CN115304781B - Amplification-free high-efficiency electrochemiluminescence probe based on MOF framework and preparation method and application thereof - Google Patents
Amplification-free high-efficiency electrochemiluminescence probe based on MOF framework and preparation method and application thereof Download PDFInfo
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Abstract
The application discloses an amplification-free high-efficiency electrochemiluminescence probe based on an MOF framework, and a preparation method and application thereof. In the MOF frame synthesis process, adding a high-efficiency electrochemiluminescence agent of tris (4, 4-dicarboxyl bipyridine) ruthenium chloride, wherein carboxyl groups on the high-efficiency electrochemiluminescence agent can be added into the MOF frame through coordination, so that a single MOF frame stably loads a large amount of electrochemiluminescence agent, and a high-efficiency electrochemiluminescence nanocrystal is obtained; modifying a layer of polydopamine film on the surface of the nanocrystal; and then is connected with a nucleic acid probe/antibody with amino or sulfhydryl to form the amplification-free high-efficiency electrochemiluminescence probe. The application has the advantages that: the amplification-free high-efficiency electrochemiluminescence probe is hopeful to simplify the detection process of nucleic acid and protein markers, shorten the detection time and ensure high-sensitivity analysis performance, and has important scientific value and practical significance for timely detection of infectious pathogens.
Description
Technical Field
The application relates to the technical field of biological detection, in particular to an electrochemiluminescence probe technology.
Background
The frequency and spread of infectious diseases represented by new crowns presents a great challenge to global economy and public health safety. Along with the continuous variation of viruses, the prevention and control means depending on vaccination are restricted, and the rapid pathogen marker detection is still the most effective means for accurately preventing and controlling infectious diseases. PCR and ELISA are the most commonly used marker detection methods at present, and have the advantages of high sensitivity, good specificity and the like, but an enzymatic process is inevitably required to obtain higher sensitivity, so that the test cost and the operation complexity are increased. The electrochemiluminescence method is an analysis method with simple operation, low cost, low background, high signal and wide linear range, and is widely applied to the technical field of biological detection, but the existing electrochemiluminescence probe has limited luminous efficiency, so that the detection sensitivity is difficult to compare favorably with that of an enzymatic method. Therefore, development of a novel low-cost, amplification-free and high-sensitivity electrochemiluminescence probe is needed, and the novel low-cost, amplification-free and high-sensitivity electrochemiluminescence probe has important scientific value and practical significance for real-time high-sensitivity detection of infectious diseases.
Disclosure of Invention
The application aims to provide an amplification-free high-efficiency electrochemiluminescence probe based on an MOF frame and a preparation method and application thereof, so as to solve the problems of low luminescence efficiency, high cost, insensitive reaction and the like of the electrochemiluminescence probe in the prior art.
In order to achieve the above purpose, the application adopts the following technical scheme:
the application provides an amplification-free high-efficiency electrochemiluminescence nanocrystal based on an MOF framework, which contains an electrochemiluminescence group of tris (4, 4-dicarboxy bipyridine) ruthenium chloride.
The application also provides a preparation method of the amplification-free high-efficiency electrochemiluminescence nanocrystal based on the MOF framework, which comprises the following steps: in the MOF frame synthesis process, the efficient electrochemiluminescence agent of tris (4, 4-dicarboxyl bipyridine) ruthenium chloride is added, and carboxyl on the efficient electrochemiluminescence agent can be added into the MOF frame through coordination, so that a single MOF frame stably loads a large amount of electrochemiluminescence agent, and the efficient electrochemiluminescence nanocrystal which is named as MOF nanocrystal is obtained.
Further, the method comprises the following steps:
2-amino terephthalic acid and a part of ferric chloride hexahydrate in the prescription amount are uniformly stirred in a solvent to form a core; then adding tris (4, 4-dicarboxy bipyridine) ruthenium chloride and the balance ferric chloride hexahydrate, heating and reacting for growth to obtain the high-efficiency electrochemiluminescence nano crystal.
Further, the morphology and the electrochemiluminescence efficiency of the nanocrystals are controlled by adjusting the reactant feeding ratio, the reaction temperature, the pretreatment method and other conditions. The detailed steps include:
(1) Soaking the reaction vessel in aqua regia for 30min, and then cleaning and drying;
(2) Adding 30ml of N, N-dimethylformamide, 0.1-0.5mmol of 2-amino terephthalic acid and 0.01-0.05 mmol of ferric chloride hexahydrate into a reaction vessel, uniformly stirring, and carrying out ultrasonic treatment for 15min-2h;
(3) Continuously adding 0.19-0.95mmol of ferric chloride hexahydrate and 1-100 mu mol of tris (4, 4-dicarboxy bipyridine) ruthenium chloride into the reaction kettle (2), uniformly stirring, placing the reaction kettle on an oil bath kettle at 80-120 ℃, reacting for 1-5h, and cooling to room temperature; centrifuging at 8000rpm, washing, and drying at 50 ℃ to obtain the high-efficiency electrochemiluminescence nano crystal.
The application also provides an amplification-free high-efficiency electrochemiluminescence probe based on the MOF framework, which contains electrochemiluminescence groups of tris (4, 4-dicarboxy bipyridine) ruthenium chloride and the probe.
A preparation method of an amplification-free high-efficiency electrochemiluminescence probe based on an MOF framework comprises the following steps: based on the advantages of high porosity, good conductivity and the like of the MOF frame, in the synthesis process of the MOF frame, the high-efficiency electrochemiluminescence agent of tris (4, 4-dicarboxyl bipyridine) ruthenium chloride is added, and carboxyl on the high-efficiency electrochemiluminescence agent can be added into the MOF frame through coordination, so that a single MOF frame stably loads a large amount of electrochemiluminescence agent, and the high-efficiency electrochemiluminescence nanocrystal is obtained; modifying a layer of polydopamine film on the surface of the high-efficiency electrochemiluminescence nanocrystal to obtain an MOF-PDA nanocrystal; PDA has benzoquinone structure, no need of activating step, and can be connected with nucleic acid probe/antibody with amino or sulfhydryl based on Schiff base reaction or Michael addition reaction, and the PDA is used as connecting medium to make the nucleic acid probe or antibody stably connected on the MOF-PDA nanocrystal under the condition of no activation so as to form the universal amplification-free high-efficiency electrochemiluminescence probe for protein nucleic acid detection.
Further, the method comprises the following steps:
2-amino terephthalic acid and a part of ferric chloride hexahydrate in the prescription amount are uniformly stirred in a solvent to form a core; continuously adding tris (4, 4-dicarboxy bipyridine) ruthenium chloride and the balance ferric chloride hexahydrate, and heating for reaction and growth to prepare the high-efficiency electrochemiluminescence nanocrystal; dispersing the high-efficiency electrochemiluminescence nanocrystals in a mixed solution, adding dopamine hydrochloride, and heating, oxidizing and polymerizing to prepare MOF-PDA nanocrystals; and incubating the MOF-PDA nanocrystals with amino or sulfhydryl modified nucleic acid probes/antibodies to prepare the amplification-free high-efficiency electrochemiluminescence probes.
Further, the detailed process includes:
(1) Soaking the reaction vessel in aqua regia for 30min, and then cleaning and drying;
(2) Adding 30ml of N, N-dimethylformamide, 0.1-0.5mmol of 2-amino terephthalic acid and 0.01-0.05 mmol of ferric chloride hexahydrate into a reaction vessel, uniformly stirring, and carrying out ultrasonic treatment for 15min-2h;
(3) Continuously adding 0.19-0.95mmol of ferric chloride hexahydrate and 1-100 mu mol of tris (4, 4-dicarboxy bipyridine) ruthenium chloride into the reaction kettle (2), uniformly stirring, placing the reaction kettle on an oil bath kettle at 80-120 ℃, reacting for 1-5h, and cooling to room temperature; centrifuging at 8000rpm, washing, and drying at 50 ℃ to obtain high-efficiency electrochemiluminescence nanocrystals;
(4) Dispersing the nanocrystals obtained in the step (3) in a mixed solution, adding 0.1-2mg/ml dopamine hydrochloride, heating and oxidizing for polymerization for 1-5h at 50-80 ℃, naturally cooling, centrifuging at 5000rpm, cleaning and re-suspending to obtain MOF-PDA nanocrystals;
(5) And (3) adding 0.2-5 mu mol of amino or sulfhydryl modified nucleic acid probe/antibody into the MOF-PDA nanocrystal in the step (4), uniformly mixing, incubating overnight at room temperature, centrifuging at 5000rpm, cleaning and re-suspending to obtain the amplification-free high-sensitivity electrochemiluminescence probe.
More preferably, the mixed solution in the step (4) is prepared according to a volume ratio EtOH: h2o=2:1.
More preferably, the nucleic acid probe in the step (5) is a thiol-modified nucleic acid probe obtained by reducing the nucleic acid probe with TCEP at room temperature for 1 hour in a molar ratio of DNA: tcep=1:10.
The application also provides an application of the amplification-free high-efficiency electrochemiluminescence probe based on the MOF framework in the field of biosensing.
The advantages of the application include:
(1) According to the application, the conditions required by MOF frame synthesis are optimized, the experimental condition requirements that a high-pressure reaction kettle and a high-temperature oven are required to be used in a hydrothermal synthesis method are reduced, the time required by the hydrothermal synthesis method is greatly shortened and the uniformity of product particles is improved through ultrasonic separation nucleation and growth processes, and the method has the advantages of simplicity in operation, high synthesis efficiency and the like;
(2) The application provides a simple nucleic acid probe modification method, which avoids the traditional functional group activation step, and can be expanded to specific antibody modification, thereby having universality;
(3) The application provides an amplification-free high-efficiency electrochemiluminescence probe, which can simplify the nucleic acid detection flow, avoid the complex and high-cost enzymatic amplification process and ensure the rapid and high-sensitivity detection performance.
(4) The POCT detection system is easy to integrate with portable detection equipment, and has the application potential of POCT detection.
(5) The application provides a universal electrochemiluminescence probe with low cost, no amplification and high sensitivity.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and constitute a part of this specification, are incorporated in and constitute a part of this specification and do not limit the application in any way, and in which:
FIG. 1 is a schematic diagram of the principle of preparing an amplification-free high-efficiency electrochemiluminescence probe based on a MOF framework;
FIGS. 2-3 are graphs of synthetic probe characterization results;
FIG. 4 is a schematic diagram showing the results of co-incubation of an amplification-free high-efficiency electrochemiluminescence probe with target nucleic acid under electrical excitation conditions after co-incubation of the electrochemiluminescence probe MOF-PDA-DNA with an experimental group with/without target nucleic acid;
FIG. 5 is a graph showing the results of using the amplification-free high-efficiency electrochemiluminescence probe for nucleic acid detection in example 2.
Detailed Description
The present application will now be described in detail with reference to the drawings and the specific embodiments thereof, which are illustrative embodiments and illustrations of the application, but are not to be construed as limiting the application.
Example 1
1. The embodiment provides a preparation method of an amplification-free high-efficiency electrochemiluminescence probe based on an MOF framework, the preparation principle is shown in figure 1, and the preparation steps are as follows:
(1) Soaking a 50ml single-neck flask in freshly prepared aqua regia for 30min, then washing with deionized water, and drying for later use;
(2) Into the flask was charged 30ml of N, N-dimethylformamide, 0.1mmol of 2-aminoterephthalic acid (BDC-NH) 2 ) And 0.01mmol of ferric chloride hexahydrate (Fe) 3+ ) Stirring uniformly, and carrying out ultrasonic treatment for 1h.
(3) To (2) was added continuously 0.19mmol of iron chloride hexahydrate (Fe) 3+ ) And 1 mu mol of terpyridyl ruthenium (Ru (dcbpy) 3 2+ ) The electrochemical luminescent agent, namely tris (4, 4-dicarboxy bipyridine) ruthenium chloride, is stirred uniformly, is placed on an oil bath pot, is heated to 100 ℃, reacts for 3 hours, and is naturally cooled to room temperature. Centrifuging at 8000rpm, washing the precipitate with N, N-dimethylformamide for three times, and placing in a vacuum drying oven, and drying at 50deg.C to obtain high-efficiency electrochemiluminescence nanocrystals, which are named MOF nanocrystals;
(4) Dispersing the nano crystal in the step (3) in a mixed solution which is EtOH 2 O (v: v) =2:1; adding 0.1mg/ml dopamine hydrochloride (DA), heating, oxidizing and polymerizing for 2 hours at 50 ℃, naturally cooling, centrifuging at 5000rpm, washing with pure water for 3 times, and re-suspending to obtain MOF-PDA nanocrystals.
(5) Taking MOF-PDA nanocrystals in (4), adding 2. Mu. Mol of sulfhydryl or amino modified nucleic acid probe (DNA-SH/NH) 2 ) Mixing uniformly, incubating overnight at room temperature, centrifuging at 5000rpm, washing 3 times with pure water, and re-suspending to obtain the amplification-free high-efficiency electrochemiluminescence probe MOF-PDA-DNA.
The thiol-modified nucleic acid probe needs to be reduced for 1h with TCEP at room temperature, and the nucleic acid probe is tcep=1:10 according to the molar ratio; the amino modified nucleic acid probe is directly incubated with MOF-PDA nanocrystals without a reduction process.
2. Amplification-free efficient electrochemiluminescence probe synthesis result characterization
Synthesizing MIL-101 (Fe) nanocrystals by referring to the steps (1) - (3) above, except that ruthenium terpyridyl is not added in the synthesis;
(a) The morphology of the synthesized and modified nanocrystals is characterized by a transmission electron microscope, DLS and Zeta potential;
(b) Testing the luminous performance of the probe by an electrochemiluminescence instrument;
the test results are shown in fig. 2-3, and each result is analyzed as follows:
FIGS. 2A-C are TEM morphology characterization of MIL-101 (Fe)/MOF/MOF-PDA nanocrystals, respectively, showing that the incorporation of Ru in the MOF framework has substantially no effect on the morphology of MIL-101 (Fe) crystals, and that PDA can successfully encapsulate MOF nanocrystals, providing active sites for subsequent probe modification.
FIG. 3D-E are graphs showing particle size and potential characteristics of MOF/MOF-PDA/MOF-PDA-DNA nanocrystals, wherein after PDA and DNA probes are combined, the particle size of hydration is obviously increased, and the potential changes from negative to positive, thus indicating the feasibility of the modification method, and the method can be used for subsequent nucleic acid detection.
FIG. 3F is a graph of the electrochemiluminescence properties of MOF/MOF-PDA/MOF-PDA-DNA nanocrystals, showing highly efficient ECL properties before and after modification of the MOF nanocrystals.
As shown in the left diagram of fig. 4, the capture probes are modified on the electrodes and can be complementarily combined with half of the sequence of the target, so that the target is captured; the other half sequence of the target is complementarily combined with the amplification-free high-efficiency electrochemiluminescence probe MOF-PDA-DNA, and under the action of electric excitation, the electrochemiluminescence probe emits 620nm light, as shown in the right graph of FIG. 4, which is the experimental result of the inventor in the process of developing the application, and the electrochemiluminescence probe MOF-PDA-DNA and the experimental group containing target nucleic acid are incubated together to find obvious electrochemiluminescence response; whereas for the experimental group without target nucleic acid, no electrochemiluminescence response occurred.
Example 2
The present example provides a method for rapid amplification-free, high-sensitivity detection of a specific fragment of the DNA polymerase I gene (polA) of treponema pallidum using the high-efficiency electrochemiluminescence probe of example 1, comprising the steps of:
(1) In the embodiment of the application, firstly, a body fluid sample of a subject is collected in a conventional collection mode, pathogenic microorganisms in the sample can be cracked through high-temperature cracking, so that nucleic acid substances are released, and nucleic acid in a biological sample is extracted by a nucleic acid instrument;
for target nucleic acids, the present example contemplates capture probes, nucleic acid probes;
the capture probe sequence is: 5'-TCATTC CAAAGA CGT CGAAGC-SH-3';
the nucleic acid probe sequence is: 5'-HS-AAAAAAAAAAAAA TGG TGC ATG ACA GCT TGGACA-3';
preparing an amplification-free high-efficiency electrochemiluminescence probe MOF-PDA-DNA according to reference example 1;
(2) Immobilizing a capture probe on an electrochemiluminescence test gold electrode through Au-S for capturing target nucleic acid;
(3) Dropwise adding the target nucleic acid extracted in the step (1) onto the electrode modified in the step (2);
(4) Washing the electrode in (3) with PBS 2 times;
(5) Continuously dripping the amplification-free high-efficiency electrochemiluminescence probe MOF-PDA-DNA onto the electrode in the step (4);
(6) Washing the electrode in (5) with purified water for 2 times;
(7) Dropwise adding an electrochemiluminescence auxiliary liquid tripropylamine on the electrode;
(8) And starting the electrochemiluminescence instrument for testing.
Meanwhile, a blank control group without target nucleic acid is arranged, the detection result is shown in figure 5, the electrochemiluminescence response is obvious in a system containing the target nucleic acid, and the electrochemiluminescence response is not obvious in a system without the target nucleic acid. The test result proves that the method can be used for rapid amplification-free high-sensitivity detection of nucleic acid.
The application provides an amplification-free high-efficiency electrochemiluminescence probe based on a luminescence group enrichment strategy, which is expected to simplify the detection process of nucleic acid and protein markers, shorten the detection time and ensure high-sensitivity analysis performance, and has important scientific value and practical significance for timely detection of infectious pathogens.
The foregoing has described in detail the technical solutions provided by the embodiments of the present application, and specific examples have been applied to illustrate the principles and implementations of the embodiments of the present application, where the above description of the embodiments is only suitable for helping to understand the principles of the embodiments of the present application; meanwhile, as for those skilled in the art, according to the embodiments of the present application, there are variations in the specific embodiments and the application scope, and the present description should not be construed as limiting the present application.
Claims (6)
1. A preparation method of an amplification-free efficient electrochemiluminescence probe based on an MOF framework is characterized by comprising the following steps of:
the method comprises the following steps: in the MOF frame synthesis process, adding a high-efficiency electrochemiluminescence agent of tris (4, 4-dicarboxyl bipyridine) ruthenium chloride, wherein carboxyl groups on the high-efficiency electrochemiluminescence agent can be added into the MOF frame through coordination, so that a single MOF frame stably loads a large amount of electrochemiluminescence agent, and a high-efficiency electrochemiluminescence nanocrystal is obtained; modifying a layer of polydopamine film on the surface of the high-efficiency electrochemiluminescence nanocrystal to obtain an MOF-PDA nanocrystal; and then is connected with a nucleic acid probe/antibody with amino or sulfhydryl to form the amplification-free high-efficiency electrochemiluminescence probe.
2. The method for preparing the amplification-free high-efficiency electrochemiluminescence probe based on the MOF framework, which is characterized by comprising the following steps of:
the method comprises the following steps:
2-amino terephthalic acid and a part of ferric chloride hexahydrate in the prescription amount are uniformly stirred in a solvent to form a core; continuously adding tris (4, 4-dicarboxy bipyridine) ruthenium chloride and the balance ferric chloride hexahydrate, and heating for reaction and growth to prepare the high-efficiency electrochemiluminescence nanocrystal; dispersing the high-efficiency electrochemiluminescence nanocrystals in a mixed solution, adding dopamine hydrochloride, and heating, oxidizing and polymerizing to prepare MOF-PDA nanocrystals; and incubating the MOF-PDA nanocrystals with amino or sulfhydryl modified nucleic acid probes/antibodies to prepare the amplification-free high-efficiency electrochemiluminescence probes.
3. The method for preparing the amplification-free high-efficiency electrochemiluminescence probe based on the MOF framework, which is characterized by comprising the following steps of:
the detailed process comprises the following steps:
(1) Soaking the reaction vessel in aqua regia for 30min, and then cleaning and drying;
(2) Adding 30ml of N, N-dimethylformamide, 0.1-0.5mmol of 2-amino terephthalic acid and 0.01-0.05 mmol of ferric chloride hexahydrate into a reaction vessel, uniformly stirring, and carrying out ultrasonic treatment for 15min-2h;
(3) Continuously adding 0.19-0.95mmol of ferric chloride hexahydrate and 1-100 mu mol of tris (4, 4-dicarboxy bipyridine) ruthenium chloride into the reaction kettle (2), uniformly stirring, placing the reaction kettle on an oil bath kettle at 80-120 ℃, reacting for 1-5h, and cooling to room temperature; centrifuging at 8000rpm, washing, and drying at 50 ℃ to obtain high-efficiency electrochemiluminescence nanocrystals;
(4) Dispersing the nanocrystals obtained in the step (3) in a mixed solution, adding 0.1-2mg/ml dopamine hydrochloride, heating and oxidizing for polymerization for 1-5h at 50-80 ℃, naturally cooling, centrifuging at 5000rpm, cleaning and re-suspending to obtain MOF-PDA nanocrystals;
(5) And (3) adding 0.2-5 mu mol of amino or sulfhydryl modified nucleic acid probe/antibody into the MOF-PDA nanocrystal in the step (4), uniformly mixing, incubating overnight at room temperature, centrifuging at 5000rpm, cleaning and re-suspending to obtain the amplification-free high-sensitivity electrochemiluminescence probe.
4. The method for preparing the amplification-free high-efficiency electrochemiluminescence probe based on the MOF framework, which is characterized in that:
the mixed solution in the step (4) is prepared from the following components in volume ratio EtOH to H 2 O=2:1;
the nucleic acid probe in the step (5) is a sulfhydryl-modified nucleic acid probe, which is obtained by reducing the nucleic acid probe with TCEP for 1h at room temperature according to the molar ratio of DNA: tcep=1:10.
5. An amplification-free, high-efficiency electrochemiluminescence probe based on a MOF framework obtained by the method of any one of claims 1 to 4, characterized in that:
it contains electrochemical luminescent group tri (4, 4-dicarboxy bipyridine) ruthenium chloride and probe.
6. Use of an amplification-free high-efficiency electrochemiluminescence probe based on a MOF framework obtained by the preparation method according to any one of claims 1 to 4 in the field of biosensing.
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