Preparation method of electrochemical probe microspheres and application thereof in electrochemical immunosensor

Abstract

The application belongs to the field of electrochemical immunosensor, and particularly discloses a preparation method of an electrochemical probe microsphere and application of the electrochemical probe microsphere in an electrochemical immunosensor. The ferrocene methanol is directly mixed in the initial feed, and the thermodynamic nucleation kinetics under the monomer concentration of 1% is skillfully utilized to realize the 'one-pot in-situ locking' of the electrochemically active probe molecule from the center of the microsphere to the whole skeleton level of the shell. The process is extremely continuous and simplified, and the industrial reproducibility is excellent. The prepared probe microsphere is not only accurately controlled in the 400-500 nm nanometer high-activity interval, but also naturally rich in active carboxyl on the surface, and can directly and mildly form a covalent amide bond with a signal antibody in one step. When applied to an integrated screen printing electrode chip, the probe microsphere exhibits high stability, zero leakage, plug-and-play, extremely low detection limit and great potential for clinical instant on-site detection.

Description

Technical Field

[0001] This invention relates to the field of electrochemical immunosensors, and in particular to a method for preparing electrochemical probe microspheres and their application in electrochemical immunosensors. Background Technology

[0002] Electrochemical immunosensors have been widely used in clinical diagnostics, food safety, and environmental monitoring due to their advantages such as high sensitivity, low cost, portability, and ease of integration. In the construction of sandwich-type electrochemical immunosensors, the performance of the electrochemical probe (i.e., signal tag) directly determines the sensor's sensitivity and stability.

[0003] Traditional electrochemical probes often employ free small-molecule electrochemically active materials, such as ferrocene and methylene blue. However, free probes suffer from inherent drawbacks, including easy loss, uneven adsorption on electrode surfaces, and limited signal amplification. To overcome these problems, researchers have begun using microspheres (such as silica microspheres and polystyrene microspheres) as carriers to construct nanoscale signal probes by loading a large number of electrochemically active molecules onto their surfaces, aiming to achieve signal amplification.

[0004] Existing technologies have reported the preparation of polymer microspheres using emulsion polymerization or dispersion polymerization and their use as electrochemical probe carriers. For example, Chinese patent CN103342767B discloses a method for preparing polystyrene-coated ferrocene microspheres, which uses emulsion polymerization with polyvinylpyrrolidone as a dispersant. First, styrene monomer is polymerized to form polystyrene microspheres, and then ferrocene is physically embedded inside the microspheres. However, this method has the following drawbacks: First, this method is a multi-step process, involving polymerization followed by encapsulation, resulting in a complex process and poor industrial reproducibility. Second, ferrocene is only physically encapsulated within the microspheres, making it prone to leakage in subsequent applications, especially in aqueous detection environments, leading to signal instability and short sensor lifespan. Third, the polystyrene microspheres lack active functional groups on their surface, preventing direct covalent coupling with antibodies. Additional surface functionalization steps (such as carboxylation and amination) are required, increasing process complexity and potentially affecting antibody bioactivity. Fourth, this method typically requires dissolving the polystyrene microspheres in an organic solvent to release the ferrocene, preventing in-situ release-free detection and limiting its application in point-of-care testing (POCT). Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for preparing electrochemical probe microspheres and their application in electrochemical immunosensors.

[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution: The first technical solution provided by this invention is a method for preparing electrochemical probe microspheres, comprising the following steps: S1. The electrochemically active probe molecules, functional monomers, and crosslinking agents are completely dispersed in an organic solvent in one step. An initiator is added, and the mixture is thoroughly mixed and deoxygenated. The volume ratio of the functional monomer to the crosslinking agent is 1:4. The amount of initiator added is 2 wt% of the total mass of the functional monomers. The mass ratio of the electrochemically active probe molecules to the functional monomers is 0.15-0.25. S2. Under mechanical stirring, heat to the boiling point of the organic solvent and carry out a one-step distillation precipitation polymerization reaction for 1-2 hours. During the polymerization process, collect and distill off 20%-30% of the total volume of organic solvent and then stop the reaction. Utilize the hydrogen bond space self-assembly of the hydroxyl groups in the ferrocene methanol structure and the methacrylic acid monomer during the copolymerization nucleation stage to allow the ferrocene methanol to grow with the copolymerization of the polymer network. It is directly woven in situ and physically locked inside the porous mesh framework of polymethacrylic acid-divinylbenzene. At the same time, free active carboxyl groups are naturally retained on the surface of the microspheres. The electrochemical probe microsphere emulsion with a particle size of 400-500 nm and high density of ferrocene methanol is spontaneously precipitated in situ within the system. S3. After the reaction is completed, the mixture is allowed to cool naturally. The resulting electrochemical probe microsphere emulsion is then centrifuged. The solid obtained is washed repeatedly with anhydrous ethanol and ultrapure water, and then dried to obtain electrochemical probe microsphere nanoparticles.

[0007] Furthermore, the electrochemically active probe molecule is ferrocene methanol, the functional monomer is methacrylic acid, the crosslinking agent is divinylbenzene, the initiator is azobisisobutyronitrile, and the organic solvent is anhydrous acetonitrile.

[0008] Furthermore, in step S1, deoxygenation involves introducing nitrogen gas for 5 minutes to remove dissolved oxygen from the system.

[0009] Furthermore, in step S3, the centrifugal separation is performed by centrifuging at 8000 r / min for 10 min in a centrifuge.

[0010] Furthermore, in step S3, the drying is performed at a constant temperature of 50°C for 24 hours.

[0011] The second technical solution provided by the present invention is an electrochemical probe microsphere prepared by the above method, wherein the particle size of the electrochemical probe microsphere is 400-500 nm.

[0012] The third technical solution provided by the present invention is a biosignal tag comprising the above-mentioned electrochemical probe microspheres and a signaling antibody covalently coupled to the surface of the electrochemical probe microspheres.

[0013] The third technical solution provided by the present invention is a method for preparing the above-mentioned biosignal tag, characterized in that the above-mentioned electrochemical probe microspheres are dispersed in a phosphate buffer solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to activate the carboxyl groups on the surface of the microspheres, then a signal antibody solution is added to carry out a covalent coupling reaction, and then bovine serum albumin solution is added to block unreacted non-specific active sites to obtain the biosignal tag.

[0014] The fourth technical solution provided by the present invention is an electrochemical immunosensor, comprising a screen-printed electrode chip and the aforementioned biosignal tag. The screen-printed electrode chip integrates a working electrode, a counter electrode, and a reference electrode. The surface of the working electrode is modified with a capture antibody, and the biosignal tag binds to the surface of the working electrode through specific immune recognition.

[0015] The fifth technical solution provided by this invention is the application of the above-mentioned electrochemical immunosensor in electrochemical detection for non-diagnostic purposes. The sample to be tested is dropped onto the working electrode surface of the screen-printed electrode chip, so that the target antigen in the sample specifically binds to the capture antibody. Then, the above-mentioned biosignal tag is dropped for sandwich immunorecognition. Then, phosphate buffer solution detection droplets are dropped onto the screen-printed electrode area. Electrochemical scanning is performed using square wave voltammetry to read the redox current response peak of the electrochemically active probe molecules, thereby realizing the quantitative detection of the target antigen.

[0016] Compared with existing technologies, this invention achieves "one-step, one-pot in-situ locking" of electrochemically active probe molecules from the center to the shell of microspheres by directly mixing ferrocene methanol in the initial feed and ingeniously utilizing the thermodynamic nucleation kinetics at a monomer concentration of 1%. The process is extremely continuous and simplified, with excellent industrial reproducibility. The prepared probe microspheres not only have a particle size precisely controlled within the 400-500 nm nanometer range of high activity, but also have a naturally rich surface of active carboxyl groups, which can directly and gently form covalent amide bonds with signal antibodies in one step. When applied to integrated screen-printed electrode chips, it exhibits high stability, zero leakage, plug-and-play capability, extremely low detection limit, and great potential for clinical point-of-care testing. Attached Figure Description

[0017] Figure 1 This is a scanning electron microscope (SEM) image of the electrochemical probe microspheres prepared in Example 1 of the present invention.

[0018] Figure 2 and Figure 3 The images shown are the elemental surface distribution analysis diagram and energy dispersive spectroscopy (EDS) image of the electrochemical probe microspheres prepared in Example 1 of this invention.

[0019] Figure 4This is a particle size distribution diagram of the electrochemical probe microspheres prepared in Example 1 of the present invention.

[0020] Figure 5 The graph shows the electrochemical probe microsphere material prepared in Example 1 of this invention after 20 cyclic voltammetric scans in phosphate buffer solution, with a scan rate of 0.05 V / s.

[0021] Figure 6 The graph shows the square wave voltammetry (SWV) response curves of the electrochemical immunosensor in Example 3 of this invention to different concentrations of thyroglobulin antigen (Tg), with a concentration range of 0-100 ng / mL.

[0022] Figure 7 This is a linear fitting graph of the peak current of the electrochemical immunosensor and the concentration of thyroglobulin antigen in Example 3 of the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0024] Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available.

[0025] Example 1: Preparation of electrochemical probe microspheres 80 mL of anhydrous acetonitrile was injected into a three-necked flask, followed by a rapid addition of 300 mg of the electrochemically active probe molecule ferrocene methanol, 0.16 mL of the functional monomer methacrylic acid (MAA), and 0.64 mL of the crosslinking agent divinylbenzene (DVB). Next, 0.015 g of azobisisobutyronitrile (AIBN, 2 wt% of the total monomer mass) was added as an initiator to ensure complete mixing and dissolution of the raw materials. The mixture was stirred for 5 min under a nitrogen atmosphere to remove dissolved oxygen. Then, mechanical stirring was initiated at 300 rpm, and the temperature was raised to the boiling point of acetonitrile (81 °C) within 30 min to initiate a one-step distillation precipitation polymerization reaction. Thermodynamic condensation was used to control the reaction; after collecting and distilling off 20-30 mL of acetonitrile solvent within 1-2 h, the reaction was immediately stopped, allowing the system to spontaneously stabilize at a nanocritical state with a total monomer mass fraction of 1%. After the reaction, the mixture was allowed to cool naturally, and then centrifuged at 8000 rpm for 10 min, discarding the supernatant. The obtained solid was washed five times alternately with anhydrous ethanol and ultrapure water to thoroughly remove free ferrocene methanol adsorbed on the surface of the microspheres. Finally, it was placed in a vacuum drying oven and dried at 50°C for 24 h to obtain ferrocene-functionalized electrochemical probe microspheres directly integrated in one step. They were characterized as spherical nanoparticles with excellent monodispersity and a particle size precisely controlled between 400-500 nm.

[0026] The prepared electrochemical probe microspheres were observed by scanning electron microscopy (SEM), and the results are as follows: Figure 1 As shown. By Figure 1 It can be clearly seen that the prepared microspheres are regular spherical in shape and have excellent monodispersity. Figure 2 and Figure 3 The images show the elemental surface distribution analysis (mapping) and energy dispersive spectroscopy (EDS) plots of the electrochemical probe microspheres, respectively. Figure 2 and Figure 3 It can be seen that ferrocene methanol has been successfully integrated into the interior of the microspheres. Figure 4 This is a particle size distribution diagram of electrochemical probe microspheres, from... Figure 4 It can be seen that the particle size of the electrochemical probe microspheres prepared in this embodiment is precisely controlled within the range of 400-500 nm.

[0027] The prepared electrochemical probe microspheres were dispersed in phosphate buffered saline (PBS) and subjected to 20 cyclic voltammetric scans at a scan rate of 0.05 V / s. The results are as follows: Figure 5 As shown. By Figure 5 It can be seen that the cyclic voltammetric curves of 20 cycles basically overlapped and showed no significant changes, indicating that the ferrocene methanol integrated inside the microspheres did not leak or bleed during repeated scanning, and the material has excellent electrochemical stability.

[0028] Example 2: Preparation of electrochemical probe microspheres directly covalently coupled with thyroglobulin signaling antibody tags Accurately weigh 5.0 mg of the ferrocene-functionalized electrochemical probe microspheres prepared in Example 1, and uniformly disperse them in 5 mL of 0.01 mol / L phosphate-buffered saline (PBS) solution with a pH of 7.4 by ultrasonication. Then, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, final concentration 10 mg / mL) and N-hydroxysuccinimide (NHS, final concentration 5 mg / mL) solution, and react magnetically at room temperature (25°C) for 30 min to activate the naturally occurring free carboxyl sites on the microsphere surface. After activation, centrifuge at 8000 r / min for 10 min and discard excess activator. Redisperse the activated microspheres in 5 mL of PBS buffer, add 100 μL of 100 μg / mL thyroglobulin signaling antibody (Ab2) solution, and incubate at 4°C in a constant temperature shaker with slow, dark shaking for 12 h. After the reaction was complete, 1% (v / v) bovine serum albumin (BSA) solution was added, and the mixture was incubated with shaking at room temperature for 30 min to block unreacted nonspecific active sites. Finally, the prepared biosignal tag (Ab2@Micro-Fc) was redispersed in PBS buffer after centrifugation and washing three times at 4°C and stored at 4°C for later use.

[0029] Example 3: Construction and In-situ Release-Free Detection of a Portable Screen-Printed Electrochemical Immunosensor An integrated screen-printed electrode (SPE) chip, comprising a carbon paste working electrode (WE), a carbon paste counter electrode (CE), and a silver / silver chloride reference electrode (RE), was selected as the substrate. First, 3 μL of a 100 μg / mL thyroglobulin capture antibody (Ab1) solution was dropped onto the working electrode surface and incubated at 37°C for 1 h. After washing, 3 μL of BSA solution was added and incubated for 2 h to block non-specific adsorption sites. Next, 3 μL of different concentrations of the target antigen thyroglobulin (Tg) analysis sample were dropped onto the working electrode surface and incubated at 37°C for 30 min. After gentle rinsing, 3 μL of a biosignal tag (Ab2@Micro-Fc) solution directly covalently coupled with a signal antibody, prepared in step 2, was directly added, and specific immunoincubation was performed at 37°C for 30 min. This resulted in the formation of a stable sandwich immunosensing interface on the working electrode surface through specific antigen-antibody binding.

[0030] During detection, the sensor chip surface was gently rinsed with ultrapure water. 20 μL of 0.1 mol / L pure PBS (pH 7.0) was then added in situ above the screen-printed effective reaction area containing the working, counter, and reference electrodes. The chip pins were directly inserted into a portable electrochemical workstation, and a linear voltage scan was performed using a square wave voltammetry (SWV) program, setting the scan potential range to 0.0 V to +0.6 V. Experimental results are as follows: Figures 6-7 As shown.

[0031] from Figure 6 It can be clearly seen that a super-strong in-situ current response peak appears at around +0.25V, which is generated by the in-situ reversible oxidation-reduction of ferrocene methanol deeply integrated inside the microspheres. As the concentration of the target antigen thyroglobulin in the system gradually increases (from 0 to 100 ng / mL), the number of functionalized probe microspheres specifically bound to the working electrode surface increases proportionally, resulting in a significant increasing trend in the peak value of the square wave voltammetric oxidation current. Moreover, the sensor exhibits extremely high sensitivity to a low concentration of thyroglobulin of 1 ng / mL.

[0032] from Figure 7 It can be clearly seen that the sensor exhibits excellent linearity over a wide concentration range from 1 ng / mL to 100 ng / mL. Its linear fitting equation is: I = 0.05978C + 6.05909 correlation coefficient (R²). 2 The linear response is as high as 0.9920. This excellent linear response and high sensitivity are mainly attributed to the following advantages in material architecture and electrochemical mass transfer: the 400-500 nm nanonuclei synthesized directly using a one-step one-pot method have an ultra-high specific surface area, and the ferrocene methanol is fully encapsulated and enriched in a three-dimensional network from the inside out in the early stage of monomer nucleation, providing a powerful source current amplification; the high-density active carboxyl groups naturally present on the surface of the functionalized microspheres enable one-step covalent amidation and direct coupling of the signal antibody, ensuring the structural stability (zero leakage) and ultra-high clinical quantitative reproducibility of the sensing interface under continuous scanning in pure aqueous phase.

[0033] In summary, this invention employs a "one-step, one-pot method" to completely mix and feed ferrocene methanol (Fc-OH), methacrylic acid (MAA), divinylbenzene (DVB), and initiator from the very beginning. The principle is as follows: utilizing the spatial hydrogen bonds between hydroxyl (-OH) and carboxyl (-COOH) groups for self-assembly, the ferrocene is copolymerized and grown along with the polymer backbone, thus in-situ "fully three-dimensionally weaving" and locking it inside the microspheres. By distilling off 20-30% acetonitrile solvent, the total monomer mass-volume fraction is spontaneously controlled to remain at a critical state of 1%. This demonstrates and successfully prepares 400-500 nm nanonuclei, with a specific surface area increased by tens of times, achieving ultra-high density enrichment of the ferrocene probe. The microspheres are naturally rich in high-density active carboxyl groups introduced by MAA. The principle is that, without the need for cumbersome multi-layer coatings or toxic glutaraldehyde cross-linking, they directly covalently couple with the thyroglobulin (Tg) signaling antibody (Ab2) via a mild EDC / NHS aqueous phase reaction, greatly ensuring antibody bioactivity and simplifying the process. This solvent-free probe microsphere is then labeled onto the surface of a screen-printed electrode chip (SPE) integrating carbon and silver / silver chloride. The principle is that during detection, only 20 μL of pure PBS is added in situ, and square wave voltammetry (SWV) scanning is used. Within the range of 1 ng / mL to 100 ng / mL, the peak oxidation response current shows an excellent linear correlation with the Tg concentration, enabling ultrasensitive, plug-and-play, point-of-care testing (POCT) of the clinically significant tumor marker thyroglobulin.

[0034] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing electrochemical probe microspheres, characterized in that, Includes the following steps: S1. The electrochemically active probe molecules, functional monomers, and crosslinking agents are completely dispersed in an organic solvent in one step. An initiator is added, and the mixture is thoroughly mixed and deoxygenated. The volume ratio of the functional monomer to the crosslinking agent is 1:

4. The amount of initiator added is 2 wt% of the total mass of the functional monomers. The mass ratio of the electrochemically active probe molecules to the functional monomers is 0.15-0.

25. S2. Under mechanical stirring, heat to the boiling point of the organic solvent to carry out a one-step distillation precipitation polymerization reaction for 1-2 hours. During the polymerization process, collect and distill off 20%-30% of the total volume of organic solvent, and then stop the reaction to obtain an electrochemical probe microsphere emulsion. S3. After the reaction is completed, the mixture is allowed to cool naturally. The resulting electrochemical probe microsphere emulsion is then centrifuged. The solid obtained is washed repeatedly with anhydrous ethanol and ultrapure water, and then dried to obtain electrochemical probe microsphere nanoparticles.

2. The method for preparing electrochemical probe microspheres according to claim 1, characterized in that, The electrochemically active probe molecule is ferrocene methanol, the functional monomer is methacrylic acid, the crosslinking agent is divinylbenzene, the initiator is azobisisobutyronitrile, and the organic solvent is anhydrous acetonitrile.

3. The method for preparing electrochemical probe microspheres according to claim 1, characterized in that, In step S1, deoxygenation involves introducing nitrogen gas for 5 minutes to remove dissolved oxygen from the system.

4. The method for preparing electrochemical probe microspheres according to claim 1, characterized in that, In step S3, centrifugation is performed by centrifuging at 8000 r / min for 10 min in a centrifuge.

5. The method for preparing electrochemical probe microspheres according to claim 1, characterized in that, In step S3, drying is performed at a constant temperature of 50°C for 24 hours.

6. An electrochemical probe microsphere prepared by the method according to any one of claims 1-5, characterized in that, The electrochemical probe microspheres have a particle size of 400-500 nm.

7. A biosignal tag, characterized in that, It comprises the electrochemical probe microspheres of claim 7 and a signaling antibody covalently coupled to the surface of the electrochemical probe microspheres.

8. A method for preparing a biosignal tag as described in claim 7, characterized in that, The electrochemical probe microspheres of claim 6 are dispersed in a phosphate buffer solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to activate the carboxyl groups on the surface of the microspheres. Then, a signal antibody solution is added to carry out a covalent coupling reaction, and bovine serum albumin solution is added to block unreacted non-specific active sites to obtain the biosignal tag.

9. An electrochemical immunosensor, characterized in that, The invention includes a screen-printed electrode chip and the biosignal tag of claim 7, wherein the screen-printed electrode chip integrates a working electrode, a counter electrode, and a reference electrode, the surface of the working electrode is modified with a capture antibody, and the biosignal tag binds to the surface of the working electrode through specific immune recognition.

10. The application of the electrochemical immunosensor as described in claim 9 in electrochemical detection for non-diagnostic purposes, characterized in that, The sample to be tested is dropped onto the working electrode surface of the screen-printed electrode chip, so that the target antigen in the sample specifically binds to the capture antibody. Then, the biosignal tag described in claim 7 is dropped for sandwich immune recognition. Phosphate buffer solution detection droplets are then dropped onto the screen-printed electrode area. Electrochemical scanning is performed using square wave voltammetry to read the redox current response peak of the electrochemically active probe molecules, thereby achieving quantitative detection of the target antigen.

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