to 120min, and more preferably 60 min.
In the present invention, the mode of removing the unconjugated substance is preferably washing with a phosphate buffer containing tween-20.
After the fourth incubation product is obtained, adding a chromogenic substrate into the fourth incubation product for chromogenic reaction, and testing the absorbance peak value of the obtained chromogenic solution at 500-800 nm.
In the present invention, the chromogenic substrate is preferably 3,3',5,5' -tetramethylbenzidine. In the present invention, when the chromogenic substrate is 3,3',5,5' -tetramethylbenzidine, the chromogenic reaction is preferably carried out in an acetic acid buffer solution, and the pH of the acetic acid buffer solution is preferably 4. In the invention, the temperature of the color reaction is preferably 37 ℃, and the time is preferably 15-60 min, and more preferably 30 min.
In the invention, when the chromogenic substrate is 3,3',5,5' -tetramethyl benzidine, the absorbance peak value of the chromogenic solution obtained by the test of the invention at 500-800 nm is detected. The present invention preferably uses an ultraviolet spectrophotometer to test the absorbance peak of the resulting color developing solution.
Alternatively, in the present invention, the chromogenic substrate is preferably 4-AP or 2, 4-DP. In the present invention, the mass ratio of 4-AP to 2,4-DP is preferably 1: 1. In the present invention, when the chromogenic substrates are 4-AP and 2,4-DP, the color reaction is preferably carried out in MES buffer, and the pH of the MES buffer is preferably 6.8. In the invention, the temperature of the color development reaction is preferably 60 ℃, and the time is preferably 15-60 min, and more preferably 30 min.
In the invention, when the chromogenic substrates are 4-AP and 2,4-DP, the absorbance peak value of the obtained chromogenic solution at 400-650 nm is tested by the invention. The present invention preferably uses an ultraviolet spectrophotometer to test the absorbance peak of the resulting color developing solution.
After the absorbance peak value is obtained, the concentration of the lipocalin-2 in the sample to be detected is obtained according to a preset standard curve and the absorbance peak value; the standard curve is a linear relationship curve of the logarithm of the lipocalin-2 concentration to the peak of the absorbance.
As a specific embodiment of the present invention, the method for obtaining the standard curve preferably includes the following steps:
a lipocalin-2 standard solution providing a gradient concentration comprising 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 316 ng/mL.
The lipocalin-2 standard solution with gradient concentration is used as a sample to be detected, the detection is carried out according to the detection method of the invention, the absorbance peak value corresponding to the lipocalin-2 standard solution with gradient concentration is obtained, the logarithm of the lipocalin-2 is taken as the abscissa, the absorbance value is taken as the ordinate, and the linear relation curve of the logarithm of the beta bungarotoxin concentration and the absorbance value is obtained.
Specifically, when the chromogenic substrate is 3,3',5,5' -tetramethylbenzidine, the data associated with the standard curve are shown in Table 1.
TABLE 1 Standard Curve for 3,3',5,5' -tetramethylbenzidine as chromogenic substrate
In the invention, when the chromogenic substrate is 3,3',5,5' -tetramethyl benzidine, the detection range of lipocalin-2 is 0.0001-316 ngmL-1The lowest detection limit was 33 fg/mL.
When the chromogenic substrates were 4-AP and 2,4-DP, the data relating to the standard curve is shown in Table 2.
TABLE 2 Standard curves for the chromogenic substrates 4-AP and 2,4-DP
In the invention, when the chromogenic substrate is 3,3',5,5'The detection range of lipocalin-2 is 0.0001-316 ngmL in the case of (tetramethylbenzidine)-1The lowest detection limit was 33 fg/mL.
The following examples are provided to illustrate the copper peroxide/hydrogen bond organic framework nanomaterial and probe thereof, and a kit for detecting lipocalin-2 provided by the present invention in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
(I) preparation of lipocalin-2 detection probes
(1) 150mg of 1,3,6, 8-tetra (4-carboxyphenyl) pyrene (H)4TBAPy) is dissolved in 22.5mLN, N-dimethylformamide solution, 90mL of methanol is added and stirred for 10min, after standing for 12h at room temperature, the mixture is purified by ethanol and acetone for a plurality of times to obtain hydrogen bond organic framework material (HOF);
(2) dissolving 10mg of copper chloride dihydrate in 5mL of water, adding 0.5g of polyvinylpyrrolidone and 1mg of HOF prepared in step (1), and stirring for 30min to mix uniformly. Then, to the above mixture was added sodium hydroxide (5mL, 0.02M) and H in that order2O2(100. mu.L). Stirring for 30 minutes, and washing with water, ethanol and acetone for several times to obtain the composite material (HOF-CuO) of the hydrogen bond organic framework and the copper peroxide nanoparticles2)；
10mg of copper chloride dihydrate was dissolved in 5mL of water, and then 0.5g of polyvinylpyrrolidone was added thereto, and stirred for 30min to mix well. Then, to the above mixture was added sodium hydroxide (5mL, 0.02M) and H in that order2O2(100. mu.L). Stirring for 30 minutes, washing with water, ethanol and several times to obtain copper peroxide nanoparticles (CuO)2)；
(3) Taking 1mg of HOF-CuO prepared in the step (2)2Dispersing in 1mL of phosphate buffer solution, adding 100. mu.L of lipocalin-2 specific antigen affinity peptide with a concentration of 1.0mg/mL, stirring at 4 ℃ for 12h, centrifuging at 8000rpm for 15min, and washing three times with phosphate buffer solution with pH 7.2 to obtain the bioconjugated probe of the peptide.
The obtained HOF and CuO2And HOF-CuO2The X-ray diffraction pattern of (A) is shown in FIG. 1. From FIG. 1The HOF material has a strong crystal diffraction peak at the position of 2 theta 4.8, which is consistent with the literature report, and the HOF material is a polycrystalline structure formed by self-assembly of organic ligands, thereby proving that the HOF material is successfully synthesized; then the main phase of the copper oxide synthesized by us is proved to be copper peroxide according to standard card PDF # 48-1548; finally according to HOF-CuO2The spectra show that the x-ray diffraction peak after the composition has both HOF and CuO2The diffraction peak of (a) indicates successful synthesis of the composite material.
The obtained HOF and CuO2And HOF-CuO2The infrared spectrum of (a) is shown in FIG. 2. From the infrared spectrum of HOF, it can be seen that the infrared spectrum is in 1606 and 1563cm-1The skeleton of the pyrene ring in the HOF skeleton vibrates; the peaks at these two positions did not change after recombination, indicating HOF-CuO2The skeleton of the HOF in the composite material was not destroyed, further illustrating the successful synthesis of the material.
The obtained HOF and CuO2And HOF-CuO2The transmission electron micrograph of (a) is shown in FIG. 3. According to TEM results, HOF is a micron-sized rod-shaped structure which is regular and CuO2The load of (a) provides a template; from HOF-CuO2TEM of (D) shows that CuO2The nano particles are uniformly loaded on the surface of the HOF, so that a large specific surface area and high reactive sites are provided for the subsequent catalytic reaction.
The resulting CuO2The particle size distribution of (A) is shown in FIG. 4.
(II) method for the detection of lipocalin-2 for non-diagnostic purposes
(1) 100 μ L lipocalin-2 Ab at a concentration of 1 μ g/mL1Adding the mixture into a 96-well enzyme label plate, and incubating overnight at 4 ℃;
(2) washing the ELISA plate in the step (1) for three times by using phosphate buffer solution containing Tween-20, and adding 1% bovine serum albumin solution to block the unbound antibody; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20;
(3) respectively dropwise adding lipocalin-2 solution with the concentration of 0.0001-316 ng/mL into the elisa plate treated in the step (2), incubating for 1h at room temperature, and washing the elisa plate hole with phosphate buffer solution containing Tween-20 for three times after incubation is completed;
(4) dripping 100 mu L of peptide biological conjugate probe into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate for three times by using a phosphate buffer solution containing Tween-20 after the incubation is finished;
(5) adding 150 mu L of 3,3',5,5' -tetramethylbenzidine and 150 mu L of acetic acid buffer solution into an enzyme label plate, and reacting for 30min at 37 ℃ to obtain a TMB colorimetric epidemic biosensor for detecting lipocalin-2 with different concentrations;
placing a color developing solution in an enzyme label plate in a micro cuvette, and placing the cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 800-500 nm;
recording absorbance peaks corresponding to lipocalin-2 at different concentrations;
detecting lipocalin-2 solutions with different concentrations by using a standard curve method, wherein the detection range is 0.0001-316 ng/mL, and the lower limit of detection (LOD) is as low as 33fgmL-1(S/N＝3)。
(6)75 mu.L of 4-AP (1mg/mL), 75 mu.L of 2,4-DP (1mg/mL) and 100 mu.L of LMES (pH 6.8) solution are added into an enzyme label plate and reacted for 30min at 65 ℃, so as to obtain the laccase colorimetric immune biosensor for detecting different concentrations of lipocalin-2.
Placing a color developing solution in an enzyme label plate in a micro cuvette, and placing the cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 650-400 nm;
recording absorbance peaks corresponding to lipocalin-2 at different concentrations;
detecting lipocalin-2 solutions with different concentrations by using a standard curve method, wherein the detection range is 0.0001-316 ng/mL, and the lower limit of detection (LOD) is as low as 33fgmL-1(S/N＝3)。
Example 2
(I) preparation of lipocalin-2 detection probes
(1) Dissolving 150mg of 1,3,6, 8-tetra (4-carboxyphenyl) pyrene in a 22.5mLN, N-dimethylformamide solution, adding 90mL of methanol, stirring for 10min, standing at room temperature for 12h, and purifying with ethanol and acetone for several times to obtain a hydrogen bond organic framework material (HOF);
(2) dissolving 10mg of copper chloride dihydrate in 5mL of water, adding 0.5g of polyvinylpyrrolidone and 1mg of HOF prepared in step (1), and stirring for 30min to mix uniformly. Then, to the above mixture was added sodium hydroxide (5mL, 0.02M) and H in that order2O2(100. mu.L). Stirring for 30 minutes, and washing with water, ethanol and acetone for several times to obtain the composite material (HOF-CuO) of the hydrogen bond organic framework and the copper peroxide nanoparticles2)；
(3) Taking 1mg of HOF-CuO prepared in the step (2)2Dispersing in 1mL of phosphate buffer solution, adding 100. mu.L of lipocalin-2 specific antigen affinity peptide with a concentration of 1.0mg/mL, stirring at 4 ℃ for 12h, centrifuging at 8000rpm for 15min, and washing three times with phosphate buffer solution with pH 7.2 to obtain the bioconjugated probe of the peptide.
(II) method for the detection of lipocalin-2 for non-diagnostic purposes
(1) 100 μ L lipocalin-2 Ab at a concentration of 1 μ g/mL1Adding the mixture into a 96-well enzyme label plate, and incubating overnight at 4 ℃;
(2) washing the ELISA plate in the step (1) for three times by using phosphate buffer solution containing Tween-20, and adding 1% bovine serum albumin solution to block the unbound antibody; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20;
(3) respectively dropwise adding lipocalin-2 solution with the concentration of 0.0001-316 ng/mL into the elisa plate treated in the step (2), incubating for 1h at room temperature, and washing the elisa plate hole with phosphate buffer solution containing Tween-20 for three times after incubation is completed;
(4) dripping 100 mu L of peptide biological conjugate probe into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate for three times by using a phosphate buffer solution containing Tween-20 after the incubation is finished;
(5) adding 150 mu L of 3,3',5,5' -tetramethylbenzidine and 150 mu L of acetic acid buffer solution into an enzyme label plate, and reacting for 30min at 37 ℃ to obtain the TMB colorimetric epidemic biosensor for detecting the lipocalin-2 with different concentrations.
The application method of the TMB colorimetric epidemic biosensor comprises the following specific steps:
(1) placing a color developing solution in an enzyme label plate in a micro cuvette, and placing the cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 800-500 nm;
(2) recording absorbance peaks corresponding to lipocalin-2 at different concentrations;
(3) detecting lipocalin-2 solutions with different concentrations by using a standard curve method, wherein the detection range is 0.0001-316 ng/mL, and the lower limit of detection (LOD) is as low as 33fgmL-1(S/N＝3)。
FIG. 5 shows the UV absorption spectrum of different concentrations of lipocalin-2 and a standard curve, wherein (A) in FIG. 5 is the UV-visible absorption spectrum of different concentrations of lipocalin-2 developed by TMB, and (B) is the standard curve. The constructed ELISA was used to detect different concentrations of lipocalin-2 under 652nm conditions using a microplate reader, and as can be seen in FIG. 5 (A), the absorbance value gradually increased with the increase of the concentration of lipocalin-2 toxin. As can be seen from FIG. 5 (B), the absorbance has a good linear relationship with the logarithm of the lipocalin-2 concentration, the correlation coefficient (R)2) 0.9920, LOD 33 fg. mL-1(S/N ═ 3), showing good linearity and lower LOD values.
Example 3
(I) preparation of lipocalin-2 detection probes
(1) Dissolving 150mg of 1,3,6, 8-tetra (4-carboxyphenyl) pyrene in a 22.5mLN, N-dimethylformamide solution, adding 90mL of methanol, stirring for 10min, standing at room temperature for 12h, and purifying with ethanol and acetone for several times to obtain a hydrogen bond organic framework material (HOF);
(2) dissolving 10mg of copper chloride dihydrate in 5mL of water, adding 0.5g of polyvinylpyrrolidone and 1mg of HOF prepared in step (1), and stirring for 30min to mix uniformly. Then, to the above mixture was added sodium hydroxide (5mL, 0.02M) and H in that order2O2(100. mu.L). Stirring for 30 minutes, and washing with water, ethanol and acetone for several times to obtain the composite material (HOF-CuO) of the hydrogen bond organic framework and the copper peroxide nanoparticles2)；
(3) Taking 1mg of HOF-Cu prepared in the step (2)O2Dispersing in 1mL of phosphate buffer solution, adding 100. mu.L of lipocalin-2 specific antigen affinity peptide with a concentration of 1.0mg/mL, stirring at 4 ℃ for 12h, centrifuging at 8000rpm for 15min, and washing three times with phosphate buffer solution with pH 7.2 to obtain the bioconjugated probe of the peptide.
(II) method for the detection of lipocalin-2 for non-diagnostic purposes
(1) 100 μ L lipocalin-2 Ab at a concentration of 1 μ g/mL1Adding the mixture into a 96-well enzyme label plate, and incubating overnight at 4 ℃;
(2) washing the ELISA plate in the step (1) for three times by using phosphate buffer solution containing Tween-20, and adding 1% bovine serum albumin solution to block the unbound antibody; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20;
(3) respectively dropwise adding lipocalin-2 solution with the concentration of 0.0001-316 ng/mL into the elisa plate treated in the step (2), incubating for 1h at room temperature, and washing the elisa plate hole with phosphate buffer solution containing Tween-20 for three times after incubation is completed;
(4) dripping 100 mu L of peptide biological conjugate probe into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate for three times by using a phosphate buffer solution containing Tween-20 after the incubation is finished;
(5) 75 μ L of 4-AP (1mg/mL), 75 μ L of 2,4-DP (1mg/mL) and 100 μ L of LMES (pH 6.8) solution were added to the plate and reacted at 65 ℃ for 30min to obtain laccase colorimetric immune biosensors for detecting different concentrations of lipocalin-2.
The application method of the laccase colorimetric immune biosensor of the lipocalin-2 comprises the following specific steps:
(1) taking a color development solution in an enzyme label plate, placing the color development solution in a micro cuvette, and placing the cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 650-400 nm;
(2) recording absorbance peaks corresponding to lipocalin-2 at different concentrations;
(3) detecting lipocalin-2 solutions with different concentrations by using a standard curve method, wherein the detection range is 0.0001-316 ng/mL, and the lower limit of detection (LOD)) Down to 33fgmL-1(S/N＝3)。
FIG. 6 shows the UV absorption spectrum of different concentrations of lipocalin-2 and a standard curve, wherein (A) in FIG. 6 is the UV-visible absorption spectrum of different concentrations of lipocalin-2 developed by TMB, and (B) is the standard curve. The constructed ELISA was used to detect different concentrations of lipocalin-2 under 510nm conditions using a microplate reader, and as can be seen from (A) in FIG. 6, the absorbance value gradually increased with the increase of the concentration of lipocalin-2 toxin. As can be seen in FIG. 6 (B), the absorbance has a good linear relationship with the logarithm of the lipocalin-2 concentration, the correlation coefficient (R)2) 0.9960, LOD 33 fg. mL-1(S/N ═ 3), showing good linearity and lower LOD values.
Example 4
(I) preparation of lipocalin-2 detection probes
(1) Dissolving 150mg of 1,3,6, 8-tetra (4-carboxyphenyl) pyrene in a 22.5mLN, N-dimethylformamide solution, adding 90mL of methanol, stirring for 10min, standing at room temperature for 12h, and purifying with ethanol and acetone for several times to obtain a hydrogen bond organic framework material (HOF);
(2) dissolving 10mg of copper chloride dihydrate in 5mL of water, adding 0.5g of polyvinylpyrrolidone and 1mg of HOF prepared in step (1), and stirring for 30min to mix uniformly. Then, to the above mixture was added sodium hydroxide (5mL, 0.02M) and H in that order2O2(100. mu.L). Stirring for 30 minutes, and washing with water, ethanol and acetone for several times to obtain the composite material (HOF-CuO) of the hydrogen bond organic framework and the copper peroxide nanoparticles2)；
(3) Taking 1mg of HOF-CuO prepared in the step (2)2Dispersing in 1mL of phosphate buffer solution, adding 100. mu.L of lipocalin-2 specific antigen affinity peptide with a concentration of 1.0mg/mL, stirring at 4 ℃ for 12h, centrifuging at 8000rpm for 15min, and washing three times with phosphate buffer solution with pH 7.2 to obtain the bioconjugated probe of the peptide.
(II) anti-interference assay for detecting lipocalin-2
(1) 100 μ L of lipocalin-2 Ab1 with a concentration of 1 μ g/mL was added to a 96-well microplate and incubated overnight at 4 ℃;
(2) washing the ELISA plate in the step (1) for three times by using phosphate buffer solution containing Tween-20, and adding 1% bovine serum albumin solution to block the unbound antibody; after incubation for 1h at room temperature, washing three times with phosphate buffer solution containing tween-20;
(3) 100 μ L of sodium ion (Na) at a concentration of 1 μ g/mL+) Potassium ion (K)+) Glucose (Glu), Ascorbic Acid (AA), Bovine Serum Albumin (BSA), Prostate Specific Antigen (PSA), Human Chorionic Gonadotropin (HCG), Lysozyme (LZ) and lipocalin-2 of 1ng/mL are respectively dripped into the ELISA plate treated in the step (2), incubation is carried out for 1h at room temperature, and after the incubation is finished, the ELISA plate holes are washed three times by phosphate buffer solution containing Tween-20;
(4) dripping 100 mu L of peptide biological conjugate probe into the ELISA plate treated in the step (3), incubating for 1h at room temperature, and washing the ELISA plate for three times by using a phosphate buffer solution containing Tween-20 after the incubation is finished;
(5) adding 150 mu L of 3,3',5,5' -tetramethyl benzidine and 150 mu L of acetic acid buffer solution into an enzyme label plate, and reacting for 30min at 37 ℃ to obtain an anti-interference experiment for analyzing and detecting the lipocalin-2 immune biosensor.
Taking a color development solution in an enzyme label plate, placing the color development solution in a micro cuvette, placing the cuvette in an ultraviolet spectrophotometer for testing, wherein the scanning range is 800-500nm, and recording the absorbance value at 652 nm;
record Na+、K+Glu, AA, BSA, PSA, HCG, LZ and lipocalin-2 samples. The results are shown in FIG. 7. From FIG. 7, it can be seen that the absorbance ratio of the common interferents is low, which indicates that the immune biosensor has good specificity and potential for clinical application.
Example 5
Test for recovery with addition of standard
In order to verify the feasibility and the practicability of the constructed immune biosensor, a labeling recovery experiment is also carried out. A blank sample of human serum was taken, and lipocalin-2 standards of known concentrations of 0.1ng/mL, 1ng/mL, and 10ng/mL were added thereto, respectively, and detection was performed using the immunobiosensor constructed by us and the recovery rate was calculated, and the results are shown in Table 1.
TABLE 1 results of Lipocalin-2 spiking recovery experiments in human serum samples
As can be seen from table 1: the recovery rate of the immune biosensor for detecting lipocalin-2 provided by the invention is 98.42-101.71%, and the standard deviation is 2.12-3.77%, which shows that the analysis accuracy and reliability of the immune biosensor for detecting lipocalin-2 in actual samples are acceptable, and the immune biosensor has potential value in clinical application.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of Yunnan
<120> copper peroxide/hydrogen bond organic framework nano material, probe thereof and kit for detecting lipocalin-2
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Asp Arg Trp Val Ala Arg Asp Pro Ala Ser Ile Phe Gly Gly Gly Gly
1 5 10 15
Ser Cys
Claims (10)
1. Peroxo-acidThe copper/hydrogen bond organic framework nano material comprises a hydrogen bond organic framework material and CuO growing on the surface of the hydrogen bond organic framework material in situ2Nanoparticles; the organic ligand of the hydrogen bond organic framework material is 1,3,6, 8-tetra (4-carboxyphenyl) pyrene.
2. The copper peroxide/hydrogen bond organic framework nanomaterial of claim 1, wherein the CuO is2The mass ratio of the nano particles to the hydrogen bond organic framework material is 5 (0.5-10).
3. The method for preparing the copper peroxide/hydrogen bond organic framework nano material as claimed in claim 1 or 2, which comprises the following steps:
dissolving 1,3,6, 8-tetra (4-carboxyphenyl) pyrene in an alcohol-containing organic solvent for self-assembly to obtain a hydrogen bond organic framework material;
dissolving soluble cupric salt aqueous solution, dispersant and H2O2And mixing inorganic strong base with the hydrogen bond organic framework material to perform coordination reaction to obtain the copper peroxide/hydrogen bond organic framework nano material.
4. The production method according to claim 3, wherein the alcohol-containing organic solvent is a mixed solvent of N, N-dimethylformamide and methanol.
5. A probe for detecting lipocalin-2, which comprises lipocalin-2 specific antigen affinity peptide and the copper peroxide/hydrogen bond organic framework nano material of any one of claims 1-4 in coordination combination with the lipocalin-2 specific antigen affinity peptide.
6. The probe for detecting lipocalin-2 according to claim 5, wherein the lipocalin-2-specific antigen affinity peptide has the amino acid sequence shown in SEQ ID No. 1.
7. The probe of claim 5 or 6, wherein the mass ratio of the lipocalin-2 specific antigen affinity peptide to the copper peroxide/hydrogen bond organic framework nanomaterial is 1: (1-20).
8. Use of the probe for the detection of lipocalin-2 according to any one of claims 5-7 in the preparation of a kit for the detection of lipocalin-2.
9. A kit for detecting lipocalin-2, comprising the probe for detecting lipocalin-2 according to any one of claims 5-7, a first antibody against lipocalin-2, a non-specific protein, and a chromogenic substrate.
10. The kit for detecting lipocalin-2 according to claim 9, wherein the chromogenic substrate is 3,3',5,5' -tetramethylbenzidine;
or 4-AP and 2, 4-DP.
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Non-Patent Citations (2)
| Title |
|---|
| GAVIN J. BEACH ET AL.: ""Inclusion of alkyl alcohol guest molecules into a copper (II)-based hydrogen-bonded metal-organic framework"", 《JOURNAL OF MOLECULAR STRUCTURE》 * |
| 谢丹妍等: ""多孔氢键金属-有机框架材料对乙炔和二氧化碳的吸附分离"", 《化工学报》 * |
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