Search

CN-122016957-A - Method for detecting in-vitro marker based on hydrogel material and electrochemiluminescence technology

CN122016957ACN 122016957 ACN122016957 ACN 122016957ACN-122016957-A

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 electrochemiluminescence technology. The method comprises the steps of constructing a core-shell structure double-function hydrogel film, constructing an ECL micro-fluidic sensing chip based on the core-shell structure double-function hydrogel film, detecting multi-target markers, collecting detection sample data, constructing a CNN model, and performing intelligent marker detection analysis treatment. The core-shell structure hydrogel is introduced to realize the coordination of the four functions of anti-pollution, identification, luminescence and catalysis, the bottleneck that a sensing interface is easy to attenuate signals and low in sensitivity in a complex matrix is broken through, the coordination and unification of the long-acting stability and high-sensitivity detection function of materials in a serum matrix are realized, meanwhile, the unique technical breakthrough is realized through the organic combination of a multichannel 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.

Inventors

  • HAN CUIYAN
  • GUO CHENG
  • WANG ZIAO

Assignees

  • 江苏医药职业学院

Dates

Publication Date
20260512
Application Date
20260303

Claims (9)

  1. 1. The method for detecting the in-vitro marker based on the hydrogel material and the electrochemiluminescence technology is characterized by comprising the following steps: s1, constructing a core-shell structure bifunctional hydrogel film, and carrying out characterization test, wherein a proper ligand is covalently bonded to specifically identify a tumor marker; s2, constructing an ECL microfluidic sensing chip based on a core-shell structure double-function hydrogel film, and detecting a multi-target marker; S3, performing a serum sample test experiment on the ECL microfluidic sensing chip, and evaluating the detection effect; s4, collecting detection sample data, constructing a CNN model, and performing intelligent marker detection analysis treatment.
  2. 2. The method for detecting an in-vitro marker based on a hydrogel material and an electrochemiluminescence technology according to claim 1, wherein the core-shell structure dual-function hydrogel film in S1 comprises a core layer and a shell layer; The core layer is a catalytic-luminous layer, wherein a polyacrylamide/polycarboxybetaine gel network forms a luminous unit through carboxyl copolymerization of luminol, and meanwhile, platinum-loaded nano particles catalyze and enhance luminous intensity of a luminol-hydrogen peroxide system.
  3. 3. The shell layer is an anti-pollution-recognition layer, wherein the poly carboxyl betaine has an anti-pollution function, and utilizes electrostatic repulsion and strong hydration to inhibit nonspecific adsorption of proteins, microorganisms and the like, and simultaneously, an amino-modified aptamer is combined with the shell layer to realize specific recognition of tumor markers.
  4. 4. The method for detecting an in-vitro marker based on a hydrogel material and an electrochemiluminescence technology according to claim 2, wherein the method for constructing the nuclear layer comprises the following steps: s1.1, copolymerizing acrylamide, carboxyl betaine and luminol monomers by adopting a free radical polymerization method to form a polyacrylamide/carboxyl betaine gel network; S1.2, loading platinum nano particles (Pt NPs) on a hydrogel network by an in-situ reduction method; S1.3, controlling reaction conditions to adjust the particle size of Pt NPs; s1.4, introducing a dynamic covalent cross-linking agent to adjust the cross-linking density through cross-linking optimization.
  5. 5. The method for detecting an in vitro marker based on a hydrogel material and an electrochemiluminescence technology according to claim 2, wherein the amino modified aptamer in S1 is bound to the shell layer through an amidation reaction.
  6. 6. The method for detecting an in vitro marker based on a hydrogel material and an electrochemiluminescence technology according to claim 4, wherein the method for performing characterization test on the core-shell structure dual-function hydrogel film in S1 comprises the following steps: s1.10, confirming the chemical structure of the synthesized hydrogel material by utilizing infrared spectrum and nuclear magnetic resonance; S1.11, observing the size and the distribution of the nano particles by means of a scanning electron microscope; s1.12, evaluating the thermal stability of the gel through thermogravimetric analysis, and tracking a catalytic reaction path and an active species by using a Raman spectrum to further define a catalytic mechanism.
  7. 7. The method for detecting an in-vitro marker based on the hydrogel material and the electrochemiluminescence technology according to claim 1, wherein the method for constructing the ECL microfluidic sensing chip by S2 comprises the following steps: s2.1, processing a polydimethylsiloxane micro-fluidic chip by adopting a photoetching technology; s2.2, designing 4-8 independent detection channels, wherein the width of each channel is 200 mu m, and the depth is 100 mu m; s2.3, embedding a core-shell hydrogel modified glassy carbon electrode into the bottom of the microchannel, wherein the electrode spacing is 500 mu m; s2.4, covalently crosslinking the aptamer for recognizing different tumor markers on the hydrogel shell layer; S2.5, setting an automatic sample injection unit, and sequentially sending samples into each detection area through an external micro pump for multi-channel detection.
  8. 8. The method for detecting an in vitro marker based on a hydrogel material and an electrochemiluminescence technology according to claim 1, wherein the evaluation method of the serum sample test experiment in S3 comprises the following steps: s3.1, detecting protein deposition and particle blocking conditions on the surface of the electrode by utilizing an X-ray photoelectron spectroscopy and a scanning electron microscope; S3.2, monitoring by 24 hours continuous testing or periodic cycling for key contaminants.
  9. 9. The method for detecting an in-vitro marker based on a hydrogel material and an electrochemiluminescence technology according to claim 1, wherein the method for constructing a CNN model in S4 comprises the following steps: s4.1, obtaining the relation between the luminous intensity of the luminol luminous probe and a time curve through electrochemiluminescence detection, and marking the relation as an ECL response curve; S4.2, performing cyclic voltammetry and electrochemical impedance spectroscopy analysis on the conductivity and the electron transfer speed of a hydrogel interface to determine the ECL signal enhancement effect of Pt NPs; S4.3, taking the ECL response curve and the spectrum information as input characteristics, and performing model training through a pre-acquired training set; S4.4, cleaning and normalizing the data, and then inputting multidimensional data such as time/channel and the like into a CNN model to output the concentration or peak intensity of the target object.

Description

Method for detecting in-vitro marker based on hydrogel material and electrochemiluminescence technology Technical Field 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 electrochemiluminescence technology. Background Today, the serious threat of tumors to human health promotes the autonomous innovation of early diagnosis and early treatment and high-throughput biomarker detection technology, which is a key link for improving the early screening capability of cancers. Tumor markers are used as a class of biomolecules closely related to tumor occurrence and development, such as prostate specific antigen, human chorionic gonadotrophin, carcinoembryonic antigen and the like, and are widely applied to auxiliary diagnosis, curative effect monitoring and prognosis evaluation of tumors. However, although the conventional detection methods such as enzyme-linked immunosorbent assay, polymerase chain reaction and radiobiological assay are mature in technology, obvious bottlenecks still exist in detection sensitivity, multiple detection capability, cost control and convenience in field application, and a key breakthrough in the design of a sensing interface and a signal amplification mechanism is needed. How to realize rapid, sensitive and accurate synchronous detection of various tumor markers in a blood complex matrix has become a key technical requirement for early diagnosis of cancer development conditions. In order to cope with the above problems, development of a new strategy capable of rapidly, sensitively and accurately detecting multiple tumor markers simultaneously is particularly urgent. Methods based on hydrogel materials and electrochemiluminescence techniques provide new viable paths for this. Disclosure of Invention The invention aims to provide a method for detecting an in-vitro marker based on a hydrogel material and an electrochemiluminescence technology, which realizes the coordination of four functions of anti-pollution, identification, luminescence and catalysis by introducing a hydrogel with a core-shell structure, breaks through the bottleneck that a sensing interface is easy to attenuate signals and low in sensitivity in a complex matrix, realizes the coordination and unification of long-acting stability and high-sensitivity detection functions of the material in a serum matrix, and simultaneously realizes the technical breakthrough with unique characteristics by organically combining a multichannel microfluidic technology and a deep learning algorithm so as to solve the problems proposed in the background technology, namely: The traditional detection method still has obvious bottlenecks in detection sensitivity, multiple detection capability, cost control and convenience in field application. To achieve the above object, a method for detecting an in vitro marker based on a hydrogel material and an electrochemiluminescence technology is provided, comprising the steps of: s1, constructing a core-shell structure bifunctional hydrogel film, and carrying out characterization test, wherein a proper ligand is covalently bonded to specifically identify a tumor marker; s2, constructing an ECL microfluidic sensing chip based on a core-shell structure double-function hydrogel film, and detecting a multi-target marker; S3, performing a serum sample test experiment on the ECL microfluidic sensing chip, and evaluating the detection effect; s4, collecting detection sample data, constructing a CNN model, and performing intelligent marker detection analysis treatment. Firstly, a dual-functional hydrogel film with a core-shell structure is constructed, wherein a core layer adopts a free radical polymerization method to copolymerize acrylamide, carboxyl betaine and luminol monomers, platinum nano particles (Pt NPs) are loaded by an in-situ reduction method to realize the luminescence and catalysis effects, and a shell layer adopts the carboxyl betaine to provide an anti-fouling function and inhibit the nonspecific adsorption of proteins, microorganisms and the like through electrostatic repulsion and strong hydration effects. The aptamer is combined with the shell layer through amino modification, so that the specific identification of the tumor marker is realized, and meanwhile, the mechanical strength and the chemical stability of the gel are enhanced by utilizing a regulatable crosslinking strategy, so that the regulation and control of the hydrogel structure are completed. Further, the anti-fouling/catalytic mechanism of the double-functional hydrogel film with the core-shell structure is explored by monitoring the chemical structure, microstructure, physical characteristics and mechanical properties of the double-functional hydrogel film, namely, the anti-fouling mechanism of the hydrogel film is explored by anti-protein adsorption, bacterial adhesion and organic macromolecule interference expe