CN116376537A - PFODBT@MSX electrochemical luminescent material with highly controllable quenching path, and preparation method and application thereof - Google Patents
PFODBT@MSX electrochemical luminescent material with highly controllable quenching path, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a PFODBT@MSX electrochemiluminescence material with a highly controllable quenching path, and a preparation method and application thereof, and belongs to the technical field of sensor materials. Organic semiconductor Polymer (PFODBT) is encapsulated in Mesoporous Silica Xerogel (MSX) with medium particle size by in-situ synthesis method with the help of surfactant to form the PFODBT@MSX electrochemiluminescence material. The material has excellent water solubility and ultrahigh electron transfer quenching interference resistance, eliminates quenching caused by electron transfer, and can be quenched by Au-NPs energy transfer, so that the material has a highly controllable quenching path. The quenching path of the Electrochemiluminescence (ECL) biosensor constructed based on the material is also highly controllable, so that the background signal of the sensor can be greatly reduced, the sensitivity of the sensor is improved, and a powerful guarantee is provided for improving the precision of the ECL biosensor for detecting low-abundance substances such as miRNA-21.
Description
Technical Field
The invention belongs to the technical field of sensor materials, and relates to a PFODBT@MSX electrochemical luminescent material with a highly controllable quenching path, and a preparation method and application thereof.
Background
Electrochemiluminescence (ECL) refers to a luminescence phenomenon caused by that a luminophor (namely an electrochemiluminescence material) on the surface of an electrode undergoes oxidation-reduction reaction under the action of an applied voltage to form an excited state, and the unstable excited state returns to a ground state. The ECL biosensor established based on the phenomenon has the advantages of low background signal, wide linear range, good reproducibility and the like, and has wide application prospect and market competitiveness in the aspects of bioassay and clinical detection. ECL biosensors are designed to allow the sensing of biologically relevant markers by curing various electrochemiluminescent materials on the surface of a working electrode and then modifying again. Among them, the choice of the electrochemiluminescence material has a crucial influence on the performance of ECL biosensors, determining its practical application value. For example, some electrochemiluminescent materials in an aggregated state or under the influence of other electroactive interferents can induce strong intermolecular electron or energy transfer, h-aggregates or excimer and other processes or species unfavorable for luminescence, and the molecules in an excited state are mostly attenuated to a ground state in a non-radiative transition manner, so that a quenching luminescence effect is caused. Usually, electron transfer quenching or energy transfer quenching is mainly used. Generally, electron transfer quenching is initiated by the electrochemical reaction of an electroactive interferent with a free radical of an electrochemiluminescent material; energy transfer quenching is initiated by overlapping donor and acceptor spectra, but most energy transfer quenching processes are inevitably accompanied by electron transfer quenching. However, electron transfer quenching interferes with the detection signal of ECL biosensors, thereby decreasing the sensitivity and accuracy of ECL biosensor detection, which is detrimental to detection of low abundance species. Therefore, how to realize a controllable quenching path and obtain a high-precision signal is a remarkable research direction.
By examining various electrochemiluminescence materials, researchers have found that polymer electrochemiluminescence materials can suppress non-radiative transitions and effectively prevent quenching of excitons. Meanwhile, the polymer has a longer main chain and a longer branched chain, and is easy to form a coil, so that a rigid structure is endowed to the polymer, and the polymer is likely to be a good candidate for quenching the ECL luminophor by resisting electron transfer. However, most polymers are poorly hydrophilic and compatible, which is a bottleneck limiting their use in ECL biosensors. Thus, to address this problem and further enhance its electron transfer quenching resistance, the present application developed an electrochemiluminescent material with a highly controllable quenching path. The ECL biosensor constructed based on the material can well realize the sensitive detection of miRNA-21 and other low-abundance substances related to various cancers.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for preparing pfodbt@msx electrochemiluminescence material with highly controllable quenching path; the second object of the invention is to provide a PFODBT@MSX electrochemiluminescence material with a highly controllable quenching path; the third object of the present invention is to provide an application of an electrochemiluminescence material with a highly controllable quenching path in an electrochemiluminescence biosensor; the fourth object of the present invention is to provide an electrochemiluminescence biosensor; the fifth object of the present invention is to provide a method for preparing an electrochemiluminescence biosensor.
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. a method for preparing a PFODBT@MSX electrochemiluminescence material with a highly controllable quenching path, which comprises the following steps:
mixing aqueous solution of hexadecyl trimethyl ammonium bromide with tetrahydrofuran of poly [2,7- (9, 9' -dioctyfluorene) -alt-4,7-bis (thiophen-2-yl) benzol-2, 1,3-thiadiazole ] (PFODBT), heating at 75-80 ℃ for 20-30 min, sequentially adding aqueous solution of Pluronic F-127, tetraethyl orthosilicate and ammonia water, reacting at 80 ℃ for 2-3 h, centrifuging and washing.
Preferably, the mass-to-volume ratio of the PFODBT, the cetyl trimethylammonium bromide, the Pluronic F-127, the tetraethyl orthosilicate and the ammonia water is 0.5:0.492:5.5:0.5, mg g: mg: mL.
Preferably, the mass volume ratio of the cetyltrimethylammonium bromide to the water in the aqueous solution of the cetyltrimethylammonium bromide is 0.492:7.5, g/mL; the mass volume ratio of the Pluronic F-127 to water in the aqueous solution of the Pluronic F-127 is 5.5:16.5, mg:mL; the mass percentage of the ammonia water is 25%.
Preferably, the rotational speed of the centrifugation is 11000-13000 rpm and the time is 15-20 min.
2. The PFODBT@MSX electrochemiluminescence material with the highly controllable quenching path is prepared by the method.
3. The PFODBT@MSX electrochemiluminescence material is applied to an electrochemiluminescence biosensor.
4. An electrochemiluminescence biosensor, wherein the sensor comprises a potassium persulfate base solution and a glassy carbon electrode, and the surface of the glassy carbon electrode is loaded with the PFODBT@MSX electrochemiluminescence material.
5. The preparation method of the electrochemiluminescence biosensor comprises the following steps:
(1) Preparing a modified electrode: dispersing the PFODBT@MSX electrochemiluminescence material in deionized water, and then dripping the PFODBT@MSX electrochemiluminescence material on the surface of a clean glassy carbon electrode;
the load capacity of the PFODBT@MSX electrochemiluminescence material on the glassy carbon electrode is 5 multiplied by 10 -6 mg/mm 2 ；
(2) Preparing a modification interface: dropwise adding 0.02% chitosan aqueous solution on the surface of the modified electrode in the step (1);
the chitosan has a loading capacity of 1.6X10 on the modified electrode -4 mg/mm 2 ；
(3) Preparing an electrode with a surface modified Au-tetrahedral DNA structure: reacting the tetrahedral DNA marked with the mercapto group and the Au-NPs modified by the T5 DNA for 2 to 3 hours at 37 ℃ to form a solution, then mixing the solution with 4- (N-maleimidomethyl) -cyclohexane-1-carboxilic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) to obtain a mixed solution, and then dripping the mixed solution on the modified interface in the step (2);
(4) Preparing an electrochemiluminescence biosensor: blocking the electrode with the surface modified Au-tetrahedral DNA structure in the step (3) by 0.25% bovine serum albumin with the mass fraction at 37 ℃ and then dripping and washing by deionized water.
Preferably, the preparation method of the T5 DNA modified Au-NPs in the step (3) is as follows:
mixing Au-NPs with the diameter of 5nm with T5 DNA with the concentration of 20 mu mol/L, then placing the mixture on a shaking table for reaction to obtain a reaction solution, adding PBS buffer solution into the reaction solution to enable the final concentration of the PBS buffer solution to reach 0.1mol/L, and aging at room temperature;
the volume ratio of the Au-NPs to the T5 DNA is 1:1;
the nucleotide sequence of the T5 DNA is shown as SEQ ID NO: 1.
Preferably, the thiol-labeled tetrahedral DNA of step (3) is prepared as follows:
the S1 chain without the 5 'modified sulfhydryl and the S2 chain, the S3 chain and the S4 chain DNA with the 5' modified sulfhydryl are kept for 10min at 95 ℃ in PCR, and then cooled at 4 ℃;
the nucleotide sequence of the S1-S4 chain DNA is shown as SEQ ID NO:2 to 5.
The invention has the beneficial effects that: the invention provides a PFODBT@MSX electrochemiluminescence material with a highly controllable quenching path. The material is prepared by encapsulating an organic semiconductor Polymer (PFODBT) in Mesoporous Silica Xerogel (MSX) with medium particle size by an in-situ synthesis method under the assistance of a surfactant. Compared with PFODBT, the formed PFODBT@MSX electrochemiluminescence material realizes the conversion from hydrophobicity to hydrophilicity and has excellent water solubility. The experimental results show that: (1) the material cannot be quenched by electron transfer, and has extremely high electron transfer interference resistance; (2) the material can be quenched by gold nanoparticle energy transfer. Namely, the material realizes electron transfer quenching resistance and highly depends on energy transfer quenching, eliminates the interference of electron transfer quenching in the general quenching process, and realizes the control of the quenching process. The preparation method is simple and is suitable for expanded production.
The invention also provides an ECL biosensor constructed based on the PFODBT@MSX electrochemiluminescence material. The material has an ultrahigh electron transfer quenching resistance effect, so that the background signal of the ECL biosensor can be greatly reduced, the sensitivity of the sensor is improved, and a powerful guarantee is provided for improving the precision of the ECL biosensor for detecting low-abundance substances such as miRNA-21. Meanwhile, because gold nanoparticles can control the occurrence of energy transfer quenching of the material, the ECL biosensor belongs to an ECL biosensor with a highly controllable quenching path.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and other advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the specification.
Drawings
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in the following preferred detail with reference to the accompanying drawings, in which:
FIG. 1 is a graph showing the measurement results of the anti-ionic substance interference ability of the PFODBT@MSX electrochemiluminescence material in example 1;
FIG. 2 is a graph showing the measurement results of the anti-redox interference ability of the PFODBT@MSX electrochemiluminescence material in example 1;
FIG. 3 is a stability test result for the ECL biosensor in example 2;
FIG. 4 is a graph showing the results of selective detection of ECL biosensors in example 2;
FIG. 5 is a graph showing response values of ECL biosensor in example 2 at different concentrations of miRNA-21;
FIG. 6 is a standard graph of the response value corresponding to the ECL biosensor in example 2 versus the logarithmic value of the miRNA-21 concentration.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be noted that the illustrations provided in the following embodiments merely illustrate the basic idea of the present invention by way of illustration, and the following embodiments and features in the embodiments may be combined with each other without conflict.
Example 1
The PFODBT@MSX electrochemiluminescence material with the highly controllable quenching path is prepared by encapsulating an organic semiconductor Polymer (PFODBT) in Mesoporous Silica Xerogel (MSX) with medium particle size with the aid of a surfactant. The preparation method comprises the following steps:
(1) Dissolving 0.5mg of PFODBT in 1mL of tetrahydrofuran, and performing ultrasonic treatment for 10min to form a uniform solution;
(2) Adding 0.492g of cetyltrimethylammonium bromide (CTAB) into 7.5mL of water, uniformly mixing the mixture with the uniform solution in the step (1), stirring and heating the mixture at 78 ℃ for 20min, sequentially adding 16.5mL of Pluronic F-127 aqueous solution, 1mL of tetraethyl orthosilicate and 0.5mL of ammonia water with mass fraction of 25%, transferring the mixture into a three-necked flask, stirring and refluxing the mixture at 80 ℃ for 2h to obtain a reaction product, centrifuging the obtained reaction product at a rotating speed of 12000rpm for 20min, washing the reaction product with deionized water for 5 times to remove redundant surfactants (CTAB and Pluronic F-127) until the pH value of a washing liquid is close to neutral, and obtaining the PFODBT@MSX electrochemical luminescent material;
(3) Dispersing the PFODBT@MSX electrochemiluminescence material in the step (2) in 4mL of deionized water, and preserving at 4 ℃ for standby.
Example 2
ECL biosensor constructed based on pfodbt@msx electrochemiluminescent material prepared in example 1, the specific preparation method is as follows:
(1) Preparing a modified electrode: diluting the prepared aqueous solution of the PFODBT@MSX electrochemical luminescent material prepared in the example 1 by 20 times, and then taking 5 mu L of the diluted aqueous solution and dripping the diluted aqueous solution onto the surface of a clean glassy carbon electrode to obtain a modified electrode;
(2) Preparing a modification interface: dropwise adding 10 mu L of chitosan solution with the mass fraction of 0.02% on the surface of the modified electrode in the step (1), and airing at room temperature to obtain a modified interface;
(3) Preparation of ANP-T5 DNA: mixing 500 mu L of Au-NPs with the diameter of 5nm with 500 mu L of T5 DNA with the concentration of 20 mu mol/L (wherein the nucleotide sequence of the T5 DNA is SEQ ID NO:1: AAAAAAAAAAGTGATGATGGTGCGAGCTAA) and then placing on a shaking table for reaction for 16 hours to obtain a reaction solution; adding PBS buffer solution with the concentration of 1mol/L into the reaction solution for 5 times to enable the final concentration of the PBS buffer solution in the reaction solution to reach 0.1mol/L, aging for 40 hours at room temperature, and then preserving at 4 ℃ for later use;
(4) Preparing an electrode with a surface modified Au-tetrahedral DNA structure: heating S1 chain without 5 'modified sulfhydryl and S2 chain, S3 chain and S4 chain DNA without 5' modified sulfhydryl to 95 ℃ in PCR (wherein the nucleotide sequence of the S1 chain DNA is SEQ ID NO:2:ACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAG TA;S2 chain DNA, the nucleotide sequence of the S1 chain DNA is SEQ ID NO:3:TATCACCAGGCAGTTGACAGTGTAGCAAG CTGTAATAGATGCGAGGGTCCAATAC;S3 chain DNA, the nucleotide sequence of the 4:TCAAC TGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC;S4 chain DNA is SEQ ID NO:5:TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGT ATTGGACCCTCGCAT), holding for 10min, rapidly cooling at 4 ℃ to obtain a tetrahedral DNA structure, hybridizing with ANP-T5 DNA in the step (3) to form a solution, mixing the solution with sulfo-SMCC, and then dripping the mixture on a modification interface in the step (2) to obtain the electrode with the surface modified Au-tetrahedral DNA structure;
(5) Preparation of ECL biosensor: and (3) sealing the electrode with the surface modified Au-tetrahedral DNA structure in the step (4) by using 0.25% bovine serum albumin with the mass fraction at 37 ℃, taking out and washing with water after sealing for 40min, and sealing the non-specific binding site to obtain the ECL biosensor with the highly controllable quenching path.
Performance testing
1. Anti-interference capability verification of PFODBT@MSX electrochemiluminescence material prepared in example 1
Firstly, diluting the prepared aqueous solution of the PFODBT@MSX electrochemiluminescence material prepared in the embodiment 1 by 20 times, then taking 5 mu L of the diluted aqueous solution and dripping the diluted aqueous solution onto the surface of a clean glassy carbon electrode to obtain modified electrodes (11 electrodes are prepared and marked as No. 1 to 11 respectively), and then carrying out an anti-interference capability experiment on the modified electrodes, wherein the experiment is divided into a control group and an experiment group, and the specific test process is as follows:
control group 1 (Blank): the surface of the No. 1 modified electrode is not dripped with any substance solution;
experiment group 1: 2-9 modified electrode was added dropwise with 10. Mu.L of ferrocene (Fc) solution, niCl, each having a concentration of 10. Mu. Mol/L, respectively 2 Solution, cuSO 4 Solution, [ Fe (CN) 6 ] 3- Solutions, dopamine (DA) solutions, citric Acid (CA) solutions, cysteine (Cys) solutions, ascorbic Acid (AA) solutions;
experiment group 2: dropwise adding Au-NPs (nano gold with the diameter of 5 nm) solution which is diluted by 20 times on the surface of the No. 10 modified electrode;
experiment group 3: two sets of mixed solutions were added dropwise to the surface of the No. 11 modified electrode. One of them is Fc-containing and NiCl 2 、CuSO 4 、[Fe(CN) 6 ] 3- Solutions of Au-NPs; the other group is a solution containing DA, CA, cys, AA, au-NPs.
The experimental and control groups were tested using a three electrode system. Wherein, the three-electrode system is divided into a working electrode, a platinum wire counter electrode and an Ag/AgCl (saturated KCl solution) reference electrode which are constructed by the ECL biosensor. The test conditions were: continuous ECL scan was performed on the working electrode in PBS buffer solution containing potassium persulfate, the potential scan speed was set at 100mV/s, the photoelectric booster tube was set at 800V, the scan voltage range was set at-2 to 0V (vs. Ag/AgCl), and ECL response values obtained by stable scan were recorded, and the results are shown in FIGS. 1 and 2. FIG. 1 is a graph showing the detection result of the capability of the PFODBT@MSX electrochemiluminescence material in example 1 to resist interference of ionic substances; FIG. 2 shows the results of the detection of the resistance of the PFODBT@MSX electrochemiluminescence material in example 1 to redox species interference. It can be seen from fig. 1 and 2 that neither ionic nor redox species quench ECL signal of pfodbt@msx electrochemiluminescent material well when Au-NPs is not contained in the solution. Only when Au-NPs are included in the solution, the ECL signal of the material can be quenched. The gold nanoparticles can control quenching, so that the PFODBT@MSX electrochemical luminescent material prepared by the method has a highly controllable quenching path.
2. Selective detection of ECL biosensor prepared in example 2
In order to research the practical value of the ECL biosensor constructed based on the PFODBT@MSX electrochemiluminescence material, the sensor is adopted to detect the interference miRNA-155, the interference miRNA-221, the interference miRNA-222 and the miRNA-21. The experiment is divided into a control group and an experimental group, and the specific detection steps are as follows:
control (Blank): the ECL biosensor in example 2 was not added with any RNA detection solution;
experiment group 1: a. mixing 10 mu L of interference miRNA-155 solution with the concentration of 100pmol/L with hairpin H1 and H2 with the concentration of 100nmol/L to execute HCR to obtain an activator; b. this activator was mixed with 5. Mu.L of a solution containing 150nmol/L LbCAs12a, 750nmol/L crRNA and 1×tolo buffer4 and placed in the ECL biosensor in example 2 to react for 120min at 37 ℃ (the above experimental procedure was repeated 3 times, the experimental result was the average of 3 times);
experiment group 2: only the interference miRNA-155 solution in the a is replaced by the interference miRNA-221 solution, and other conditions are unchanged;
experiment group 3: only replacing the interference miRNA-155 solution in the a with the interference miRNA-222 solution, and keeping other conditions unchanged;
experiment group 4: only the interfering miRNA-155 solution with the concentration of 100pmol/L in the a is replaced by the miRNA-21 solution with the concentration of 10fmol/L, and other conditions are unchanged;
experimental group 5: only the mixed solution of the interfering miRNA-155 solution with the concentration of 100pmol/L, the interfering miRNA-221 solution with the concentration of 100pmol/L and the interfering miRNA-222 solution with the concentration of 100pmol/L is replaced by the miRNA-21 solution with the concentration of 10fmol/L, and other conditions are unchanged.
The experimental and control groups were tested using a three electrode system. Wherein the three-electrode system is a working electrode, a platinum wire counter electrode and an Ag/AgCl (saturated KCl solution) reference electrode which are respectively constructed by the ECL biosensor. The test conditions were: continuous ECL scan was performed on the working electrode in PBS buffer solution containing potassium persulfate, the potential scan speed was set at 100mV/s, the photoelectric booster tube was set at 800V, the scan voltage range was set at-2 to 0V (vs. Ag/AgCl), and ECL response values obtained by stable scan were recorded, and the results are shown in FIGS. 3 and 4. Fig. 3 is a stability test result of the ECL biosensor in example 2, and it can be seen from fig. 3 that the intensity of the sensor remains almost unchanged in twenty scanning cycles, rsd=0.99%, indicating that the sensor has good stability; fig. 4 shows the selective detection results of ECL biosensor in example 2, and it is understood from fig. 4 that the ECL response values of the sensor to the mixed solution of interfering miRNA-155, interfering miRNA-221, interfering miRNA-222 and miRNA-21 hardly changed as compared with the solution of individually testing miRNA-21. It is demonstrated that even in the case where the interfering RNA solution concentration is greater than the miRNA-21 solution concentration, the ECL biosensor prepared by the invention does not have a significant effect on the test results of miRNA-21, indicating that the sensor has excellent selectivity.
3. Detection of actual sample on ELC biosensor prepared in example 2
In order to further study the practical value of the ECL biosensor constructed based on the PFODBT@MSX electrochemiluminescence material, the invention utilizes a labeled recovery method to detect an actual sample, and the specific detection process is as follows:
(1) Diluting miRNA-21 by using human serum to obtain miRNA-21 solutions with the concentration of 10000pmol/L, 1000pmol/L, 100pmol/L and 10pmol/L respectively;
(2) Respectively placing the four miRNA-21 solutions with different concentrations in the step (1) into the ELC biosensor in the example 2, incubating for 120min at 37 ℃, and washing with deionized water after the reaction is completed;
(3) And detecting the sample by adopting a three-electrode system (a working electrode constructed by an ECL biosensor, a platinum wire counter electrode and an Ag/AgCl (saturated KCl solution) reference electrode). Test conditions: continuous ECL scanning is carried out on the working electrode in PBS buffer solution containing potassium persulfate, the potential scanning speed is set to be 100mV/s, the photoelectric voltage doubling booster tube is set to be 800V, the scanning voltage range is-2-0V (vs. Ag/AgCl), and ECL response values obtained by stable scanning are recorded.
In order to better evaluate the detection effect of the ELC biosensor prepared in the present application on an actual sample, it is necessary to construct a relationship between ECL response value-concentration. During the experiment, the ECL signal detected by the sensor is found to increase with the increase of the miRNA-21 concentration (as shown in figure 5), and is proportional to the logarithm of the miRNA-21 concentration, so that a standard curve can be drawn (as shown in figure 6). The response value of the ECL biosensor detected the actual sample is corresponding to the standard curve, so that the concentration and recovery rate of miRNA-21 in the actual sample can be calculated, and the experimental result is shown in Table 1.
As can be seen from Table 1, the actual addition concentration of miRNA-21 and the concentration detected by the ECL biosensor prepared in example 2 are not greatly different, and the recovery rate is between 98% and 113.6%, which indicates that the ECL biosensor prepared by the invention has high sensitivity and can be better used for detection and analysis of actual samples.
TABLE 1 detection results of ECL biosensor on actual samples
| Sample label | concentration/(pmol/L) of the mixture | Measured concentration/(pmol/L) | Recovery/% |
| 1 | 10 | 9.80 | 98.0 |
| 2 | 100 | 97.89 | 97.89 |
| 3 | 1000 | 1136 | 113.6 |
| 4 | 10000 | 10337 | 103.4 |
In summary, the invention provides a PFODBT@MSX electrochemiluminescence material with a highly controllable quenching path. The material is prepared by encapsulating organic semiconductor Polymer (PFODBT) in Mesoporous Silica Xerogel (MSX) with medium particle size with the aid of surfactant. The material has excellent water solubility, can not be quenched by electron transfer, can be quenched by gold nanoparticle energy transfer, and has a highly controllable quenching path. The ECL biosensor constructed based on the material can greatly reduce the background signal of the ECL biosensor, thereby improving the sensitivity of the sensor and providing powerful guarantee for improving the precision of the ECL biosensor for detecting low-abundance substances such as miRNA-21 and the like.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the claims of the present invention.
Claims (10)
1. A preparation method of a PFODBT@MSX electrochemiluminescence material with a highly controllable quenching path is characterized by comprising the following steps: the preparation method comprises the following steps:
mixing aqueous solution of hexadecyl trimethyl ammonium bromide with tetrahydrofuran solution of poly [2,7- (9, 9' -dioctyfluorene) -alt-4,7-bis (thiophen-2-yl) benzol-2, 1,3-thiadiazole ], heating at 75-80 ℃ for 20-30 min, sequentially adding aqueous solution of Pluronic F-127, tetraethyl orthosilicate and ammonia water, reacting at 80 ℃ for 2-3 h, centrifuging and washing with water.
2. The method for preparing the pfodbt@msx electrochemiluminescence material according to claim 1, wherein: the mass-volume ratio of poly [2,7- (9, 9' -dioctyfluorene) -alt-4,7-bis (thiophen-2-yl) benzol-2, 1,3-thiadiazole ], cetyltrimethylammonium bromide, pluronic F-127, tetraethyl orthosilicate and ammonia water is 0.5:0.492:5.5:0.5, mg: g: mg: mL.
3. The method for preparing the pfodbt@msx electrochemiluminescence material according to claim 1, wherein: the mass volume ratio of the cetyl trimethyl ammonium bromide to the water in the aqueous solution of the cetyl trimethyl ammonium bromide is 0.492:7.5, g:ml; the mass volume ratio of the Pluronic F-127 to water in the aqueous solution of the Pluronic F-127 is 5.5:16.5, mg:mL; the mass percentage of the ammonia water is 25%.
4. The method for preparing the pfodbt@msx electrochemiluminescence material according to claim 1, wherein: the rotational speed of the centrifugation is 11000-13000 rpm, and the time is 15-20 min.
5. Pfodbt@msx electrochemiluminescent material with highly controllable quenching path prepared according to the method of any one of claims 1 to 4.
6. The use of the pfodbt@msx electrochemiluminescent material of claim 5 in an electrochemiluminescent biosensor.
7. An electrochemiluminescence biosensor, said sensor comprising a potassium persulfate base solution and a glassy carbon electrode, characterized in that: the pfodbt@msx electrochemiluminescence material of claim 5 is loaded on the surface of the glassy carbon electrode.
8. The method for preparing the electrochemiluminescence biosensor of claim 7, wherein: the preparation method comprises the following steps:
(1) Preparing a modified electrode: dispersing the PFODBT@MSX electrochemiluminescence material of claim 5 in deionized water, and then dripping the mixture on the surface of a clean glassy carbon electrode;
the load capacity of the PFODBT@MSX electrochemiluminescence material on the glassy carbon electrode is 5 multiplied by 10 -6 mg/mm 2 ；
(2) Preparing a modification interface: dropwise adding 0.02% chitosan aqueous solution on the surface of the modified electrode in the step (1);
the chitosan has a loading capacity of 1.6X10 on the modified electrode -4 mg/mm 2 ；
(3) Preparing an electrode with a surface modified Au-tetrahedral DNA structure: reacting the tetrahedral DNA marked with the mercapto and the Au-NPs modified by the T5 DNA for 2-3 hours at 37 ℃ to form a solution, then mixing the solution with 4- (N-maleimidomethyl) -cyclohexane-1-carboxic acid 3-sulfoo-N-hydroxysuccinimide ester sodium salt to obtain a mixed solution, and then dripping the mixed solution on the modified interface in the step (2);
(4) Preparing an electrochemiluminescence biosensor: blocking the electrode with the surface modified Au-tetrahedral DNA structure in the step (3) by 0.25% bovine serum albumin with the mass fraction at 37 ℃ and then dripping and washing by deionized water.
9. The method for manufacturing an electrochemiluminescence biosensor according to claim 8, wherein: the preparation method of the T5 DNA modified Au-NPs in the step (3) comprises the following steps:
mixing Au-NPs with the diameter of 5nm with T5 DNA with the concentration of 20 mu mol/L, then placing the mixture on a shaking table for reaction to obtain a reaction solution, adding PBS buffer solution into the reaction solution to enable the final concentration of the PBS buffer solution to reach 0.1mol/L, and aging at room temperature;
the volume ratio of the Au-NPs to the T5 DNA is 1:1;
the nucleotide sequence of the T5 DNA is shown as SEQ ID NO: 1.
10. The method for manufacturing an electrochemiluminescence biosensor according to claim 8, wherein: the thiol-labeled tetrahedral DNA of step (3) is prepared as follows:
the S1 chain without the 5 'modified sulfhydryl and the S2 chain, the S3 chain and the S4 chain DNA with the 5' modified sulfhydryl are kept for 10min at 95 ℃ in PCR, and then cooled at 4 ℃;
the nucleotide sequence of the S1-S4 chain DNA is shown as SEQ ID NO:2 to 5.
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