Immune electrochemical sensing material and preparation method thereof

Abstract

The present application relates to a kind of immunoelectrochemical sensing material and its printing preparation method, which uses redox group functionalized polysaccharide aqueous solution as ink, and is directly printed and deposited on the surface of electrode;The redox group functionalized polysaccharide macromolecular material can be electronically conductive, has high sensitive sensitivity, anti-non-specific interference, non-labeled sensitive, solution processing characteristics, and can be used for non-labeled immunoelectrochemical sensor to rapidly detect trace markers in blood, tissue fluid, sweat and other body fluids;Redox group functionalized polysaccharide macromolecule is prepared by printing and depositing, and the immunoelectrochemical sensing material and electrode are prepared by using the characteristics of redox group functionalized polysaccharide macromolecule aqueous solution dissolving but drying, i.e.water phase stability, without crosslinking treatment, to obtain immunoelectrochemical sensing material and sensing electrode, simplify the preparation process, easy to realize low cost, and has wide popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of polymer materials and relates to an immunoelectrochemical sensing material based on polysaccharide polymers and its printing preparation. Background Technology

[0002] Developing wearable biomarker detection and analysis devices based on body fluids to monitor biomarker concentrations in body fluids in real time can help doctors make accurate diagnoses and timely treatments. Wearable detection technology has the following requirements: 1. Rapid and simple detection; 2. Miniaturization and flexibility of the detection system to meet the requirements of wearable devices; 3. Low-cost fabrication of sensing materials for wearable devices to meet the needs of large-scale production. Currently, these three points represent the technological bottlenecks facing wearable biosensors.

[0003] Immunoelectrochemical sensing technology features small device size, portability, low cost, and high sensitivity, making it a promising candidate for wearable detection of biomolecules. CN202110365347 discloses an electrochemical immunosensing material and sensor. This material utilizes gold nanoparticles to modify screen-printed carbon electrodes, and then couples a monoclonal antibody against the capsid protein of norovirus to the gold nanoparticles using protein A to prepare an immunoelectrochemically sensitive material capable of detecting norovirus. CN202110931186 discloses a methylated DNA immunoelectrochemical sensor, which uses gold nanoparticles to modify gold electrodes, grafts anti-5-mC antibody onto the gold nanoparticles, and uses enzyme-labeled secondary antibodies to label and amplify the markers. Recently, significant progress has also been made in immunoelectrochemical sensing targeting cortisol. CN201810968649 discloses a method for preparing an immunoelectrochemical sensing material. Using a modified silk fibroin blend as a substrate, a gold nanoparticle-modified electrode is used as the conductive electrode. Zinc oxide nanorods are electrochemically deposited to modify the gold nanorods, improving detection accuracy. Monoclonal glucose oxidase and cortisol antibodies are grafted onto the zinc oxide nanorods, and redox ion pairs in solution are used to achieve sensitive sensing of cortisol. CN202111441662 invents a PET-based flexible cortisol sensor. Mxene and MWCNTs are sequentially deposited on a flexible PET substrate. After modification with a cortisol antibody and blocking non-specific interactions using bovine serum albumin, potassium ferricyanide redox pairs in solution are used to achieve sensitive sensing of cortisol. CN106796223A discloses a cortisol immunosensing electrode that uses zero-length crosslinking agents N-(3-dimethylaminopropyl)-N-ethylcarbodiimide and 10mMn-hydroxysulfosuccinimide to covalently link monoclonal antibodies to a 16-mercaptohexadecanoic acid-functionalized gold working electrode. Cortisol is detected using ferrocyanide reagent in phosphate-buffered saline.

[0004] As the background document above shows, immunoelectrochemical sensing materials either have low sensing sensitivity, require secondary labeling for amplification, or necessitate the addition of redox pairs to the detection sample. Furthermore, they all require the use of bovine serum to seal the surface of the sensing material to reduce interference from complex biological environments. Moreover, the preparation of these sensing materials is complex and costly, hindering large-scale commercial applications. Those skilled in the art understand that these techniques make it difficult to achieve in-situ body fluid sensing for wearable applications, and also prevent the large-scale, efficient preparation of sensing materials and sensor devices. Summary of the Invention

[0005] Addressing the issues of low sensitivity, poor resistance to nonspecific interference, and labeling limitations in immunoelectrochemical sensing materials, the inventors, through extensive and systematic research, discovered that introducing redox probe molecules into the chain of polysaccharide polymers allows for direct and sensitive detection of biomarker molecules in body fluids without the need for adding redox probes to samples or secondary labeling markers. Furthermore, the excellent hydrophilicity of polysaccharide polymers endows the sensing material with superior resistance to nonspecific interference. Additionally, those skilled in the art of polysaccharide polymers generally believe that they lack electronic conductivity due to their insulating properties, making it difficult to achieve redox reactions under volume effects. However, the inventors unexpectedly discovered that under suitable redox groups, appropriate linking structures, and optimized grafting densities, diffusion-dominated rapid electron transitions exist within the polysaccharide polymer and its molecular chains, exhibiting volume-effect redox reactions. This drives high-current ion entry and exit, significantly enhancing immunosensing sensitivity.

[0006] Similarly, the introduction of side groups into polysaccharide polymers typically weakens their water solubility. Through extensive innovative work, the inventors discovered, designed, synthesized, and developed a series of water-soluble redox probe-functionalized polysaccharide polymers. Experimental verification has fully demonstrated that these functionalized polysaccharide polymers are soluble in slightly acidic aqueous solutions and exhibit good printing performance. Furthermore, after drying into films, they demonstrate good morphological stability in body fluids. Based on this technology, the inventors further developed their printing technique for fabricating immunoelectrochemical biosensors, significantly simplifying the sensor manufacturing process.

[0007] The present invention has two objectives: firstly, to provide a solution-processable, label-free polysaccharide polymer-based immunoelectrochemical sensitive material that simultaneously possesses high sensitivity, resistance to non-specific interference, and label-free sensitivity; and secondly, to provide a printing technology suitable for large-scale and efficient preparation.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] A label-free electrochemical biomolecular sensing material uses a polysaccharide polymer or its derivatives grafted with redox groups as an electrochemical transducer, and further couples it with a probe molecule that specifically recognizes the biomarker to achieve label-free electrochemical sensitivity to the biomarker.

[0010] As a preferred technical solution:

[0011] The polysaccharide polymer-based label-free electrochemical biomolecular sensing material described above has the following characteristics: grafting redox groups onto at least one of arabinoxylan, chitosan, chitin, cellulose, pectin, alginic acid, starch, chondroitin sulfate, and the above polysaccharide derivatives as an electrochemical transducer, and further coupling it with probe molecules that can specifically recognize markers to achieve label-free electrochemical sensitivity to markers.

[0012] As a preferred technical solution:

[0013] The polysaccharide polymer-based label-free electrochemical biomolecular sensing material described above has the following characteristics: grafting redox groups onto at least one of chitosan, cellulose, pectin, alginate, chondroitin sulfate, and the above-mentioned polysaccharide derivatives as an electrochemical transducer, and further coupling it with probe molecules that can specifically recognize markers to achieve label-free electrochemical sensitivity to body fluid markers.

[0014] As a preferred technical solution:

[0015] The polysaccharide polymer-based label-free electrochemical biomolecular sensing material described above has the following characteristics: grafting redox groups onto at least one of chitosan, cellulose, alginate, chondroitin sulfate, and the above-mentioned polysaccharide derivatives as an electrochemical transducer, and further coupling probe molecules to achieve specific label-free electrochemical sensitivity to at least one of small molecules, proteins, peptides, DNA, RNA, viruses, and cells.

[0016] As a preferred technical solution:

[0017] The polysaccharide polymer-based label-free electrochemical biomolecular sensing material described above has the following characteristics: it contains the chemical structure shown in formula (I):

[0018]

[0019] Wherein, M is at least one of chitosan, cellulose, alginate, and the above-mentioned polysaccharide derivative monomer units;

[0020] a, b, and c are positive integers, and 0 ≤ a ≤ 5000, 0 ≤ b ≤ 5000, and 0 ≤ c ≤ 5000;

[0021] -L1- is at least one of -O-, -NH-C(O)-, -C(O)-NH-, -C(O)-O-, and -OC(O)-;

[0022] -L2- is at least one of -(CH2)x-, -(CH2)xO-(CH2)y-, -(CH2)x-(CH2-CH2-O)y-, -(CH2)xO-(CH2-CH2-O)y-, and -(CH2)2-SS-(CH2)2-, where x and y are integers and 0 ≤ x ≤ 20 and 0 ≤ y ≤ 20;

[0023] -L3- is at least one of -O-, -NH-C(O)-, -C(O)-NH-, -C(O)-O-, and -OC(O)-;

[0024] -L4- is at least one of -O-, -NH-, -C(O)-, and -C(O)-O-;

[0025] -L5- is at least one of -(CH2)x-, -(CH2)xO-(CH2)y-, -C(O)-(CH2)x-, -C(O)-(CH2)xO-(CH2)y-, -NH-(CH2)x-, -NH-(CH2)xO-(CH2)y-, where x is an integer and 0≤x≤20;

[0026] -R1 is a probe molecule that can specifically recognize at least one of cortisol, interferon, tumor necrosis factor α, interleukin-6, interleukin-8, interleukin-10, 1g-M, 1g-A, circulating tumor cells, and SARS-CoV-2.

[0027] -R2 is At least one of its derivatives.

[0028] As a preferred technical solution:

[0029] Considering solubility, solution processability, and sensing performance, the polysaccharide polymer-based label-free electrochemical biomolecular sensing material shown in formula (I) contains chitosan or its derivative monomer units:

[0030]

[0031] Where a, b, and c are positive integers, and 0 ≤ a ≤ 5000, 0 ≤ b ≤ 5000, and 0 ≤ c ≤ 5000;

[0032] -L1- is at least one of -O- and -NH-C(O)-;

[0033] -L2- is at least one of -(CH2)x-(CH2-CH2-O)y-, -(CH2)xO-(CH2-CH2-O)y-, and -(CH2)2-SS-(CH2)2-, where x and y are integers and 0≤x≤20, 0≤y≤20;

[0034] -L3- is at least one of -NH-C(O)- and -C(O)-NH-;

[0035] -L4- is at least one of -O- and -NH-;

[0036] -L5- is at least one of -(CH2)x-, -(CH2)xO-(CH2)y-, -C(O)-(CH2)x-, and -C(O)-(CH2)xO-(CH2)y-, where x is an integer and 0≤x≤20;

[0037] -R1 is a probe molecule that can specifically recognize at least one of cortisol, interferon, IgM, IgA, circulating tumor cells, and SARS-CoV-2.

[0038] -R2 is At least one of its derivatives.

[0039] As a preferred technical solution:

[0040] Considering the influence of redox groups on improving sensing performance and sensitivity, the polysaccharide polymer-based label-free electrochemical biomolecular sensing material shown in formula (I) contains chitosan or its derivative monomer units:

[0041]

[0042] Where a, b, and c are positive integers, and 0 ≤ a ≤ 5000, 0 ≤ b ≤ 5000, and 0 ≤ c ≤ 5000;

[0043] -L1- is -NH-C(O)-;

[0044] -L2- is -(CH2)2-SS-(CH2)2-;

[0045] -L3- is -NH-C(O)-;

[0046] -L4- is -NH-;

[0047] -L5- is -(CH2)x-, where x is an integer and 0≤x≤20;

[0048] -R1 is a probe molecule that can specifically recognize at least one of cortisol, interferon, IgG-M, and IgG-A;

[0049] -R2 is and its derivatives.

[0050] The preparation method of the polysaccharide polymer-based label-free electrochemical biomolecular sensing material is characterized by the following: the aqueous solution of the material is directionally deposited onto the electrode surface via printing, without the need for cross-linking and curing treatment; the polysaccharide polymer-based label-free electrochemical biomolecular sensing material and electrode are directly obtained, as detailed below.

[0051] Using a 0.1-10% (v / v) acetic acid solution, a redox-functionalized polysaccharide polymer solution with a concentration of 0.01-30 mg / mL was prepared as printing ink. The polymer was then directionally deposited onto the electrode surface using appropriate printing parameters and conditions. A biocoupler solution was prepared by adding it dropwise to a marker recognition molecule solution with a concentration of 0.1-1000 μg / mL to achieve a concentration of 0.1-1000 mM. The mixture was activated for 1-120 min, and then an antibody solution was added dropwise to achieve a concentration of 0.1-1000 mM. The mixture was then thoroughly mixed to obtain a sensitive material and sensing electrode with specific recognition of marker molecules.

[0052] Compared with the prior art, the present invention has the following obvious and prominent substantive features and significant advantages:

[0053] 1. The present invention provides a polysaccharide polymer-based immunoelectrochemical sensitive material, which has the characteristics of high sensitivity, resistance to non-specific interference, label-free sensitivity, and solution processability; it can be used to prepare label-free immunoelectrochemical sensors to achieve rapid detection of various trace body fluid biomarkers.

[0054] 2. The present invention provides a method for preparing a polysaccharide polymer-based immunoelectrochemical sensitive material, which involves printing and directionally depositing the material onto the electrode surface. Utilizing the weakly acidic aqueous phase solubility but dry-drying stability of the polysaccharide polymer-based immunoelectrochemical sensitive material, the preparation process is simplified, making it easy to achieve continuous, large-scale, and low-cost production, and thus has broad application value. Attached Figure Description

[0055] Figure 1 The detection mechanism is described in the preferred embodiment of the present invention.

[0056] Figure 2 The CV electrochemical curve of ferrocene-functionalized chitosan as a selective sensor material is shown in the preferred embodiment 1 of the present invention.

[0057] Figure 3 The DPV detection curves at different concentrations are shown in the preferred embodiment of the present invention. Detailed Implementation

[0058] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0059] This invention uses redox-functionalized polysaccharide polymers to print and prepare the immunoelectrochemical sensing material. An aqueous solution of the redox-functionalized polysaccharide polymers is prepared, the pH value is adjusted, the printing parameters are adjusted, and the material is printed and directionally deposited on the electrode surface. The biomolecules that recognize the marker molecules are further chemically modified to prepare the immunoelectrochemical sensing material and the sensing electrode.

[0060] The above solution will be further described below with reference to specific embodiments. The preferred embodiments of the present invention are described in detail below:

[0061] Example 1

[0062] Weigh 1.2 mg of polysaccharide derivative 1 and add it to 1 ml of 0.5% (v / v) acetic acid solution to prepare a 1.2 mg / ml solution of polysaccharide derivative 1. Spray the polysaccharide derivative 1 solution onto the working electrode using inkjet printing. After evaporating the solvent at 51 °C, prepare a 10 mM crosslinking agent solution and a 10 μg / ml cortisol recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 30 min, add the antibody solution dropwise, and mix using a vortex mixer for 1.7 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for cortisol in this sensing application reaches 0.07 ng / ml.

[0063] Example 2

[0064] 1 mg of polysaccharide derivative 2 was weighed and added to 1 ml of 0.6% (v / v) acetic acid solution to prepare a 1 mg / ml solution of polysaccharide derivative 2. The polysaccharide derivative 2 solution was then inkjet printed onto the working electrode. After the solvent was evaporated at 55°C, a 50 mM crosslinking agent solution and a 50 μg / ml tumor necrosis factor α recognition molecule solution were prepared. The crosslinking agent solution was added dropwise, and the mixture was activated for 30 min. The antibody solution was then added dropwise and mixed using a vortex mixer for 2.3 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for cortisol-tumor necrosis factor α in this sensing application reached 0.90 ng / ml.

[0065] Example 3

[0066] Weigh 0.6 mg of polysaccharide derivative 3 and add it to 1 ml of 0.7% (v / v) acetic acid solution to prepare a 0.6 mg / ml solution of polysaccharide derivative 3. Spray the polysaccharide derivative 3 solution onto the working electrode using inkjet printing. After evaporating the solvent at 47°C, prepare a 30 mM crosslinking agent solution and a 30 μg / ml interferon recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 20 min, add the antibody solution dropwise, and mix using a vortex mixer for 2.2 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for interferon in this sensing application reaches 0.3 ng / ml.

[0067] Example 4

[0068] Weigh 0.3 mg of polysaccharide derivative 4 and add it to 1 ml of 0.8% (v / v) acetic acid solution to prepare a 0.3 mg / ml solution of polysaccharide derivative 4. Spray the polysaccharide derivative 4 solution onto the working electrode using inkjet printing. After evaporating the solvent at 53°C, prepare a 100 mM crosslinking agent solution and a 100 μg / ml cortisol recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 20 min, add the antibody solution dropwise, and mix using a vortex mixer for 2 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for cortisol in this sensing application reaches 5 ng / ml.

[0069] Example 5

[0070] Weigh 0.1 mg of polysaccharide derivative 5 and add it to 1 ml of 0.9% (v / v) acetic acid solution to prepare a 0.1 mg / ml solution of polysaccharide derivative 5. Spray the polysaccharide derivative 5 solution onto the working electrode using inkjet printing. After evaporating the solvent at ℃, prepare a 10 mM crosslinking agent solution and a 10 μg / ml 1g-M recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 30 min, add the antibody solution dropwise, and mix using a vortex mixer for 2.1 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for 1g-M in this sensing application reaches 7 ng / ml.

[0071] Example 6

[0072] Weigh 0.06 mg of polysaccharide derivative 6 and add it to 1 ml of 1.0% (v / v) acetic acid solution to prepare a 0.06 mg / ml solution of polysaccharide derivative 6. Spray the polysaccharide derivative 6 solution onto the working electrode using inkjet printing. After evaporating the solvent at 43°C, prepare a 10 mM crosslinking agent solution and a 10 μg / ml lg-A recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 30 min, add the antibody solution dropwise, and mix using a vortex mixer for 2.3 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for lg-A in this sensing application reaches 3 ng / ml.

[0073] Example 7

[0074] Weigh 1.5 mg of polysaccharide derivative 7 and add it to 1 ml of 0.5% (v / v) acetic acid solution to prepare a 1.5 mg / ml solution of polysaccharide derivative 7. Spray the polysaccharide derivative 7 solution onto the working electrode using inkjet printing. After evaporating the solvent at 54 °C, prepare a 10 mM crosslinking agent solution and a 10 μg / ml interleukin-8 recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 30 min, add the antibody solution dropwise, and mix using a vortex mixer for 2.9 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for interleukin-8 in this sensing application reaches 0.05 ng / ml.

[0075] Example 8

[0076] Weigh 1.7 mg of polysaccharide derivative 8 and add it to 1 ml of 0.6% (v / v) acetic acid solution to prepare a 1.7 mg / ml solution of polysaccharide derivative 8. Spray the polysaccharide derivative 8 solution onto the working electrode using inkjet printing. After evaporating the solvent at 60°C, prepare a 10 mM crosslinking agent solution and a 10 μg / ml interleukin-10 recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 30 min, add the antibody solution dropwise, and mix using a vortex mixer for 1.7 h to obtain a label-free electrochemical biomolecular sensing application. This sensing application achieves a detection limit of 1 ng / ml for interleukin-10.

[0077] Example 9

[0078] 1.9 mg of polysaccharide derivative 9 was weighed and added to 1 ml of 0.7% (v / v) acetic acid solution to prepare a 1.9 mg / ml solution of polysaccharide derivative 9. The polysaccharide derivative 9 solution was then inkjet printed onto the working electrode. After the solvent was evaporated at 56 °C, a 10 mM crosslinking agent solution and a 10 μg / ml circulating tumor cell recognition molecule solution were prepared. The crosslinking agent solution was added dropwise, and the mixture was activated for 30 min. The antibody solution was then added dropwise and mixed using a vortex mixer for 2.5 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for circulating tumor cells in this sensing application reached 0.15 ng / ml.

[0079] Example 10

[0080] 2.1 mg of polysaccharide derivative 10 was weighed and added to 1 ml of 0.8% (v / v) acetic acid solution to prepare a 2.1 mg / ml solution of polysaccharide derivative 10. The polysaccharide derivative 10 solution was then inkjet printed onto the working electrode. After the solvent was evaporated at 50°C, a 10 mM crosslinking agent solution and a 10 μg / ml tumor necrosis factor α recognition molecule solution were prepared. The crosslinking agent solution was added dropwise, and the mixture was activated for 30 min. The antibody solution was then added dropwise and mixed using a vortex mixer for 2.4 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for tumor necrosis factor α reached 0.11 ng / ml.

[0081] Example 11

[0082] Weigh 2.3 mg of polysaccharide derivative 11 and add it to 1 ml of 0.9% (v / v) acetic acid solution to prepare a 2.3 mg / ml solution of polysaccharide derivative 11. Spray the polysaccharide derivative 11 solution onto the working electrode using inkjet printing. After evaporating the solvent at 48°C, prepare a 10 mM crosslinking agent solution and a 10 μg / ml interferon recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 30 min, add the antibody solution dropwise, and mix using a vortex mixer for 1.5 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for interferon in this sensing application reaches 0.13 ng / ml.

[0083] Example 12

[0084] Weigh 2.5 mg of polysaccharide derivative 12 and add it to 1 ml of 1.0% (v / v) acetic acid solution to prepare a 2.5 mg / ml solution of polysaccharide derivative 12. Spray the polysaccharide derivative 12 solution onto the working electrode using inkjet printing. After evaporating the solvent at 57°C, prepare a 10 mM crosslinking agent solution and a 10 μg / ml SARS-CoV-2 recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 30 min, then add the antibody solution dropwise and mix using a vortex mixer for 2 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for this sensing application for SARS-CoV-2 reaches 9 ng / ml.

[0085] Example 13

[0086] 2.7 mg of polysaccharide derivative 13 was weighed and added to 1 ml of 0.5% (v / v) acetic acid solution to prepare a 2.7 mg / ml solution of polysaccharide derivative 13. The polysaccharide derivative 13 solution was then inkjet printed onto the working electrode. After the solvent was evaporated at 46 °C, a 10 mM crosslinking agent solution and a 10 μg / ml lg-A recognition molecule solution were prepared. The crosslinking agent solution was added dropwise, and the mixture was activated for 30 min. The antibody solution was then added dropwise and mixed using a vortex mixer for 2.6 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for lg-A in this sensing application reached 0.1 ng / ml.

[0087] Example 14

[0088] 2.9 mg of polysaccharide derivative 14 was weighed and added to 1 ml of 0.6% (v / v) acetic acid solution to prepare a 2.9 mg / ml solution of polysaccharide derivative 14. The polysaccharide derivative 14 solution was then inkjet printed onto the working electrode. After evaporating the solvent at 42°C, a 10 mM crosslinking agent solution and a 10 μg / ml interleukin-6 recognition molecule solution were prepared. The crosslinking agent solution was added dropwise, and the mixture was activated for 30 min. The antibody solution was then added dropwise and mixed using a vortex mixer for 1.8 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for interleukin-6 in this sensing application reached 10 ng / ml.

[0089] Example 15

[0090] Weigh 3.1 mg of polysaccharide derivative 15 and add it to 1 ml of 0.7% (v / v) acetic acid solution to prepare a 3.1 mg / ml solution of polysaccharide derivative 15. Spray the polysaccharide derivative 15 solution onto the working electrode using inkjet printing. After evaporating the solvent at 58°C, prepare a 10 mM crosslinking agent solution and a 10 μg / ml 1g-M recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 30 min, add the antibody solution dropwise, and mix using a vortex mixer for 1.9 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for 1g-M in this sensing application reaches 0.09 ng / ml.

[0091] Example 16

[0092] 3.3 mg of polysaccharide derivative 16 was weighed and added to 1 ml of 0.8% (v / v) acetic acid solution to prepare a 3.3 mg / ml solution of polysaccharide derivative 16. The polysaccharide derivative 16 solution was then inkjet printed onto the working electrode. After the solvent was evaporated at 52°C, a 10 mM crosslinking agent solution and a 10 μg / ml circulating tumor cell recognition molecule solution were prepared. The crosslinking agent solution was added dropwise, and the mixture was activated for 30 min. The antibody solution was then added dropwise and mixed using a vortex mixer for 2.7 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for circulating tumor cells in this sensing application reached 0.03 ng / ml.

[0093] Example 17

[0094] Weigh 3.5 mg of polysaccharide derivative 17 and add it to 1 ml of 0.9% (v / v) acetic acid solution to prepare a 3.5 mg / ml solution of polysaccharide derivative 17. Spray the polysaccharide derivative 17 solution onto the working electrode using inkjet printing. After evaporating the solvent at 41°C, prepare a 10 mM crosslinking agent solution and a 10 μg / ml interleukin-6 recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 30 min, add the antibody solution dropwise, and mix using a vortex mixer for 2.8 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for interleukin-6 in this sensing application reaches 0.01 ng / ml.

[0095] Example 18

[0096] Weigh 3.7 mg of polysaccharide derivative 18 and add it to 1 ml of 1.0% (v / v) acetic acid solution to prepare a 3.7 mg / ml solution of polysaccharide derivative 18. Spray the polysaccharide derivative 18 solution onto the working electrode using inkjet printing. After evaporating the solvent at 45°C, prepare a 10 mM crosslinking agent solution and a 10 μg / ml SARS-CoV-2 recognition molecule solution. Add the crosslinking agent solution dropwise, activate for 30 min, then add the antibody solution dropwise and mix using a vortex mixer for 1.6 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for this sensing application for SARS-CoV-2 reaches 0.5 ng / ml.

[0097] Example 19

[0098] 3.9 mg of polysaccharide derivative 19 was weighed and added to 1 ml of 0.5% (v / v) acetic acid solution to prepare a 3.9 mg / ml solution of polysaccharide derivative 19. The polysaccharide derivative 19 solution was then inkjet printed onto the working electrode. After evaporating the solvent at 59°C, a 10 mM crosslinking agent solution and a 10 μg / ml interleukin-8 recognition molecule solution were prepared. The crosslinking agent solution was added dropwise, and the mixture was activated for 30 min. The antibody solution was then added dropwise and mixed using a vortex mixer for 2.6 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for interleukin-8 in this sensing application reached 1.1 ng / ml.

[0099] Example 20

[0100] 4.1 mg of polysaccharide derivative 20 was weighed and added to 1 ml of 0.6% (v / v) acetic acid solution to prepare a 4.1 mg / ml solution of polysaccharide derivative 20. The polysaccharide derivative 20 solution was then inkjet printed onto the working electrode. After the solvent was evaporated at 44°C, a 10 mM crosslinking agent solution and a 10 μg / ml interleukin-10 recognition molecule solution were prepared. The crosslinking agent solution was added dropwise, and the mixture was activated for 30 min. The antibody solution was then added dropwise and mixed using a vortex mixer for 3 h to obtain a label-free electrochemical biomolecular sensing application. The detection limit for interleukin-10 in this sensing application reached 0.7 ng / ml.

[0101] Table 1. Materials and structural formulas used in preferred embodiments of the present invention.

[0102]

[0103] Table 2. Printing and Sensing Results Used in Preferred Embodiments of the Invention

[0104]

[0105] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Various changes can be made according to the purpose of the invention. Any changes, modifications, substitutions, combinations or simplifications made based on the spirit and principle of the technical solution of the present invention shall be equivalent substitutions. As long as they meet the purpose of the invention and do not deviate from the technical principle and inventive concept of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. A polysaccharide polymer-based label-free electrochemical biomolecular sensing material, characterized in that: Grafting redox groups onto polysaccharide polymers or their derivatives as electrochemical transducers, and further coupling with probe molecules that can specifically recognize solution markers to achieve label-free electrochemical sensitivity to the markers; the polysaccharide polymer-based label-free electrochemical biomolecular sensing material contains the chemical structure shown in formula (I): （I） Wherein, M is at least one of chitosan, cellulose, alginate, and the above-mentioned polysaccharide derivative monomer units; a, b, and c are positive integers, and 0 ≤ a ≤ 5000, 0 ≤ b ≤ 5000, and 0 ≤ c ≤ 5000; -L1- is at least one of -O-, -NH-CO-, -CO-NH-, -CO-O-, and -O-CO-; -L2- is at least one of -(CH2)x-, -(CH2)xO-(CH2)y-, -(CH2)x-(CH2-CH2-O)y-, -(CH2)xO-(CH2-CH2-O)y-, and -(CH2)2-SS-(CH2)2-, where x and y are integers and 0 ≤ x ≤ 20 and 0 ≤ y ≤ 20; -L3- is at least one of -O-, -NH-CO-, -CO-NH-, -CO-O-, and -O-CO-; -L4- is at least one of -O-, -NH-, -CO-, and -CO-O-; -L5- is at least one of -(CH2)x-, -(CH2)xO-(CH2)y-, -CO-(CH2)x-, -CO-(CH2)xO-(CH2)y-, -NH-(CH2)x-, -NH-(CH2)xO-(CH2)y-, where x is an integer and 0≤x≤20; -R1 is a probe molecule that specifically recognizes at least one of cortisol, interferon, tumor necrosis factor α, interleukin-6, interleukin-8, interleukin-10, 1g-M, 1g-A, circulating tumor cells, and SARS-CoV-2. -R2 is , , , At least one of its derivatives.

2. The polysaccharide polymer-based label-free electrochemical biomolecular sensing material according to claim 1, wherein the polysaccharide polymer-based label-free electrochemical biomolecular sensing material of formula (I) contains chitosan or its derivative monomer units: （I） in, a, b, c are positive integers, and 0 ≤ a ≤ 5000, 0 ≤ b ≤ 5000, 0 ≤ c ≤ 5000; -L1- is at least one of -O- and -NH-CO-; -L2- is at least one of -(CH2)x-(CH2-CH2-O)y-, -(CH2)xO-(CH2-CH2-O)y-, and -(CH2)2-SS-(CH2)2-, where x and y are integers and 0≤x≤20, 0≤y≤20; -L3- is at least one of -NH-CO- and -CO-NH-; -L4- is at least one of -O- and -NH-; -L5- is at least one of -(CH2)x-, -(CH2)xO-(CH2)y-, -CO-(CH2)x-, and -CO-(CH2)xO-(CH2)y-, where x is an integer and 0≤x≤20; -R1 is a probe molecule that specifically recognizes at least one of cortisol, interferon, IgG-M, IgG-A, circulating tumor cells, and SARS-CoV-2. -R2 is , At least one of its derivatives.

3. The polysaccharide polymer-based label-free electrochemical biomolecular sensing material according to claim 2, wherein the polysaccharide polymer-based label-free electrochemical biomolecular sensing material shown in formula (I) contains chitosan or its derivative monomer units: （I） in, a, b, c are positive integers, and 0 ≤ a ≤ 5000, 0 ≤ b ≤ 5000, 0 ≤ c ≤ 5000; -L1- is -NH-CO-; -L2- is -(CH2)2-SS-(CH2)2-; -L3- is -NH-CO-; -L4- is -NH-; -L5- is -(CH2)x-, where x is an integer and 0≤x≤20; -R1 is a probe molecule that specifically recognizes at least one of cortisol, interferon, IgG-M, and IgG-A; -R2 is and its derivatives.

4. The method for preparing the polysaccharide polymer-based label-free electrochemical biomolecular sensing material according to any one of claims 1-3, characterized in that: A 0.01-30 mg / mL solution of redox functionalized polysaccharide polymer was prepared using a 0.1-10% (v / v) acetic acid solution as printing ink. The polymer was then directionally deposited onto the electrode surface using appropriate printing parameters and conditions. A biocoupler solution was prepared by adding it dropwise to a 0.1-1000 μg / mL biomarker recognition molecule solution to achieve a concentration of 0.1-1000 mM. The mixture was activated for 1-120 min, and then an antibody solution was added dropwise to achieve a concentration of 0.1-1000 mM. The mixture was then thoroughly mixed to obtain a sensing material with specific recognition capabilities for biomarker molecules.

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