CN116183897A - Chemiluminescent immunosensor and application thereof - Google Patents
Chemiluminescent immunosensor and application thereof Download PDFInfo
- Publication number
- CN116183897A CN116183897A CN202310163508.2A CN202310163508A CN116183897A CN 116183897 A CN116183897 A CN 116183897A CN 202310163508 A CN202310163508 A CN 202310163508A CN 116183897 A CN116183897 A CN 116183897A
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- CN
- China
- Prior art keywords
- chemiluminescent
- immunosensor
- fluorouracil
- resin beads
- solution
- Prior art date
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Classifications
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
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- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
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Abstract
The invention belongs to the field of chemical biosensors, and particularly relates to a chemiluminescent immunosensor and application thereof. The invention is based on a flow injection chemiluminescence immunoassay method of Fenton-like effect monoatomic cobalt nano enzyme, and is used for rapidly and sensitively detecting 5-fluorouracil in human serum. The antibody is directly coupled with Co-N bond to form stable immune probe. In addition, the carboxyl resin beads have the advantages of good biocompatibility, large specific surface area and the like, and are used for loading antigens. Based on the competitive immune principle, 5-fluorouracil standard solution and 5-fluorouracil coating antigen loaded on carboxyl resin beads compete with limited binding sites on the 5-fluorouracil monoclonal antibody together, and the 5-fluorouracil content can be detected in an ultrasensitive way. The chemiluminescent immunosensor is used for detecting 5-fluorouracil in human serum of an actual sample for the first time, and provides a novel analysis method for detecting 5-fluorouracil in blood or urine of a clinical user and similar small molecules.
Description
Technical Field
The invention belongs to the field of chemical biosensors, and particularly relates to a chemiluminescent immunosensor and application thereof.
Background
Fluoropyrimidines, particularly 5-fluorouracil (5-Fu), capecitabine, tegafur, and cytarabine, are the third most commonly used anticancer drugs currently used to treat solid cancers, including colorectal and breast cancers, with an estimated over 200 thousands of patients receiving fluoropyrimidine treatment each year. 5-fluorouracil, a derivative in which the hydrogen at the 5-position of uracil has been replaced by fluorine, was first introduced as an artificially synthesized anticancer agent 30 years ago. It is widely used for treating breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer and the like, which are common malignant tumors. 5-fluorouracil is advantageous but also disadvantageous. On the one hand, it can be converted to 5-fluorouracil deoxynucleotide (5F-dUMP) in cells to inhibit deoxythymidylate synthase, prevent methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP), and thus affect DNA synthesis. In vivo it can be converted to 5-fluorouracil nucleoside (5-FUR), which is incorporated into RNA to interfere with protein synthesis. If too much 5-fluorouracil target is contained in the human body, there are such problems as loss of appetite, nausea and vomiting, leukopenia and thrombocytopenia, alopecia, nail pigmentation, decline of kidney and cardiac muscle functions and a small number of them are accompanied by cerebellar degeneration and ataxia. On the other hand, low doses of 5-fluorouracil in the blood also reduce the therapeutic efficiency. Therefore, the detection of 5-fluorouracil in human serum or urine is particularly important.
In recent years, flow Injection Chemiluminescence (FICL) technology has been attracting more and more attention because of its simple equipment, convenient operation, high sensitivity and high precision. The flow injection technology is used for pipelining analysis flows of spectrophotometry, fluorescence spectrophotometry, atomic absorption spectrophotometry, nephelometry and ion selective electrode analysis, so that a large amount of complicated manual operation in the original analysis is removed, the intermittent flow is changed into continuous automatic analysis, the artificial error in the experiment is avoided, and the analysis frequency is high because the reaction is not required to be measured after the equilibrium is reached. The Immunoassay (IA) has higher specificity due to the specific binding of antigen and antibody, and is widely used because the reaction factor is relatively small and the reaction control is easy. The flow injection chemiluminescence immunoassay (FI-CLIA) established by the method has the advantages of strong specificity, high sensitivity, wide linear range, no need of a light source, simple instrument structure, convenient operation, high analysis speed and the like. The combination of immunoassay and flow injection chemiluminescence improves the stability of a chemiluminescence system, the sensitivity and reproducibility of analysis results, and the sensitivity can be improved by at least 2 to 3 orders of magnitude, which becomes an important research and application field in modern trace analysis.
Disclosure of Invention
Currently, detection of 5-fluorouracil is mainly focused on chromatography, such as gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography-tandem mass spectrometry, ultra-high performance liquid chromatography-tandem mass spectrometry, and the like. The traditional chromatography is simple to operate and rapid to detect, but has the defects of high technical requirements, expensive equipment, low selectivity and sensitivity and the like, and is insufficient to meet the demands of people. It is therefore desirable to construct a faster, more sensitive and more selective assay for the detection of 5-fluorouracil and similar small molecule substances. Therefore, it is necessary to establish a method for detecting 5-fluorouracil based on flow injection chemiluminescence immunoassay to improve the detection sensitivity and reduce the operation difficulty.
In order to solve the problems, the invention aims to provide a chemiluminescent immunosensor and application thereof in detection of 5-fluorouracil. The invention establishes a competitive chemiluminescence immunosensor based on a flow injection technology, and uses carboxyl resin beads and Co SAzyme as an antigen-loaded carrier and an immune probe respectively for efficiently and rapidly detecting 5-fluorouracil in human serum.
The invention provides a chemiluminescent immunosensor comprising a chemiluminescent probe and a chemiluminescent immunosensor substrate; the chemiluminescent probe is prepared by the following steps:
s11: dissolving zinc nitrate hexahydrate and 2-methylimidazole in alcohol, and reacting to obtain ZIF-8 nano particles;
s12: dissolving the ZIF-8 nano particles in an alcohol aqueous solution, regulating the pH to 10-12, adding cetyltrimethylammonium bromide and tetraethyl orthosilicate, and reacting to obtain ZIF-8@SiO 2 A nanoparticle;
s13: the ZIF-8@SiO is subjected to 2 Dissolving the nano particles in water, and adding a mixed aqueous solution containing cobalt chloride, ammonium chloride and ammonia water for reaction to obtain Co SAzyme precipitate;
s14: incubating the Co SAzyme precipitate with a 5-fluorouracil (5-Fu) antibody to obtain the chemiluminescent probe;
the chemiluminescent immunosensor substrate is prepared by the following steps:
s21: mixing carboxyl resin beads, 2-morpholinoethanesulfonic acid (MES) and an activating reagent for reaction to obtain activated resin beads;
s22: and incubating the activated resin beads and a 5-fluorouracil (5-Fu) coated antigen, and then adding casein to obtain the chemiluminescent immunosensor substrate.
The chemiluminescent immunosensor substrate comprises carboxyl resin beads, the carboxyl resin beads are provided by Nanjing Michaelis high-efficiency separation carrier limited company, a large number of carboxyl functional groups are modified on the carboxyl resin beads, and the carboxyl resin beads are connected with a 5-fluorouracil antibody through chemical bonds.
Preferably, in the step S11, the reaction time is 1.5-2.5h.
Preferably, in the step S11, the alcohol is methanol or ethanol.
Preferably, in the step S11, the precipitate is collected after the reaction and washed 2 to 4 times with methanol.
Preferably, in the step S12, the alcohol is methanol or ethanol.
Preferably, in the step S12, the precipitate is collected after the reaction, and washed with ethanol and water 2 to 4 times.
Preferably, in the step S13, the reaction time is 14-18 hours.
Preferably, in the step S13, the precipitate is collected after the reaction, washed 2 to 4 times with water, and freeze-dried.
Preferably, in the step S14, the mass ratio of the Co SAzyme precipitate to the 5-fluorouracil antibody is 100-150:1.
Preferably, in the step S14, bovine Serum Albumin (BSA) is added for blocking after incubation.
Preferably, in the step S21, the activating reagent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) or N-hydroxysuccinimide (NHS).
Preferably, in the step S21, the reaction time is 1-3 hours.
Preferably, in the step S22, casein is added for blocking after incubation.
Preferably, in the chemiluminescent immunosensor substrate, the mass ratio of the carboxyl resin beads to the 5-fluorouracil-coated antigen is 500-700:1.
Specifically, the Chemiluminescent (CL) immunosensor includes:
chemiluminescent immuno probes, including Co SAzyme linked by a chemical bond to an antibody to 5-fluorouracil;
a chemiluminescent immunosensor substrate comprising a carboxyl resin bead modified with a plurality of carboxyl functional groups that are attached to a 5-fluorouracil antibody via chemical bonds.
Further, the preparation method of the chemiluminescent immune probe comprises the following steps:
(1) Zinc nitrate hexahydrate and 2-methylimidazole were dissolved in methanol and stirred for 2 hours. Subsequently, collecting the precipitate, and washing with methanol three times to obtain ZIF-8 nanoparticles;
(2) The ZIF-8 nanoparticles prepared above were dissolved in methanol. After ultrasonic treatment, ultra-pure water was added to the ZIF-8 solution and stirred at room temperature. The pH of the mixture was adjusted to 11 with sodium hydroxide. Subsequently, hexadecyltrimethylAmmonium bromide was added to the adjusted mixture and stirred. Then, tetraethyl orthosilicate was added, followed by stirring. After standing, collecting precipitate, washing with ethanol and water for three times to obtain ZIF-8@SiOthree times 2 A nanoparticle;
(3) The ZIF-8@SiO prepared above is subjected to 2 The nanoparticles are dispersed in ultrapure water and sonicated. Simultaneously, cobalt chloride hexahydrate and ammonium chloride were added to another ultrapure water solution, and sonicated. Then ammonia is added to the mixture containing cobalt chloride hexahydrate and ammonium chloride. Immediately adding a mixture containing cobalt chloride hexahydrate, ammonium chloride and ammonia to the ZIF-8@SiO 2 In an aqueous nanoparticle solution. After stirring for 16 hours, the precipitate was washed three times with water, and the precipitate was collected by freeze-drying to give Co SAzyme;
(4) Incubating Co SAzyme with a 5-fluorouracil antibody, and connecting the Co SAzyme with the 5-fluorouracil antibody through a Co-N bond to obtain the chemiluminescent probe.
Further, in the step (2), the concentration of sodium hydroxide used was 0.1M.
Further, in the steps (1) (2) (3), the reaction temperature was room temperature.
In step (4), BSA blocking is used in order to eliminate non-specific adsorption;
further, in the chemiluminescent immunoassay probe, the mass ratio of Co SAzyme to 5-fluorouracil antibody is 125:1.
Further, the preparation method of the chemiluminescent immunosensor substrate comprises the following steps:
(S1) soaking carboxyl resin beads purchased from Nanjing Michafie high-efficiency separation Carrier Co., ltd in an ethanol-water mixture solution (V Absolute ethyl alcohol :V Water and its preparation method =1:4), placed in a refrigerator at 4 ℃ for subsequent use;
(S2) placing carboxyl resin beads in a 5mL centrifuge tube, sucking out ethanol supernatant, and washing 3 times with 2-morpholinoethanesulfonic acid (MES);
(S3) adding 2-morpholinoethanesulfonic acid solution and an activating reagent into a centrifuge tube, uniformly mixing the solution and the activating reagent in the centrifuge tube, wrapping the mixture with tinfoil paper, and then placing the mixture on an oscillator for oscillating for 2 hours to activate carboxyl groups on the resin beads;
(S4) after the activation is completed, washing the carboxyl resin beads with PBS buffer solution three times, and adding 5-fluorouracil coating antigen to enable activated carboxyl groups on the carboxyl resin beads to be connected with amino groups on the 5-fluorouracil through an amide reaction.
Further, in the step S2, the concentration of the 2-morpholinoethanesulfonic acid solution used was 0.01M and the pH was 5.8.
Further, in the step S2, 300. Mu.L of carboxyl resin beads were used.
Further, in the step S3, the activating agents are EDC and NHS.
Further, in the step S4, the concentration of the PBS buffer solution is 0.01M, and the pH is 7.4.
Further, after said step S4, the non-specific binding sites are blocked with 2% casein.
Further, in the chemiluminescent immunosensor substrate, the mass ratio of the carboxyl resin beads to the 5-fluorouracil-coated antigen is 600:1.
The invention also provides an application of the chemiluminescent immunosensor for detecting 5-fluorouracil, which comprises the following steps:
s31: mixing a chemiluminescent probe with a standard solution of 5-fluorouracil and then incubating to obtain a compound solution;
s32: adding a chemiluminescent immunosensor substrate into the complex solution, and reacting to obtain an immunosensor composition;
s33: placing the immunosensor composition in a detector, adding 3-amino-phthalhydrazide, H 2 O 2 After buffering the solution, recording a time-luminous intensity curve, and establishing a relation between the chemiluminescent intensity of the immunosensor composition and the concentration of 5-fluorouracil to obtain a linear regression equation;
s34: and according to the linear regression equation, the concentration of the 5-fluorouracil in the solution to be detected is obtained through the luminous intensity of the immunosensor composition.
Preferably, in the step S31, the incubation time is 1-2h.
Specifically, the application of the chemiluminescent immunosensor for detecting 5-fluorouracil comprises the following steps:
(a1) Centrifuging chemiluminescent probe solution at 4500rpm to remove supernatant, and using 0.001-1000ng mL -1 Dissolving and uniformly mixing the precipitate in the centrifuge tube by using 5-fluorouracil standard solutions with different concentrations, and then incubating;
(a2) Washing the chemiluminescent immunosensor substrate with PBS to block the non-specific binding sites;
(a3) Washing the chemiluminescent immunosensor substrate treated in the step a2 with PBST and PBS for three times respectively;
(a4) Adding the immunosensor probe treated in the step a1 into the chemiluminescent immunosensor substrate treated in the step a3, uniformly mixing, and placing on an oscillator for medium-speed oscillation, so that the 5-fluorouracil standard solution and the 5-fluorouracil antigen compete for a binding site on the 5-fluorouracil antibody together. The antigen and the antibody are combined through specific recognition, namely the chemiluminescent immunosensor is constructed;
(a5) C, cleaning the chemiluminescent immunosensor obtained after the treatment in the step a4 by using PBS, and dispersing the washed chemiluminescent immunosensor in PBS buffer solution for later use;
(a6) Injecting the chemiluminescent immunosensor obtained in step a5 into a glass tube, placing the glass tube in a detector, and injecting Luminol and H through 2 peristaltic pumps 2 O 2 And PBS solution, record the time that the computer outputs and shiny intensity curve, establish CL shiny intensity and 5-fluorouracil concentration pair linear relation of numerical value, get the corresponding linear regression equation;
(a7) And c, combining the linear regression equation established in the step a6, and obtaining the concentration of the 5-fluorouracil in the solution to be detected through the luminous intensity of the immunosensor.
Further, in step a1, the chemiluminescent-immuno-probe solution was at a concentration of 1.25mg mL -1 ；
Further, in step a2, the agent for blocking is 2% casein;
further, in step a4, the medium speed oscillation time is 1h, i.e. competing for 1h;
further, in step a5, a process for formulating Luminol and H 2 O 2 Is Tris-HCl buffer solution;
further, in step a5, lumineol and H 2 O 2 The concentration of (C) is 0.1M and 0.8mM, respectively;
further, in step a5, the pump speeds of the 2 peristaltic pumps are 2mLmin, respectively -1 And 0.5mLmin -1 ；
Further, in step a5, the photomultiplier tube high voltage in the detector is-620V, and the amplification factor is 2.
When the chemiluminescent immunosensor is used for detecting 5-fluorouracil, the quantitative basis is that 5-fluorouracil standard solution competes with 5-fluorouracil antigen for limited 5-fluorouracil antibody.
When 5-fluorouracil is not added, there is no standard solution competing with the antigen for the binding site on the antibody, so the 5-fluorouracil coated antigen binds well to the antibody, thus showing a strong CL signal. As the concentration of 5-fluorouracil standard solution increases, the amount of immuno-probe attached to the immunosensor substrate decreases, and the CL signal decreases. The immunosensor has excellent sensitivity, stability and reproducibility, and has important significance for detection of 5-fluorouracil and similar small molecules.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the chemiluminescent immunosensor comprises an immunosensor substrate and a chemiluminescent immunosensor probe, wherein carboxyl resin beads are used as carriers of the immunosensor substrate to load 5-fluorouracil coating antigen, co SAzyme and 5-fluorouracil antibody complex are used as the immunosensor probe, and a flow injection chemiluminescent immunoassay method for detecting the 5-fluorouracil with high sensitivity is developed based on the detection. Based on the principle of competitive immunity, the specific immune reaction between the 5-fluorouracil monoclonal antibody and the antigen is used for quantitatively detecting the 5-fluorouracil. The invention improves the selectivity and sensitivity of the target detection object, and the immunosensor using equipment has the advantages of simple structure, simple and convenient operation, easy automatic continuous analysis, high precision, small reagent sample consumption, wide applicability and the like, has high sensitivity and wide detection range, and is suitable for detecting small molecules of 5-fluorouracil and other similar structures.
The foregoing description is only an overview of the present invention, and is presented in terms of preferred embodiments of the present invention and the following detailed description of the invention in conjunction with the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of the preparation process principle and the detection device diagram of an immunosensor for detecting 5-fluorouracil.
Fig. 2 is a TEM image of Co SAzyme nanomaterials.
FIG. 3 is a graph of CL-time curves and standard curves of luminescence intensity versus logarithm of 5-fluorouracil concentration for different 5-fluorouracils.
Fig. 4 is an SEM image of carboxyl resin beads.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
In the following examples of the invention, both the 5-Fu coated antigen and the 5-Fu monoclonal antibody were given to the university of Suzhou Deng Anping professor task group. Wherein, the 5-Fu monoclonal antibody is a monoclonal antibody secreted by 5-Fu hybridoma cell line 4G11-A11 (preserved in China center for type culture collection, with the preservation number of CCTCC No. C2020249, with the preservation address of university of Wuhan in China and the preservation date of 2020, 11 months and 24 days). The preparation of the antibody firstly determines proper modification sites in the 5-Fu molecular structure according to hapten molecular design theory and 5-Fu molecular structure characteristics, synthesizes two 5-Fu modifications with different connecting arm lengths and carboxyl end groups, is connected with carrier protein, and then prepares the monoclonal antibody with high sensitivity and high specificity for resisting the 5-Fu through the steps of animal immunization, fusion, screening and the like by applying hybridoma antibody preparation technology. The IC50 value of ELISA method for measuring 5-Fu established based on the antibody is 20ng/mL, and the cross reaction rate with uracil, cytosine, thymine, 5-bromouracil, 5-fluoro-1, 3-dimethylpyrimidine, uridine, 5-bromo-2' -deoxyuridine, capecitabine and gimeracil is less than 0.1%.
Example 1
The detailed preparation scheme of the chemiluminescent immunosensor based on the monoatomic cobalt nano-enzyme is as follows:
(1) Preparation of Co SAzyme nanomaterial
Synthesis of ZIF-8 NPs: zn (NO) 3 ) 2 ·6H 2 O (3.0 g) and 2-methylimidazole (3.5 g) were dissolved in methanol (125 mL), and then the two solutions were mixed and stirred for 2h, followed by collecting the precipitate and washing with methanol three times.
ZIF-8@SiO 2 Synthesis of NPs: ZIF-8@SiO 2 NPs are obtained by the Stober method. The ZIF-8NPs prepared were dissolved in methanol (25 mL). Ultrasonic treatment was performed for 15min, and ultra-pure water (200 mL) was added to the ZIF-8 solution and stirred at room temperature for 5min. The pH of the mixture was then adjusted to 11 by adding the desired 0.1M sodium hydroxide. Subsequently, cetyltrimethylammonium bromide (0.25 g) was added to the adjusted mixture and stirred for 30min. Then, tetraethyl orthosilicate (1.25 mL) was added and stirred for an additional 30min. Standing for 15min, collecting precipitate, and washing with ethanol and water three times.
Synthesis of Co SAzyme: co SAzyme was synthesized by a room temperature etching strategy. The ZIF-8@SiO prepared above is subjected to 2 The nanoparticles were dispersed in ultrapure water (15 mL) and sonicated for 15min. At the same time, coCl 2 ·6H 2 O (0.476 g,2 mmol) and ammonium chloride (0.401 g,7.5 mmol) were added to another 15mL of ultra-pure water and dissolved by sonication. Then, will contain CoCl 2 ·6H 2 A mixture of O and ammonium chloride was added to 15mL of NH 3 ·H 2 O (wt.28%, 3.5 mL). Will contain CoCl 2 ·6H 2 O, ammonium chloride and NH 3 ·H 2 The mixture of O was immediately added with ZIF-8@SiO 2 In an aqueous solution of NPs. The precipitate was stirred for 16h, washed three times with water and then collected by freeze drying.
(2) Preparation of chemiluminescent immuno probes
First, the freeze-dried Co SAzymes were precisely weighed and dissolved with ultra-pure water to prepare nanoparticle solutions of a certain concentration. Next, 99. Mu.L of the nanoparticles were added to a 0.5mL centrifuge tube and left to stand by. Will 1.12mg mL -1 The 5-Fu antibody stock solution of (2) was taken out from a refrigerator at 0℃to extract 10. Mu.L, and diluted to 100. Mu.gmL with 0.01M PBS buffer solution -1 Then to 1.25mgmL -1 To the nanoparticle solution of (2) was added 11. Mu.L of the above antibody solution. After mixing the two lightly, the mixture was placed in a refrigerator at 4℃and incubated overnight. The following day, 6.8 μl of 5% Bovine Serum Albumin (BSA) was added to each centrifuge tube to block non-specific binding sites. After 2h, unbound BSA and antibody were removed by centrifugation (4500 rpm,5 min). Finally, redissolving the mixture by using a 5-fluorouracil standard solution with a certain concentration, and placing the mixture in a refrigerator with the temperature of 4 ℃ for storage for later use.
(3) Preparation of chemiluminescent immunosensor substrates
First, carboxyl resin beads were dissolved in an ethanol-water mixed solution (V Absolute ethyl alcohol :V Water and its preparation method =1:4), placed in a refrigerator at 4 ℃ for subsequent use. 300. Mu.L of carboxyl resin beads were placed in a 5mL centrifuge tube, the ethanol supernatant was aspirated off, and washed 3 times with 0.01 mM 2-morpholinoethanesulfonic acid (MES) pH 5.8. To the centrifuge tube was added 0.01M MES,8mg mL -1 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and 8mgmL -1 N-hydroxysuccinimide (NHS) was mixed well in a test tube, wrapped with tinfoil and protected from light. Then, the mixture was placed on an oscillator, and the mixture was oscillated at a medium-high speed for 2 hours, thereby activating the carboxyl groups on the resin beads. Then 2.6mg mL of the solution was buffered with 0.01M PBS -1 Is diluted to 5 mu gmL -1 . Finally, 1mL of diluted coated antigen stock solution is added to the activated resin beads, and the mixture is placed in a refrigerator at 4 ℃ for incubation overnight. The next day, the antigen-modified resin beads were washed with 0.01M PBS and blocked by adding 1mL of 2% casein to each centrifuge tube. After blocking for 2h, the mixture was washed 3 times with PBST and PBS, respectively, and finally dispersed in 0.01MPBS buffer solution, and placed in a refrigerator at 4℃for subsequent useIs used.
Example 2
The chemiluminescent immunosensor prepared in example 1 was quantitatively detected for 5-fluorouracil as follows:
(1) After the supernatant was first removed by centrifugation of 99. Mu.L of Ab-Co SAzyme complex solution, the lower precipitate was then taken and added to 200. Mu.L of different concentrations (0.001 ng mL) -1 ，0.01ng mL -1 0.1ng mL-1,1ng mL-1,10ng mL-1,100ng mL-1 and 1000ng mL-1) of the 5-fluorouracil standard solution, and then adding the complex solution to the prepared immunosensor substrate, wherein both 5-Fu-Ae and 5-Fu can bind to the 5-Fu-Ab. After 1h, ab-Co SAzyme that did not bind to 5-Fu-Ae and excess 5-Fu were washed out with PBS. Finally, a solution containing 0.1M Luminol (Luminol, also known as Luminol, chemical name 3-amino-phthalhydrazide, CAS number 521-31-3) and 8mM H 2 O 2 Is used to position the immunosensor by controlling the speed of the peristaltic pump. The chemiluminescent signal detected by the photomultiplier (-620V, ×2) is linearly inversely correlated with the concentration of the 5-Fu standard solution, and the signal is converted by a computer to be displayed as an amplified electrical signal, and a time-luminous intensity curve is recorded. Establishing a linear relation between CL luminous intensity and 5-fluorouracil concentration logarithmic value, wherein a linear equation is I CL ＝-2278.44logC+10884.01(R 2 =0.998). Therefore, the immunosensor can detect a signal to noise ratio of 3 in a range of 0.001 to 1000ng mL-1, and a LOD of 0.31pg mL-1.
A, B of FIG. 3 is a graph of luminescence signal intensity versus time and a standard curve of luminescence intensity versus logarithm of 5-fluorouracil concentration for different concentrations of 5-fluorouracil, respectively, and in FIG. 3A, the concentrations of 5-fluorouracil standard solutions corresponding to each adjacent three peaks are 0.001ng mL-1,0.01ng mL-1,0.1ng mL-1,1ng mL-1,10ng mL-1,100ng mL-1, and 1000ng mL-1, in order from left to right.
(2) Analog detection of 5-fluorouracil in human serum
In order to evaluate the detection capability of the immunosensor for an actual sample, a labeled recovery experiment for human serum was performed. Added samples of 0.001, 0.01, 0.1 and 1ng mL-1 human-containing serum were prepared. As shown in the table, the recovery rate of the immunosensor is between 98.5% and 112.1%, and the errors are all within an acceptable range. The experimental results show that the adopted analysis method and the CL immunosensor have feasibility and good practical application value for detecting the 5-Fu in the human serum sample.
TABLE 1CL immunosensor for 5-fluorouracil in actual sample labeled recovery measurement results
N.d. =undetected.
The invention develops a flow injection chemical immunoassay method based on Fenton-like effect Co SAzyme, and is applied to ultrasensitive detection of 5-fluorouracil in human serum for the first time. In one aspect, unlike many methods reported in the literature that require pyrolysis to prepare monoatomic nanoenzymes, co SAzyme was synthesized by a novel non-high temperature treated in situ etching strategy. Compared with natural peroxidase HRP used in the previous experiment, co SAzyme has high loading capacity, stable property and simple preparation. The method also simplifies experimental steps, does not need modification, can be directly attached to the antibody through Co-N bond, and is easy to fix with the antibody. Moreover, the material has high Fenton-like activity and can effectively catalyze the decomposition of hydrogen peroxide, thereby amplifying Luminol-H 2 O 2 CL signal of the system. On the other hand, the carboxyl resin beads with good biocompatibility and large specific surface area are used as substrates to load the 5-fluorouracil coating antigen, so that the loading efficiency is high and the property is stable. The invention provides a novel detection method for detecting the 5-fluorouracil in the human serum, and has good practical value and practical significance.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. A chemiluminescent immunosensor, wherein the chemiluminescent immunosensor comprises a chemiluminescent probe and a chemiluminescent immunosensor substrate; the chemiluminescent probe is prepared by the following steps:
s11: dissolving zinc nitrate hexahydrate and 2-methylimidazole in alcohol, and reacting to obtain ZIF-8 nano particles;
s12: dissolving the ZIF-8 nano particles in an alcohol aqueous solution, regulating the pH to 10-12, adding cetyltrimethylammonium bromide and tetraethyl orthosilicate, and reacting to obtain ZIF-8@SiO 2 A nanoparticle;
s13: the ZIF-8@SiO is subjected to 2 Dissolving the nano particles in water, and adding a mixed aqueous solution containing cobalt chloride, ammonium chloride and ammonia water for reaction to obtain Co SAzyme precipitate;
s14: incubating the Co SAzyme precipitate and a 5-fluorouracil antibody to obtain the chemiluminescent probe;
the chemiluminescent immunosensor substrate is prepared by the following steps:
s21: mixing carboxyl resin beads, 2-morpholinoethanesulfonic acid and an activating reagent for reaction to obtain activated resin beads;
s22: and incubating the activated resin beads and the 5-fluorouracil coating antigen to obtain the chemiluminescent immunosensor substrate.
2. The chemiluminescent immunosensor of claim 1, wherein the reaction time in step S11 is 1.5-2.5 hours.
3. The chemiluminescent immunosensor of claim 1, wherein the reaction time in step S13 is 14-18 hours.
4. The chemiluminescent immunosensor of claim 1, wherein in step S14, the mass ratio of Co SAzyme precipitate to 5-fluorouracil antibody is 100-150:1.
5. The chemiluminescent immunosensor of claim 1, wherein in step S14, bovine serum albumin is added for blocking after incubation.
6. The chemiluminescent immunosensor of claim 1, wherein the activating reagent in step S21 is 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.
7. The chemiluminescent immunosensor of claim 1, wherein the reaction time in step S21 is 1-3 hours.
8. The chemiluminescent immunosensor of claim 1, wherein in step S22, casein is added for blocking after incubation.
9. The chemiluminescent immunosensor of claim 1, wherein the mass ratio of carboxyl resin beads to 5-fluorouracil-coated antigen in the chemiluminescent immunosensor substrate is 500-700:1.
10. Use of a chemiluminescent immunosensor according to any one of claims 1 to 9 for detecting 5-fluorouracil, comprising the steps of:
s31: mixing a chemiluminescent probe with a standard solution of 5-fluorouracil and then incubating to obtain a compound solution;
s32: adding a chemiluminescent immunosensor substrate into the complex solution, and reacting to obtain an immunosensor composition;
s33: placing the immunosensor composition in a detector, adding 3-amino-phthalhydrazide, H 2 O 2 After buffering the solution, recording the time-luminous intensity curve, and establishing immunosensoryObtaining a linear regression equation according to the relation between the chemiluminescent intensity of the composition and the concentration of the 5-fluorouracil;
s34: and according to the linear regression equation, the concentration of the 5-fluorouracil in the solution to be detected is obtained through the luminous intensity of the immunosensor composition.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310163508.2A CN116183897A (en) | 2023-02-24 | 2023-02-24 | Chemiluminescent immunosensor and application thereof |
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