Method for detecting in-vitro marker based on hydrogel material and electrochemical luminescence technology

Abstract

The invention relates to the technical field of biological detection, in particular to a method for detecting an in-vitro marker based on a hydrogel material and an electrochemical luminescence technology. The preparation method comprises the following steps: constructing a core-shell structure difunctional hydrogel film; the method comprises the following steps: constructing an ECL microfluidic sensing chip based on a core-shell structure difunctional hydrogel film, and detecting multiple target markers; data of a detection sample is collected, a CNN model is constructed, and intelligent marker detection analysis processing is carried out. The core-shell structure hydrogel is introduced to realize synergism of four functions of pollution resistance, recognition, luminescence and catalysis, so that the bottleneck that a sensing interface is easy to attenuate signals and low in sensitivity in a complex matrix is broken through, and synergism and unification of long-acting stability and high-sensitivity detection functions of the material in a serum matrix are realized; meanwhile, a unique technical breakthrough is realized through organic combination of a multi-channel microfluidic technology and a deep learning algorithm, and a novel multi-target detection method with high sensitivity, high selectivity, high accuracy and high stability is constructed.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and more specifically, to a method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology. Background Technology

[0002] Cancer poses a serious threat to human health, and promoting independent innovation in early diagnosis and treatment, as well as high-throughput biomarker detection technologies, is a key link in improving early cancer screening capabilities. Tumor markers, as a class of biomolecules closely related to the occurrence and development of tumors, such as prostate-specific antigen, human chorionic gonadotropin, and carcinoembryonic antigen, have been widely used in the auxiliary diagnosis, treatment monitoring, and prognostic assessment of tumors.

[0003] However, traditional detection methods such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and radiobioassay, while technically mature, still face significant bottlenecks in terms of detection sensitivity, multiplexing capabilities, cost control, and ease of on-site application. Key breakthroughs in sensor interface design and signal amplification mechanisms are urgently needed. Achieving rapid, sensitive, and accurate simultaneous detection of multiple tumor markers in the complex matrix of blood has become a critical technological requirement for the early diagnosis of cancer progression.

[0004] To address these challenges, developing a novel strategy for the rapid, sensitive, and accurate simultaneous detection of multiple tumor markers is urgently needed. Methods based on hydrogel materials and electrochemiluminescence technology offer a new and feasible approach for this purpose. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology. By introducing a core-shell structured hydrogel, a synergistic effect of antifouling, recognition, luminescence, and catalysis is achieved, overcoming the bottleneck of signal attenuation and low sensitivity at the sensing interface in complex matrices. This method achieves a synergistic unity of long-term material stability and high-sensitivity detection in serum matrices. Furthermore, the unique technological breakthrough achieved through the organic combination of multi-channel microfluidic technology and deep learning algorithms addresses the problems mentioned in the background art, namely: Traditional detection methods still have significant bottlenecks in terms of detection sensitivity, multiple detection capabilities, cost control, and ease of on-site application.

[0006] To achieve the above objectives, a method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology is provided, comprising the following steps: S1. Construct a core-shell bifunctional hydrogel membrane and perform characterization tests. Covalently bind aptamers to specifically recognize tumor markers. S2. Construct an ECL microfluidic sensing chip based on a core-shell bifunctional hydrogel membrane to detect multiple target markers; S3. Conduct serum sample testing experiments on the ECL microfluidic sensor chip to evaluate the detection effect; S4. Collect and test sample data, construct a CNN model, and perform intelligent marker detection and analysis.

[0007] First, a core-shell bifunctional hydrogel membrane was constructed. The core layer was copolymerized with acrylamide, carboxybetaine, and luminol monomers using a free radical polymerization method, and platinum nanoparticles (Pt NPs) were loaded using an in-situ reduction method to achieve luminescence and catalysis. The shell layer utilized polycarboxybetaine to provide antifouling function, inhibiting the non-specific adsorption of proteins, microorganisms, etc., through electrostatic repulsion and strong hydration. The aptamer bound to the shell layer through amino modification to achieve specific recognition of tumor markers. At the same time, a tunable cross-linking strategy was used to enhance the mechanical strength and chemical stability of the gel, thus completing the regulation of the hydrogel structure.

[0008] Furthermore, the antifouling / catalytic mechanism of the core-shell bifunctional hydrogel membrane was investigated by monitoring its chemical structure, microstructure, physical properties, and mechanical properties. Specifically, the antifouling mechanism of the hydrogel membrane was explored through experiments on anti-protein adsorption, bacterial adhesion, and interference from organic macromolecules. The catalytic mechanism was further clarified by tracing the catalytic reaction pathway and active species using Raman spectroscopy.

[0009] Finally, the ECL microfluidic sensor chip was constructed using PDMS lithography. A core-shell hydrogel-modified glassy carbon electrode was embedded into the chip. The anti-interference lifetime of the ECL microfluidic sensor chip in environments containing proteins, bacteria, and organic macromolecules was evaluated using serum matrix testing. Simultaneously, by collecting sample data, a CNN model was constructed for intelligent biomarker detection and analysis, including noise correction and multi-dimensional signal analysis of the sample data. Continuous 24-hour monitoring was conducted to assess the physical stability of the hydrogel interface during long-term operation, resulting in a novel multi-target detection method characterized by high sensitivity, high selectivity, high accuracy, and high stability.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: This method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology introduces a core-shell structured hydrogel to achieve a synergistic effect of four functions: anti-fouling, recognition, luminescence, and catalysis. This overcomes the bottleneck of signal attenuation and low sensitivity at the sensing interface in complex matrices, achieving a synergistic unity of long-term stability and high-sensitivity detection in serum matrices. Furthermore, the method achieves a unique technological breakthrough by organically combining multi-channel microfluidic technology with deep learning algorithms, constructing a new multi-target detection method with high sensitivity, high selectivity, high accuracy, and high stability. Attached Figure Description

[0011] Figure 1 This is a diagram illustrating the overall method steps of the present invention; Figure 2 This is a simulation diagram of the overall scheme of the present invention; Detailed Implementation

[0012] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] Please see Figure 1 As shown, a method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology is provided, including the following steps: S1. Construct a core-shell bifunctional hydrogel membrane and perform characterization tests. Covalently bind aptamers to specifically recognize tumor markers. S2. Construct an ECL microfluidic sensing chip based on a core-shell bifunctional hydrogel membrane to detect multiple target markers; S3. Conduct serum sample testing experiments on the ECL microfluidic sensor chip to evaluate the detection effect; S4. Collect and test sample data, construct a CNN model, and perform intelligent marker detection and analysis.

[0014] The details are as follows: Firstly, existing electrochemiluminescence (ECL) sensing technology has shown broad application prospects in biomolecular detection due to its high sensitivity, low detection limit, on-site analysis capability, and excellent signal-to-noise ratio. However, serum contains a large number of complex components such as proteins, macromolecular organic matter, and microorganisms, which can cause non-specific adsorption or deposition at the sensing interface, leading to signal attenuation or passivation. To address the non-specific contamination of the sensing interface by proteins, macromolecular organic matter, and microorganisms in the complex serum matrix, this approach constructs a core-shell bifunctional hydrogel membrane and combines it with aptamers for specific recognition of tumor markers.

[0015] The core layer is a catalytic-luminescent layer: a polyacrylamide / polycarboxylic betaine hydrogel network is used to generate ECL signals rapidly and efficiently through carboxylated copolymer luminol luminescent units during electrochemical excitation; at the same time, highly active platinum nanomaterials (Pt NPs) loaded on the core layer can significantly improve the reaction efficiency of the luminol-hydrogen peroxide system through catalytic hydrogen peroxide decomposition and electron transfer, thereby amplifying the ECL signal.

[0016] In constructing the core layer, acrylamide, carboxybetaine, and luminol monomers were copolymerized using free radical polymerization to form a polyacrylamide / polycarboxybetaine hydrogel network. Chloroplatinic acid was loaded onto the hydrogel network via in-situ reduction, and the Pt NPs particle size (5-20 nm) was adjusted by controlling the reaction conditions (temperature, pH). Crosslinking optimization was performed by introducing a dynamic covalent crosslinking agent (such as polyethylene glycol diacrylate) to adjust the crosslinking density (1-5 wt%), balancing the mechanical strength (elastic modulus ≥3 kPa) and swelling properties of the polyacrylamide / polycarboxybetaine hydrogel network.

[0017] The shell layer serves as an antifouling-recognition layer: Polycarboxybetaine provides antifouling properties, inhibiting the non-specific adsorption of proteins, bacteria, and other contaminants through electrostatic repulsion and strong hydration. During aptamer loading, since different aptamers detect different biomarkers, it is necessary to select the corresponding aptamer based on the biomarker during the detection process. Using an amino-carboxyamidinization reaction, aptamers for prostate-specific antigen (PSA), human chorionic gonadotropin (HCG), and carcinoembryonic antigen (CEA) are covalently anchored to the shell surface, achieving specific recognition of tumor biomarkers.

[0018] Meanwhile, in order to characterize the structure and properties of the core-shell bifunctional hydrogel membrane, infrared spectroscopy and nuclear magnetic resonance were used to confirm the chemical structure of the synthesized hydrogel material; scanning electron microscopy was used to observe the size and distribution of platinum nanoparticles; thermogravimetric analysis was used to evaluate the thermal stability of the gel; and Raman spectroscopy was used to trace the catalytic reaction pathway and active species to further clarify the catalytic mechanism.

[0019] The core-shell hydrogel structure design effectively integrates four functions: antifouling, recognition, luminescence, and catalysis. The shell layer utilizes a zwitterionic polymer of polycarboxylated betaine, which, through copolymerization with an amino-modified aptamer, achieves precise target recognition while simultaneously inhibiting the non-specific adsorption of proteins, large organic molecules, and microorganisms. The core layer retains the efficient electron transport pathways of catalytic nanoparticles (Pt NPs) and the luminol luminescent group. The research aims to clarify both the strong hydration and electrostatic repulsion mechanisms of polycarboxylated betaine at the molecular level, and the interaction between the nanoparticles and luminol. The catalytic sensitization mechanism of the hydrogen peroxide system was studied, and the core-shell structure was precisely controlled during network construction and nanoparticle loading to obtain a material system with both high antifouling ability and ECL signal amplification effect. This ensures that the antifouling layer does not affect ECL signal transmission, but rather further enhances ECL efficiency through microenvironment regulation.

[0020] Furthermore, in the complex matrix of serum, ECL signals are easily interfered with by multiple factors such as proteins, organic macromolecules, and microorganisms. Therefore, this scheme constructs an ECL microfluidic sensor chip based on a core-shell bifunctional hydrogel membrane to detect multiple target biomarkers. During the construction of the ECL microfluidic sensor chip, photolithography was used to fabricate a polydimethylsiloxane microfluidic chip, designing 4-8 independent detection channels with a channel width of 200 μm and a depth of 100 μm. Core-shell hydrogel-modified glassy carbon electrodes were embedded at the bottom of the microchannels with an electrode spacing of 500 μm. Adaptors for recognizing different tumor biomarkers were covalently cross-linked in the hydrogel shell layer, enabling parallel detection of multiple targets (PSA, HCG, and CEA, etc.). Finally, an automated sample delivery unit was set up, using an external micropump to sequentially deliver samples into each detection area, and finally discharge them to the waste outlet, achieving parallel detection across multiple channels. Scanning electron microscopy was used to examine the surface morphology and thickness distribution.

[0021] Furthermore, in order to evaluate the detection performance of the ECL microfluidic sensor chip, it is necessary to conduct serum sample testing experiments on the ECL microfluidic sensor chip to assess its detection performance. The specific evaluation method is as follows: Characterization techniques such as X-ray photoelectron spectroscopy and scanning electron microscopy are used to monitor whether protein deposition or particle blockage occurs on the electrode surface. For key pollutants, the physical stability of the hydrogel interface during long-term operation is assessed through 24-hour continuous testing or periodic cyclic monitoring.

[0022] Finally, to improve detection efficiency, a CNN model was constructed using collected sample data for intelligent biomarker detection and analysis. Cyclic voltammetry and electrochemical impedance spectroscopy were performed on the conductivity and electron transfer velocity of the hydrogel interface to determine the enhancement effect of Pt NPs on the ECL signal. During the CNN model construction, the ECL response curve and spectral information were used as input features. The model was trained using a pre-collected training set containing multi-dimensional data including different interfering agent concentrations and buffer solution conditions. The data was cleaned and normalized. Then, multi-dimensional data such as time / channel data were input into the CNN model, which outputs the target concentration or peak intensity. The model was then validated using an experimental test set. If the error was too large, the network hyperparameters (kernel size, learning rate, etc.) were adjusted or the amount of data was increased to ensure that the detection limit could be reduced to the ppt level while maintaining high stability. The CNN model was used to extract features from multi-dimensional, non-linear data, automatically separating background noise from the target signal.

[0023] Overall plan as follows Figure 2As shown, a core-shell bifunctional hydrogel membrane was first constructed. The core layer was copolymerized with acrylamide, carboxybetaine, and luminol monomers using a free radical polymerization method, and Pt NPs were loaded using an in-situ reduction method to achieve luminescence and catalysis. The shell layer provided antifouling function through polycarboxybetaine, inhibiting the non-specific adsorption of proteins, microorganisms, etc. through electrostatic repulsion and strong hydration. The aptamer binds to the shell layer through amino modification to achieve specific recognition of tumor markers. At the same time, a tunable crosslinking strategy was used to enhance the mechanical strength and chemical stability of the gel, thus completing the regulation of the hydrogel structure.

[0024] Furthermore, by monitoring the chemical structure, microstructure, physical properties, and mechanical properties of the core-shell bifunctional hydrogel membrane, we explored its antifouling / catalytic mechanism. Specifically, we investigated its antifouling mechanism through experiments on anti-protein adsorption, microbial adhesion, and interference from organic macromolecules, and further clarified the catalytic mechanism by tracing the catalytic reaction pathway and active species using Raman spectroscopy.

[0025] Finally, the ECL microfluidic sensor chip was constructed using PDMS lithography. Core-shell hydrogel-modified glassy carbon electrodes were embedded into the chip. After chip fabrication, the anti-interference lifetime of the ECL microfluidic sensor chip under conditions of protein interference and organic macromolecules was evaluated through serum matrix testing. Simultaneously, by collecting sample data, a CNN model was constructed for intelligent biomarker detection and analysis, including noise correction and multi-dimensional signal analysis of the sample data. Continuous 24-hour monitoring was conducted to assess the physical stability of the hydrogel interface during long-term operation, resulting in a novel multi-target detection method with high sensitivity, high selectivity, high accuracy, and high stability.

[0026] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology, characterized in that: Includes the following steps: S1. Construct a core-shell bifunctional hydrogel membrane and perform characterization tests. Covalently bind aptamers to specifically recognize tumor markers. S2. Construct an ECL microfluidic sensing chip based on a core-shell bifunctional hydrogel membrane to detect multiple target markers; S3. Conduct serum sample testing experiments on the ECL microfluidic sensor chip to evaluate the detection effect; S4. Collect and test sample data, construct a CNN model, and perform intelligent marker detection and analysis.

2. The method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology according to claim 1, characterized in that: The core-shell bifunctional hydrogel membrane in S1 includes a core layer and a shell layer; The core layer is a catalytic-luminescent layer: a polyacrylamide / polycarboxylic betaine hydrogel network forms luminescent units through carboxylic copolymerization of luminol; at the same time, platinum nanoparticles are loaded to catalyze and enhance the luminescence intensity of the luminol-hydrogen peroxide system.

3. The shell is an antifouling-recognition layer: polycarboxylated betaine has antifouling function, which inhibits the non-specific adsorption of proteins, microorganisms and other substances by using electrostatic repulsion and strong hydration; at the same time, the amino-modified aptamer binds to the shell to achieve specific recognition of tumor markers.

4. The method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology according to claim 2, characterized in that: The method for constructing the kernel layer includes the following steps: S1.1 Acrylamide, carboxybetaine and luminol monomers are copolymerized using free radical polymerization to form a polyacrylamide / polycarboxybetaine hydrogel network; S1.2 Platinum nanoparticles (Pt NPs) were loaded onto a hydrogel network using an in-situ reduction method; S1.3, Controlling reaction conditions to adjust Pt NPs particle size; S1.

4. Crosslinking optimization is carried out by introducing a dynamic covalent crosslinking agent to adjust the crosslinking density.

5. The method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology according to claim 2, characterized in that: The amino-modified aptamer in S1 binds to the shell via an amidation reaction.

6. The method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology according to claim 4, characterized in that: The method for characterizing and testing the core-shell bifunctional hydrogel membrane in S1 includes the following steps: S1.

10. The chemical structure of the synthesized hydrogel material was confirmed using infrared spectroscopy and nuclear magnetic resonance. S1.

11. Observe the size and distribution of nanoparticles using a scanning electron microscope; S1.

12. Thermogravimetric analysis was used to assess the thermal stability of the gel, and Raman spectroscopy was used to trace the catalytic reaction pathway and active species to further clarify the catalytic mechanism.

7. The method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology according to claim 1, characterized in that: The method for constructing the ECL microfluidic sensor chip using S2 includes the following steps: S2.

1. Polydimethylsiloxane microfluidic chips are fabricated using photolithography technology; S2.2 Design 4-8 independent detection channels, with a channel width of 200 μm and a depth of 100 μm; S2.3 Embed the core-shell hydrogel-modified glassy carbon electrode into the bottom of the microchannel with an electrode spacing of 500 μm; S2.

4. Adaptors that recognize different tumor markers are covalently cross-linked in the hydrogel shell; S2.

5. Set up an automatic sample injection unit, and use an external micro pump to sequentially send the sample into each detection area for multi-channel detection.

8. The method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology according to claim 1, characterized in that: The evaluation method for the serum sample testing experiment in S3 includes the following steps: S3.

1. X-ray photoelectron spectroscopy and scanning electron microscopy were used to detect protein deposition and particle blockage on the electrode surface; S3.

2. For key pollutants, conduct 24-hour continuous testing or periodic monitoring.

9. The method for detecting in vitro biomarkers based on hydrogel materials and electrochemiluminescence technology according to claim 1, characterized in that: The method for constructing the CNN model in S4 includes the following steps: S4.1 Obtain the relationship between the luminescence intensity of the luminol luminescent probe and time curve by electrochemiluminescence detection, and mark it as the ECL response curve; S4.

2. Cyclic voltammetry and electrochemical impedance spectroscopy were performed to analyze the conductivity and electron transfer rate of the hydrogel interface to determine the enhancement effect of Pt NPs on the ECL signal. S4.

3. Use the ECL response curve and spectral information as input features, and train the model using a pre-collected training set; S4.4 Clean and normalize the data, then input the multi-dimensional data such as time / channel into the CNN model to output the target concentration or peak intensity.

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