CN110082415B - Photoelectric chemical detection probe based on conjugated polymer nanoparticles and application thereof - Google Patents
Photoelectric chemical detection probe based on conjugated polymer nanoparticles and application thereof Download PDFInfo
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Abstract
A conjugated polymer nanoparticle-based photoelectrochemical detection probe, a preparation method and a sensor for detecting alpha fetoprotein prepared by the photoelectrochemical detection probe belong to the technical field of photoelectrochemical detection. The photoelectrochemistry detection probe consists of a conjugated polymer and a functionalized polymer and is prepared in water by a reprecipitation method. Compared with the prior art, the conjugated polymeric nanoparticles are non-toxic and have good biocompatibility, and are suitable for detecting the alpha fetoprotein with low concentration. In the photoelectrochemistry detection test, the detection limit of 0.05ng/mL is obtained by utilizing the principle of competitive immunity. The detection probe can flexibly change the molecular structure, adjust the light absorption characteristic and utilize sunlight to carry out photoelectrochemical detection research in the whole solar spectrum range. The preparation method is simple, has good repeatability, can be applied to mass production, has good application prospect in the field of photoelectrochemical detection, and can be used for preparing a sensor for detecting alpha fetoprotein.
Description
Technical Field
The invention belongs to the technical field of photoelectrochemical detection, and particularly relates to a photoelectrochemical detection probe based on conjugated polymer nanoparticles, a preparation method and a sensor for detecting alpha fetoprotein.
Background
Alpha-fetoprotein (AFP) is an important detection marker for primary liver cancer. Mainly synthesized in fetal liver and thus present in large amounts in fetal plasma, but at very low concentrations in adult plasma. Research shows that when the AFP concentration in human plasma is abnormally increased, the body of the human plasma is likely to have cancer pathological changes. The abnormal increase in AFP concentration is often observed early in cancer, and early detection of a change in AFP concentration in a patient is therefore of great significance for the early detection and early treatment of cancer patients.
Since researchers combined chemical recognition materials with electronic components to create electrochemical detection devices, new technologies based on early electrochemical detection are emerging to meet the increasing detection demands. Among the most promising detection techniques, photo-electrochemical detection, in which a photoactive substance is introduced into a detection electrode and light is applied, resulting in a significant increase in the generated electrical signal, is undoubtedly one of the most promising detection techniques. The technology can also be used in the field of biological detection and has good detection effects on enzymes, nucleic acids, antibodies and other small biological molecules due to the characteristics of high sensitivity, strong specificity, good operability, economy, portability and the like. The detection device can be divided into a photo-addressable potentiometric sensor (LAPS) and a current-type photoelectrochemical sensor according to different forms of detection electric signals. The former converts the concentration signal of the object to be detected into potential change through the photoelectric effect of the semiconductor, and the latter directly converts the concentration signal into a photocurrent signal for realizing different detection purposes. The biosensor specially designed can even automatically identify and capture target analytes for qualitative or quantitative measurement, and has wide application in the fields of disease diagnosis, environmental monitoring, medical research, safety detection of foods and cosmetics and the like. A typical photoelectrochemical biological detection system consists of an excitation light source (xenon lamp), a three-electrode system, an electrochemical workstation and a signal processing unit (computer). In which a sensing material having biological activity is disposed on working electrodes, and an electrochemical workstation is responsible for inputting a desired voltage signal between the electrodes. When the excitation light with specific wavelength irradiates the working electrode, the optical active substance connected on the electrode is excited to generate photo-generated carriers, and the carriers form special electric signals under the dual effects of voltage and bioactive substances, and then are recorded by the electrochemical workstation and transmitted to a computer for processing. In the whole process, the excitation signal (optical signal) and the receiving signal (electric signal) do not overlap and do not interfere with each other, and the accurate and stable detection is facilitated. Meanwhile, the electrochemical workstation benefits from high integration and signal processing means of the electrochemical workstation, only the working electrode and the matched electrolyte are needed to be replaced to meet different detection requirements, and the electrochemical workstation is suitable for practical application in the fields of biomass detection, medical monitoring and the like.
It is known from the working principle of the typical photoelectrochemical biological detection system described above that the photoactive material on the electrodes is the key to the overall detection system. Therefore, the development of a material with high photoelectric conversion efficiency, which has low toxicity and is easy to be biologically modified, is a research hotspot for developing a photoelectrochemical biological detection system at present. In general, inorganic semiconductor Materials (material Chemistry And Physics,2017,189,56-63), organic semiconductor Materials (RSC Advances,2016,6(74), 70691-. While developing materials with different properties, the photoelectric material can be doped with other substances to form a heterojunction so as to promote carrier separation and effective conduction of charges (Biosensors & Bioelectronics,2017,96, 8-16.). Therefore, developing a nano material with high photoelectric conversion efficiency, good biocompatibility and easy modification, establishing a new reagent for primary liver cancer detection, and being an important way for realizing rapid, convenient and sensitive photoelectrochemical detection of liver cancer tumor markers.
Disclosure of Invention
The invention aims to provide a conjugated polymer nanoparticle-based photoelectrochemical detection probe, a preparation method and a sensor for detecting alpha fetoprotein, wherein the preparation method of the photoelectrochemical detection probe is simple and easy to implement, and the particle size of the nanoparticles can be conveniently adjusted according to the feeding proportion.
The photoelectrochemistry detection probe consists of a conjugated polymer and a functionalized polymer and is prepared in water by a reprecipitation method. Wherein the functionalized polymer is used to adjust the size and surface potential of the conjugated polymer nanoparticles and also to prevent aggregation of the nanoparticles at high concentrations. Common Functionalized polymers such as amphiphilic polymer Polystyrene maleic anhydride Poly (PSMA) and amphiphilic polymer Polystyrene Graft Ethylene Oxide Functionalized with a carbon atom (PS-PEG-COOH) with carboxyl terminated polyethylene glycol grafted onto the Polystyrene backbone; the mass content of the functional polymer in the conjugated polymer nanoparticles is 1-20%, and the balance is the conjugated polymer; the size of the conjugated polymer nano particle is 5 nm-100 nm.
The invention relates to a preparation method of a photoelectrochemical detection probe based on conjugated polymer nanoparticles, which comprises the following steps:
(1) dissolving a conjugated polymer and a functional polymer in 20-100 mL of tetrahydrofuran to form a mixed solution, wherein the concentration of the conjugated polymer is 10-100 mu g/mL, and the concentration of the functional polymer is 0.1-10 mu g/mL; then, under the condition of ultrasonic treatment, quickly injecting 1-10 mL of the mixed solution into 10-100 mL of water and continuing ultrasonic treatment for 1-5 minutes;
(2) under the protection of inert gases (nitrogen, argon, helium and the like), heating the solution obtained in the step (1) to 85-95 ℃, keeping the temperature for 2-6 hours to remove tetrahydrofuran, cooling to room temperature, and filtering by using a 200-220 nm filter head to remove large particles to obtain an aqueous solution of the conjugated polymer nanoparticles; the size range of the conjugated polymer nanoparticles is 5 nm-100 nm by adjusting the concentration and the injection volume of the initial solution;
(3) diluting the prepared conjugated polymer nanoparticle solution to 30-50 ppm by using deionized water, taking 1mL of the diluted conjugated polymer nanoparticle solution, sequentially adding 20-50 muL of 1M 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) aqueous solution (with the pH value of 6.5), 20-50 muL of 5% mass fraction polyethylene glycol aqueous solution, 10-20 muL of 1mg/mL Alpha Fetoprotein (AFP) aqueous solution and 20-40 muL of 5mg/mL 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride (EDC) aqueous solution, placing the mixture in a mixing instrument, and stirring for 4-6 hours at room temperature to obtain the AFP-conjugated polymer nanoparticle aqueous solution; wherein EDC is used for activating carboxyl to effectively combine the conjugated polymer nano particles with AFP, HEPES is used as a hydrogen ion buffering agent to maintain the pH value of the solution; putting the obtained AFP-conjugated polymer nanoparticle aqueous solution into a dialysis bag with the molecular weight cutoff of 50kDa, carrying out dialysis treatment for 48-72 hours, and fixing the volume to 1mL by using a 20mM HEPES aqueous solution; then sequentially adding 20-50 mul of 1M HEPES aqueous solution (pH is 6.5), 20-50 mul of 5% mass fraction polyethylene glycol aqueous solution, 10-20 mul of 1mg/mL AFP aqueous solution and 20-50 mul of 5mg/mL EDC aqueous solution, and placing the mixture in a mixer to stir at room temperature for 4-6 hours; and then putting the obtained mixed solution into a dialysis bag with the molecular weight cutoff of 100kDa for dialysis for 48-72 hours, and then using 20mM HEPES aqueous solution to fix the volume to 1mL to obtain the photoelectrochemical detection probe based on the conjugated polymer nanoparticles.
The conjugated polymers described in the present invention include, but are not limited to, benzothiazole-containing polyfluorene derivatives, poly-p-phenylene vinylene and derivatives, polyalkylfluorene, dithienobenzothiazole-containing polyfluorene derivatives, or poly-p-phenylene ethynylene and derivatives. The obtained conjugated polymer nanoparticles are prepared by selecting different polymer materials.
Through repeated comparison and optimization selection, the polyfluorene derivative poly [ (9,9-dioctyl fluoro-2, 7-diyl) -co- (1, 4-benzol- [2, 1', 3] -thiadazole) ] (PDFDBT) containing benzothiazole is selected in the example to prepare the conjugated polymer nanoparticles, and the structural formula of the conjugated polymer nanoparticles is shown as follows:
poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-[2,1’,3]-thiadazole)](PDFDBT)
the structural formula of the conventional Functionalized polymer such as amphiphilic polymer Polystyrene maleic anhydride Poly (PSMA) and amphiphilic polymer Polystyrene grade Ethylene Oxide Functionalized with carbon atom (PS-PEG-COOH) grafted with carboxyl terminated polyethylene glycol on Polystyrene backbone is shown as follows:
n and m are positive integers and represent the number of repeating units;
the conjugated polymer nano-particles are prepared in an aqueous solution system, the preparation method adopts simple equipment and mild conditions, and the used chemical reagents are cheap and easy to obtain. The particle size of the PDFDBT nano particles is 5-100 nm, and the nano particles are spherical. The PDFDBT nano particles prepared by the method show strong absorption in ultraviolet and visible light regions, and can well utilize sunlight. Experimental results show that the probe has excellent biocompatibility and photoelectric conversion performance and shows stable and efficient performance of photoelectrochemical detection of alpha fetoprotein. Compared with the prior art, the conjugated polymeric nanoparticles are non-toxic and have good biocompatibility, and are suitable for detecting the alpha fetoprotein with low concentration. In the photoelectrochemistry detection test, the detection limit of 0.05ng/mL is obtained by utilizing the principle of competitive immunity. The conjugated polymer nanoparticle photoelectrochemical detection probe can flexibly change the molecular structure, adjust the light absorption characteristic and utilize sunlight to carry out photoelectrochemical detection research in the whole solar spectrum range. The conjugated polymer nano particle has the advantages of simple preparation method, good repeatability, large-scale production, good application prospect in the field of photoelectrochemical detection, and can be used for preparing a sensor for detecting alpha fetoprotein.
Drawings
FIG. 1: the absorption spectrogram of the AFP-PDFDBT photoelectrochemical detection probe, the AFP protein and the PDFDBT nano particle aqueous solution in the embodiment 1;
FIG. 2: dynamic Light Scattering (DLS) particle size distribution plot of PDFDBT nanoparticles in example 1;
FIG. 3: transmission electron micrograph of PPDFDBT nanoparticles of example 1;
FIG. 4: photoelectrochemical response curves of the AFP-PDFDBT photoelectrochemical detection probe in example 1 at a fixed concentration of AFP;
FIG. 5: the AFP-PDFDBT photoelectrochemical detection probe in example 1 shows a response curve in different concentrations of AFP (the response current gradually decreases as the concentration of AFP to be detected increases).
FIG. 6: DLS particle size distribution plot of PDFDBT nanoparticles in example 2;
FIG. 7: photoelectrochemical detection curve of AFP-PDFDBT photoelectrochemical detection probe in fixed concentration of AFP in example 2;
FIG. 8: the response curve of the AFP-PDFDBT photoelectrochemical detection probe in example 2 in different concentrations of AFP (the response current gradually decreases as the concentration of AFP to be detected increases); .
Detailed Description
The technical solution of the present invention is described in more detail with the following specific examples, but the examples are not to be construed as limiting the present invention.
Example 1
First, tetrahydrofuran solutions of PDFDBT conjugated polymer and polystyrene maleic anhydride (PSMA) were prepared at concentrations of 50. mu.g/mL and 10. mu.g/mL, respectively. Then, under sonication, 3mL of the mixed solution was quickly injected into 10mL of water and sonication was continued for 2 minutes. Finally, the tetrahydrofuran solvent was removed by heating on a heating table (85 ℃) for 4 hours under nitrogen blanket. Then cooling the polymer nano particle water solution to room temperature, and filtering by a 220nm filter head to remove large particles to obtain the conjugated polymer PDFDBT nano particle water solution with the particle size of 32 nm.
The prepared PDFDBT nanoparticle (about 32nm in diameter) solution was diluted to 50ppm with deionized water. 1mL of the diluted PDFDBT nanoparticle solution was taken, 20. mu.L of 1M HEPES aqueous solution (pH 6.5), 20. mu.L of 5% polyethylene glycol aqueous solution, 10. mu.L of 1mg/mL AFP aqueous solution and 20. mu.L of 5mg/mL EDC aqueous solution were sequentially added, and the mixture was stirred in a homogenizer at room temperature for 4 hours. Wherein EDC is used to activate carboxyl groups for efficient binding of PDFDBT nanoparticles to AFP; HEPES serves as a hydrogen ion buffer to maintain the pH of the solution. The obtained AFP-PDFDBT solution was placed in a dialysis bag with a molecular weight cut-off of 50kDa, dialyzed for 48 hours and made up to 1mL with 20mM HEPES aqueous solution. Then, 20. mu.L of 1M HEPES aqueous solution (pH 6.5), 20. mu.L of 5% polyethylene glycol aqueous solution, 10. mu.L of 1mg/mL AFP aqueous solution and 20. mu.L of 5mg/mL EDC aqueous solution were sequentially added thereto, and the mixture was stirred in a homogenizer at room temperature for 4 hours. And putting the obtained mixed solution into a dialysis bag with the molecular weight cutoff of 100kDa for dialysis for 48 hours, and then using 20mM HEPES aqueous solution to fix the volume to 1mL to obtain the AFP-PDFDBT photoelectrochemical detection probe.
Example 1 Performance testing
The AFP-PDFDBT photoelectrochemical detection probe is matched with an immunodetection electrode substrate for use, and an immune competition method is adopted for AFP detection. After 15. mu.L of AFP aqueous solution with different concentrations and 10. mu.L of AFP-PDFDBT photoelectrochemical detection probe aqueous solution (1ng/mL) are fully mixed, the mixture is dripped on an ITO electrode substrate containing AFP antibodies, and after incubation for 30 minutes at 37 ℃, the mixture is carefully rinsed by 0.01M PBS aqueous solution to remove unconnected antigens or probes. The resulting electrode was used as a working electrode in a three-electrode system, and a 300W xenon lamp was used as a light source for the test, and its photocurrent was measured by an electrochemical workstation, with a working bias set at 0.6V and an electrolyte solution of PBS (0.1M, pH 7.4).
FIG. 1 is an absorption spectrum diagram of AFP-PDFDBT photoelectrochemical detection probe, AFP protein and PDFDBT nano-particle aqueous solution. As shown, PDFDBT nanoparticles show relatively broad absorption in the 200nm to 550nm range, with the strongest absorption peaks at 340nm and 455 nm. AFP exhibits a relatively weak absorption in the 200nm to 300nm range; the absorption spectrum of the AFP-PDFDBT photoelectrochemical detection probe combined the respective absorption characteristics of AFP and PDFDBT, indicating that AFP and PDFDBT are effectively combined together.
FIG. 2 is a Dynamic Light Scattering (DLS) particle size distribution plot of PDFDBT nanoparticles. As shown in the figure, the diameter distribution of the prepared PDFDBT nanoparticles is in the range of 20nm to 100nm, wherein the distribution ratio of the diameter of 32nm is the largest. The distribution of diameters of the polymeric nanoparticles is related to the distribution of molecular weights of the polymeric material. Each polymer nanoparticle is formed by the curling of polymer chains in a hydrophilic environment, and when the polymer chains are not consistent in chain length, the diameters of the formed nanoparticles are distributed in the parts with longer chain length and shorter chain length.
FIG. 3 is a transmission electron micrograph of the PDFDBT nanoparticles prepared. As shown in the figure, the PPDFBT nanoparticles all showed spherical shapes, the particle size distribution was substantially consistent with the DLS data, and the diameter of the nanoparticles was 32 nm.
FIG. 4 is a graph of the photoelectrochemical response of the AFP-PDFDBT photoelectrochemical detection probe in a fixed concentration of AFP. mu.L of an AFP aqueous solution with a fixed concentration (10ng/mL) and 10. mu.L of an AFP-PDFDBT photoelectrochemical detection probe aqueous solution (1ng/mL) were mixed well and dropped on an ITO electrode substrate containing AFP antibodies, incubated at 37 ℃ for 30 minutes and carefully rinsed with 0.01M PBS aqueous solution to remove the unattached antigens or probes. The obtained electrode is used as a working electrode in a three-electrode system, and a photoelectrochemical detection system is adopted for detection. As can be seen, the detection electrode has very good response characteristics.
FIG. 5 is a graph of the detection curve for the AFP-PDFDBT photoelectrochemical detection probe. Fully mixing 15 mu L of AFP solution with different concentrations (0.05 ng/mL-100 ng/mL) and 10 mu L of AFP-PDFDBT photoelectrochemical detection probe solution (1ng/mL), dripping the mixture on an ITO electrode substrate containing an AFP antibody, and incubating for 30 minutes at 37 ℃ to obtain a working electrode for a photoelectric detection system. As shown, the photocurrent gradually decreased with increasing concentration. This is because the generation of photocurrent, mainly derived from PDFDBT nanoparticles, is competitive in the recognition of AFP-PDFDBT photoelectrochemical detection probes on the electrode when they are incubated with AFP. The more AFP antibody on the electrode binds to AFP, the less binding to the AFP-PDFDBT photoelectrochemical detection probe will be, and thus the less current will be available for photoelectrochemical detection. As can be seen from the figure, the lowest detection limit can reach 0.05 ng/mL.
Example 2:
solutions of PDFDBT conjugated polymer and PSMA in tetrahydrofuran were first prepared at concentrations of 100. mu.g/mL and 1. mu.g/mL, respectively. Then, under sonication, 1mL of the above mixed solution was rapidly injected into 20mL of water and sonication was continued for 5 minutes; finally, the tetrahydrofuran solvent was removed by heating on a heating table (95 ℃) for 3 hours under nitrogen blanket. After cooling to room temperature, filtering the polymer nano particle water solution without tetrahydrofuran by a 200nm filter head to remove large particles, and obtaining the conjugated polymer PDFDBT conjugated polymer nano particle water solution with the particle size of 50 nm. The same AFP protein modification process as in step 3) of example 1 was followed to obtain a photoelectrochemical detection probe.
Example 2 Performance testing
The same test method as in example 1 was employed for example 2. Except that 0.05ng/mL to 1ng/mL was used when different concentrations of AFP were tested.
Fig. 6 is a DLS particle size distribution plot of PDFDBT nanoparticles prepared in example 2. As shown in the figure, the diameter distribution of the prepared PDFDBT nanoparticles is in the range of 38nm to 122nm, wherein the distribution ratio of 50nm diameter is the largest.
FIG. 7 is a graph of the photoelectrochemical response of the AFP-PDFDBT photoelectrochemical detection probe in a fixed concentration of AFP. mu.L of an AFP aqueous solution with a fixed concentration (10ng/mL) and 10. mu.L of an AFP-PDFDBT photoelectrochemical detection probe aqueous solution (1ng/mL) were mixed well and dropped on an ITO electrode substrate containing AFP antibodies, incubated at 37 ℃ for 30 minutes and carefully rinsed with 0.01M PBS aqueous solution to remove the unattached antigens or probes. The obtained electrode is used as a working electrode in a three-electrode system, and a photoelectrochemical detection system is adopted for detection. As shown in the figure, the photoelectric response current obtained increases as the particle size of the PDFDBT nanoparticles increases, because the amount of the photoresponsive population contained in a single nanoparticle increases as the particle size increases, and thus a large photoelectric response current can be obtained.
FIG. 8 is a graph of the detection curve for the AFP-PDFDBT photoelectrochemical detection probe. Fully mixing 15 mu L of AFP solution with different concentrations (0.05 ng/mL-1 ng/mL) and 10 mu L of AFP-PDFDBT photoelectrochemical detection probe solution (1ng/mL), dripping the mixture on an ITO electrode substrate containing an AFP antibody, and incubating for 30 minutes at 37 ℃ to obtain a working electrode for a photoelectric detection system. As shown, the photocurrent gradually decreased with increasing concentration.
Example 3:
tetrahydrofuran solutions of PDFDBT conjugated polymer and PSMA were prepared as 100mL mixed solutions at 40. mu.g/mL and 10. mu.g/mL, respectively. Then, under sonication, 10mL of the mixed solution was quickly injected into 100mL of water and sonication was continued for 5 minutes. Finally, the tetrahydrofuran solvent was removed by heating on a heating table (95 ℃ C.) under nitrogen for 6 hours. After cooling to room temperature, the polymer nanoparticle aqueous solution was filtered through a 220nm filter to remove large particles, and a conjugated polymer PDFDBT nanoparticle aqueous solution having a particle size of 38nm (maximum distribution ratio) was obtained. The same AFP protein modification process as in step 3) of example 1 was followed to obtain a photoelectrochemical detection probe.
Example 4:
the procedure of example 1 was followed except for step 3) of example 4: the prepared conjugated polymer nanoparticle solution was diluted to 30ppm with deionized water. 1mL of the diluted conjugated polymer nanoparticles was taken, and 50. mu.L of a 1M HEPES aqueous solution (pH 6.5), 50. mu.L of a 5% mass fraction polyethylene glycol aqueous solution, 20. mu.L of a 1mg/mL AFP aqueous solution and 40. mu.L of a 5mg/mL EDC aqueous solution were sequentially added thereto, and the mixture was stirred in a homogenizer at room temperature for 6 hours. The obtained AFP-conjugated polymer nanoparticle aqueous solution was contained in a dialysis bag having a molecular weight cut-off of 50kDa, dialyzed for 72 hours and made to 1mL with 20mM HEPES aqueous solution. Then, 50. mu.L of 1M HEPES aqueous solution (pH 6.5), 50. mu.L of 5% polyethylene glycol aqueous solution, 20. mu.L of 1mg/mL AFP aqueous solution and 50. mu.L of 5mg/mL EDC aqueous solution were sequentially added thereto, and the mixture was stirred in a homogenizer at room temperature for 6 hours. The resulting mixed aqueous solution was dialyzed for 72 hours in a dialysis bag having a molecular weight cut-off of 100kDa, and made up to 1mL again with 20mM HEPES aqueous solution.
Example 5:
preparing 20mL of tetrahydrofuran solution with the concentration of PDFDBT conjugated polymer being 10 mu g/mL and the concentration of PS-PEG-COOH functionalized polymer being 0.1 mu g/mL; then, under sonication, 1mL of the above mixed solution was rapidly injected into 10mL of water and sonication was continued for 1 minute; the solution obtained above was heated to 85 ℃ under the protection of inert gas (argon), tetrahydrofuran was removed by heating for 2 hours, and after cooling to room temperature, large particles were removed by filtration with a 200nm filter, to obtain an aqueous solution of conjugated polymer PDFDBT nanoparticles having a particle size of 13nm (maximum distribution ratio). The same AFP protein modification process as in step 3) of example 1 was followed to obtain a photoelectrochemical detection probe.
Example 6:
preparing 100mL of tetrahydrofuran solution with the concentration of PDFDBT conjugated polymer being 10 mu g/mL and the concentration of PSMA functionalized polymer being 2 mu g/mL; then, in the case of sonication, 2mL of the above mixed solution was rapidly injected into 20mL of water and sonication was continued for 5 minutes; under the protection of inert gas (nitrogen), the solution obtained in the step is heated to 95 ℃ for 6 hours to remove tetrahydrofuran, and after the solution is cooled to room temperature, a 220nm filter head is used for filtering to remove large particles, so that the conjugated polymer PDFDBT nano particle aqueous solution with the particle size of 5nm (the distribution ratio is maximum) is obtained. The same AFP protein modification process as in step 3) of example 1 was followed to obtain a photoelectrochemical detection probe.
In examples 3-6, the prepared PDFDBT nanoparticles have the similar properties of photoelectrochemical detection AFP as in examples 1 and 2. The detection limit is 0.05 ng/mL.
Claims (4)
1. A preparation method of a photoelectrochemical detection probe based on conjugated polymer nanoparticles comprises the following steps:
(1) dissolving a conjugated polymer and a functional polymer in 20-100 mL of tetrahydrofuran to form a mixed solution, wherein the concentration of the conjugated polymer is 10-100 mu g/mL, and the concentration of the functional polymer is 0.1-10 mu g/mL; then, under the condition of ultrasonic treatment, quickly injecting 1-10 mL of the mixed solution into 10-100 mL of water and continuing ultrasonic treatment for 1-5 minutes; the conjugated polymer is a polyfluorene derivative containing benzothiazole, poly-p-phenylene ethylene and a derivative thereof, polyalkyl fluorene, a polyfluorene derivative containing dithienobenzothiazole or poly-p-phenylene acetylene and a derivative thereof; the functional polymer is PSMA or PS-PEG-COOH, the structural formula is shown as follows,
n and m are positive integers and represent the number of repeating units;
(2) under the protection of inert gas, heating the solution obtained in the step (1) to 85-95 ℃, keeping the temperature for 2-6 hours to remove tetrahydrofuran, cooling to room temperature, and filtering by using a 200-220 nm filter head to remove large particles to obtain an aqueous solution of conjugated polymer nanoparticles; the size range of the conjugated polymer nanoparticles is 5 nm-100 nm by adjusting the concentration and the injection volume of the initial solution;
(3) diluting the prepared conjugated polymer nanoparticle solution to 30-50 ppm by using deionized water, taking 1mL of the diluted conjugated polymer nanoparticle solution, sequentially adding 20-50 muL of 1M HEPES (high efficiency polyethylene) aqueous solution, 20-50 muL of 5% mass fraction polyethylene glycol aqueous solution, 10-20 muL of 1mg/mL AFP aqueous solution and 20-40 muL of 5mg/mL EDC aqueous solution, placing the mixture in a mixing instrument, and stirring at room temperature for 4-6 hours to obtain an AFP-conjugated polymer nanoparticle aqueous solution; putting the obtained AFP-conjugated polymer nanoparticle aqueous solution into a dialysis bag with the molecular weight cutoff of 50kDa, carrying out dialysis treatment for 48-72 hours, and fixing the volume to 1mL by using a 20mM HEPES aqueous solution; then sequentially adding 20 mu L-50 mu L of HEPES aqueous solution with the pH value of 6.5, 20 mu L-50 mu L of polyethylene glycol aqueous solution with the mass fraction of 5%, 10 mu L-20 mu L of AFP aqueous solution with the mass fraction of 1mg/mL and 20 mu L-50 mu L of EDC aqueous solution with the mass fraction of 5%, and placing the mixture in a blending instrument to stir for 4-6 hours at room temperature; then putting the obtained mixed solution into a dialysis bag with the molecular weight cutoff of 100kDa for dialysis for 48-72 hours, and then using 20mM HEPES aqueous solution to fix the volume to 1mL to obtain the photoelectrochemical detection probe based on the conjugated polymer nanoparticles;
wherein HEPES represents 4-hydroxyethylpiperazine ethanesulfonic acid, AFP represents alpha fetoprotein, and EDC represents 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
2. The method for preparing the conjugated polymer nanoparticle-based photoelectrochemical detection probe according to claim 1, wherein the conjugated polymer nanoparticle-based photoelectrochemical detection probe comprises: the conjugated polymer is PDFDBT, the structural formula of which is shown as follows,
3. a photoelectrochemical detection probe based on conjugated polymer nanoparticles is characterized in that: is prepared by the method of any one of claims 1 to 2.
4. A conjugated polymer nanoparticle-based photoelectrochemical detection probe according to claim 3, used for preparing a sensor for detecting alpha-fetoprotein.
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