An anti-radiation interference electrochemical sensor and a preparation method and application thereof

Abstract

The present application relates to the technical field of electrochemical sensor, in particular to an anti-radiation interference electrochemical sensor and a preparation method and application thereof.The electrochemical sensor comprises an electrode system, and the electrode system comprises at least a working electrode; and a damage suppression protein Dsup coating for quenching radiation-induced free radicals is fixed on the surface of the working electrode.The sensor provided by the present application actively quenches radiation-induced free radicals by using the damage suppression protein Dsup coating on the surface of the working electrode, and effectively eliminates the background current drift and background noise.The protection mechanism greatly improves the detection accuracy in a strong radiation environment, does not hinder the mass transfer of the measured substance, and perfectly balances the high anti-radiation interference capability and the high sensitivity and fast response characteristics.

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensor technology, and more specifically, to a radiation-resistant electrochemical sensor, its preparation method, and its application. Background Technology

[0002] Electrochemical sensors, as efficient analytical tools, have an urgent need for real-time monitoring in various scenarios requiring strong ionizing radiation environments. However, ionizing radiation can irradiate and decompose solvent molecules at sensitive interfaces, generating a large number of highly reactive hydroxyl radicals. These radicals not only easily lead to the functional degradation of components at the interface, but their electrochemical behavior on the electrode surface also generates significant background current drift and noise, thus causing severe background interference to the electrochemical detection signal of the target analyte.

[0003] Currently, strategies to improve the stability of electrochemical sensors under radiation environments mainly include: using thick-film encapsulation technology to physically isolate the sensitive element from the external environment, using inorganic identification materials with stronger radiation resistance to replace biometric elements, or adding a physical radiation shielding layer to the sensor's peripheral structure.

[0004] However, the aforementioned conventional strategies all have significant limitations in practical applications. Physical encapsulation layers hinder the diffusion and mass transfer of the analyte to the electrode-sensitive interface, significantly reducing the sensor's response speed and detection sensitivity; inorganic recognition materials typically have low selectivity and affinity, making it difficult to meet the requirements of high-precision detection; and adding external radiation shielding layers greatly increases the sensor's size and weight, making it difficult to implement in specific load-constrained scenarios.

[0005] In general, existing radiation protection methods are mostly passive shielding or material alternatives that sacrifice performance, and none of them can effectively solve the direct electrochemical interference generated by radiation-induced free radicals at the electrode sensitive interface. Once free radicals are generated at the electrode interface, they will cause severe background noise and background current drift, resulting in a sharp drop in the signal-to-noise ratio of the sensor. As a result, traditional electrochemical sensors are very prone to signal distortion or complete loss of function in strong radiation environments.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an anti-radiation interference electrochemical sensor, its preparation method and application. The electrochemical sensor utilizes the damage inhibition protein Dsup coating on the surface of the working electrode to actively quench radiation-induced free radicals, which significantly improves signal stability and detection accuracy in strong radiation environments without sacrificing mass transfer, sensitivity and response speed.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides an electrochemical sensor resistant to radiation interference, comprising an electrode system; The electrode system includes at least a working electrode; The surface of the working electrode is coated with a damage-inhibiting protein Dsup coating for quenching radiation-induced free radicals.

[0009] In an optional embodiment, the damage-inhibiting protein Dsup is derived from tardigrades, and its amino acid sequence is shown in SEQ ID NO:1, or it is a polypeptide or its functional active fragment that has at least 90% identity with SEQ ID NO:1 and retains hydroxyl radical quenching activity.

[0010] In an optional embodiment, the damage-inhibiting protein Dsup coating is a monolayer or sub-monolayer coating; and / or, The damage inhibition protein Dsup is coated at a concentration of 0.5 μg / cm² on the surface of the working electrode. 2 ~10μg / cm 2 .

[0011] In an optional embodiment, a specific identification element is also fixed on the surface of the working electrode; Preferably, the specific recognition element is fixed on or within the damage inhibition protein Dsup coating; Preferably, the specific recognition element is selected from one or more of antibodies, nucleic acid aptamers, enzymes, and DNA probes; Preferably, the specific recognition element is covalently linked to the damage-inhibiting protein Dsup coating via a crosslinking agent, or is directionally immobilized on the damage-inhibiting protein Dsup coating via an avidin-biotin system.

[0012] In an optional embodiment, the working electrode is made of at least one of gold, platinum, glassy carbon, carbon paste, and screen-printed carbon electrode.

[0013] In an optional embodiment, the sensor is preserved by immersion in a preservation solution or by freeze-drying and then sealing it for preservation; Preferably, the preservation solution contains a lyophilization protectant, which is selected from one or more of trehalose, sucrose, mannitol, lactose, and dextran; Preferably, the preservation solution comprises: 1%~5% (w / v) trehalose, 0.5%~2% (w / v) sucrose, 0.1%~1% (w / v) mannitol, 0.01%~0.1% (w / v) bovine serum albumin, and supporting electrolyte buffer; Preferably, the supporting electrolyte buffer is a phosphate buffer; Preferably, the preservation solution also contains sodium azide as a preservative.

[0014] Secondly, the present invention provides a method for preparing an anti-radiation interference electrochemical sensor as described in any of the foregoing embodiments, comprising: The surface of the working electrode is activated to obtain an activated working electrode; The activated working electrode is brought into contact with the damage inhibitor protein Dsup, thereby fixing the damage inhibitor protein Dsup onto the surface of the working electrode and forming the damage inhibitor protein Dsup coating.

[0015] In an optional embodiment, the activation treatment of the working electrode and the fixation are selected from any of the following methods: A. The working electrode is made of gold or platinum; the activation treatment includes: forming a self-assembled monolayer with exposed carboxyl groups at the ends on the surface of the working electrode, and activating the carboxyl groups with carbodiimide and N-hydroxysuccinimide to form an active ester intermediate; the fixation includes: reacting the amino group in the damage inhibitor protein Dsup with the active ester intermediate to form an amide bond for covalent fixation; B. The working electrode is made of glassy carbon, carbon paste, or screen-printed carbon; the activation treatment and the fixation include: adsorbing the damage inhibition protein Dsup onto the surface of the working electrode and performing cross-linking treatment using a cross-linking agent; C. The working electrode is a nickel ion modified electrode, and the damage inhibition protein Dsup is tagged with histidine; the immobilization includes: immobilization through the affinity between the histidine tag and the nickel ion modified electrode.

[0016] In an optional implementation, the contact conditions include at least one of the following: A. The reaction temperature is 4℃~25℃; B. The reaction time is 1 hour to 12 hours; C. The concentration of the damage-inhibiting protein Dsup is 0.1 mg / mL to 2.0 mg / mL; and / or, After forming the damage-inhibiting protein Dsup coating, the method further includes a step of sealing the remaining active sites on the electrode surface with a sealing agent; more preferably, the sealing agent includes at least one of bovine serum albumin, ethanolamine, and polyvinyl alcohol; and / or, After forming the damage-inhibiting protein Dsup coating, the method further includes the step of attaching a specific recognition element to the surface of the damage-inhibiting protein Dsup coating through a chemical crosslinking or affinity system.

[0017] Thirdly, the present invention provides a detection device, comprising: an electrochemical sensor resistant to radiation interference as described in the foregoing embodiments; and a signal acquisition and processing module electrically connected to the electrochemical sensor, wherein the signal acquisition and processing module is used to output a detection signal of a target analyte.

[0018] Preferably, the detection equipment is a nuclear industry radiation monitor, a nuclear waste online analyzer, a medical radiotherapy dose calibrator, or a space exploration payload analyzer.

[0019] Fourthly, the present invention provides a test kit, comprising: an electrochemical sensor resistant to radiation interference as described in the foregoing embodiments.

[0020] In an optional embodiment, the kit further includes the preservation solution described in the foregoing embodiments; the electrochemical sensor is immersed in the preservation solution for sealed packaging; or, the surface of the electrochemical sensor is coated with a lyophilized product of the preservation solution described in the foregoing embodiments and then vacuum-sealed.

[0021] Fifthly, the present invention provides an application of the radiation-resistant electrochemical sensor described in any of the foregoing embodiments in the detection of target analytes under radiation environments; Preferably, the radiation environment includes at least one of a nuclear industrial facility, a nuclear waste disposal site, a space exploration device, and a medical radiotherapy area; Preferably, the detection mode used in the detection is selected from at least one of the amperometric method, the voltammetric method, and the impedance method.

[0022] Compared with existing technologies, the electrochemical sensor provided in this application achieves active quenching of radiation-induced free radicals by fixing a damage-inhibiting protein Dsup coating on the surface of the working electrode. In a strong radiation environment, this coating can preferentially capture and neutralize highly reactive free radicals generated by the irradiation decomposition of solvent molecules, effectively preventing them from undergoing non-specific reactions with the sensitive interface, thus eliminating the source of interference that causes measurement errors at the source.

[0023] By effectively quenching free radicals, the electrochemical behavior of free radicals superimposed on the electrode surface is significantly reduced, thereby greatly reducing the drift of background current and the increase of environmental noise. This allows the sensor to maintain excellent signal-to-noise ratio and extremely high detection accuracy even under continuous high-dose irradiation conditions, avoiding the signal distortion or failure problems that are prone to occur under traditional passive shielding mechanisms.

[0024] Unlike conventional physical thick-film encapsulation or the addition of inorganic isolation layers, this protein coating provides a microscopic active protection mechanism that does not hinder the diffusion and mass transfer process of the target analyte to the electrode sensitive interface. Therefore, while achieving excellent resistance to radiation interference, the electron transfer and mass diffusion kinetics of the electrode interface are well preserved, thus ensuring that the sensor possesses extremely high inherent detection sensitivity and extremely fast response speed. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 A schematic diagram illustrating the principle of covalent immobilization of Dsup protein on a gold electrode surface, as provided in this application; Figure 2 This is a comparison of the current-concentration standard curves (IC curves) and sensitivity of the bare gold electrode and the Dsup-modified gold electrode in Example 5 of this application when detecting potassium ferricyanide standard solutions of different concentrations. Figure 3 This is a comparison of the chronoamperometric response time (It curve) of the bare gold electrode and the Dsup-modified gold electrode after adding 2 mM potassium ferricyanide standard solution to the blank buffer in Example 5 of this application. Figure 4 This is a comparison of the current response of two sets of sensors to different concentrations of glucose in Example 9 of this application. Detailed Implementation

[0027] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0028] This application provides a radiation-resistant electrochemical sensor, the main structure of which includes an electrode system. The electrode system, serving as the hardware basis for electrochemical reactions and signal transduction, includes at least one working electrode. To enable the sensor to operate stably in harsh environments with strong ionizing radiation, such as nuclear industrial facilities, nuclear waste disposal sites, space exploration equipment, and medical radiotherapy areas, the sensitive interface of the working electrode has undergone microscopic molecular reconstruction.

[0029] Specifically, the surface of the working electrode is coated with a damage-inhibiting protein Dsup coating for quenching radiation-induced free radicals.

[0030] The damage-inhibiting protein Dsup coating on the working electrode surface primarily functions through a dual active protection mechanism of free radical quenching and electric field shielding. When the sensor is exposed to ionizing radiation, radiation-induced solvent molecule decomposition generates highly reactive hydroxyl radicals (·OH) around the electrode interface. The surface of the damage-inhibiting protein Dsup, immobilized at the sensitive interface of the working electrode, is rich in aromatic and sulfur-containing amino acid residues. These residues preferentially capture and neutralize the hydroxyl radicals, converting them into stable, inactive products, thus preventing oxidative damage and non-specific interference caused by free radicals to the sensitive interface. Simultaneously, because the damage-inhibiting protein Dsup carries a large number of positively charged residues in its flexible three-dimensional conformation, it forms a localized micro-electric field at the sensitive interface of the working electrode. This localized electric field has an electric field shielding effect, significantly reducing the non-specific electrochemical redox behavior of residual free radicals on the electrode surface, thereby suppressing radiation-induced background noise and background current drift at the source.

[0031] As a further extension of the structure in this embodiment, the damage inhibition protein Dsup coating exhibits a dense monolayer or sub-monolayer architecture on the working electrode surface. Because the thickness of this bioprotective coating remains at the monomolecular level, its highly flexible conformation closely adheres to the microscopic inhomogeneous regions of the electrode surface. Therefore, while demonstrating excellent radiation resistance and free radical quenching capabilities, it does not significantly increase the transfer impedance at the electrode sensitive interface, nor does it hinder the diffusion and mass transfer process of external analytes to the electrode surface. This allows the sensor, even under high-dose cumulative irradiation, to not only reduce the concentration of hydroxyl radicals near the electrode sensitive interface by approximately 82% and control the background current drift to an extremely low level, but also perfectly retain over 96% of its inherent detection sensitivity. Furthermore, the detection response speed shows no statistically significant difference compared to the bare electrode without the coating, achieving an electrochemical synergy of radiation resistance and high-sensitivity response speed.

[0032] Furthermore, the substrate material of the working electrode can be selected from any of gold, platinum, glassy carbon, carbon paste, or screen-printed carbon electrodes, depending on the actual detection scenario. The amino acid sequence of the damage inhibitor protein Dsup can be a natural sequence, or it can be a homologous polypeptide or active fragment retaining free radical quenching function. The sensitive interface of the sensor can directly use the electrode modified with the Dsup coating to measure electrochemically active targets, or it can be further co-modified with one or more specific recognition elements such as antibodies, nucleic acid aptamers, enzymes, or DNA probes on or within the damage inhibitor protein Dsup coating through covalent cross-linking or affinity immobilization structures to achieve highly selective and high-precision real-time monitoring of specific target substances under high radiation backgrounds.

[0033] In summary, the electrochemical sensor, by immobilizing a damage-inhibiting protein Dsup coating on the working electrode surface, can actively capture and neutralize highly reactive free radicals generated by radiation, thus severing the interaction between free radicals and the sensitive interface at its source. This not only significantly reduces radiation-induced background current drift and background noise, greatly improving the signal-to-noise ratio and detection accuracy under strong radiation environments, but also ensures that the active protective coating does not hinder the mass transfer and diffusion of the target analyte. Therefore, while achieving excellent anti-radiation interference capabilities, it perfectly preserves the sensor's original high sensitivity and rapid response characteristics.

[0034] In some embodiments, the damage-inhibiting protein Dsup is derived from tardigrades, and its amino acid sequence is shown in SEQ ID NO:1, or it is a polypeptide or its functionally active fragment that has at least 90% identity with SEQ ID NO:1 and retains hydroxyl radical quenching activity.

[0035] The radiation-resistant electrochemical sensor provided in this embodiment includes an electrode system, which includes at least a working electrode. The surface of the working electrode is coated with a damage-inhibiting protein Dsup coating for quenching radiation-induced free radicals.

[0036] Specifically, the damage-inhibiting protein Dsup is derived from tardigrades, and its amino acid sequence is shown in SEQ ID NO: 1, or it is a polypeptide or its functionally active fragment that has at least 90% identity with SEQ ID NO: 1 and retains hydroxyl radical quenching activity. In this embodiment, SEQ ID NO: 1, serving as the reference sequence, possesses specific biophysical characteristics. It belongs to naturally occurring disordered proteins (IDPs) and does not have a fixed tertiary crystal structure in space, but rather exhibits a highly open and flexible linear conformation. This highly flexible conformation allows the protein molecule or its polypeptide variant to adhere closely to the microscopic sensitive surface of the working electrode with extremely high adaptability, forming a continuous protective coating with molecular-level thickness.

[0037] The surface of the damage inhibitor protein Dsup and its homologous polypeptide variants is rich in high-density aromatic amino acid residues (such as tyrosine and phenylalanine) and sulfur-containing amino acid residues (such as methionine). In a strong ionizing radiation environment, as the highly oxidizing hydroxyl radicals (·OH) generated by the decomposition of solvent molecules at the radiation-induced interface diffuse toward the electrode sensitive surface, the damage inhibitor protein Dsup or its variant polypeptides utilize side chain groups as efficient electron donors to preferentially undergo single-electron transfer chemical reactions with the hydroxyl radicals. This captures and neutralizes the highly reactive free radicals, blocking the pathway for non-specific electrochemical redox reactions of free radicals on the working electrode surface, and fundamentally eliminating background current drift and background noise interference caused by ionizing radiation.

[0038] The peptide variants with at least 90% identity include derived peptides formed by conserved amino acid residue substitutions, deletions, or additions based on the sequence of SEQ ID NO: 1. Since the free radical quenching activity of the Dsup protein mainly depends on the chemical properties and charge density distribution of specific functional residues in its flexible linear conformation, rather than a precise dependence on specific steric hindrance, those skilled in the art can foresee that when the amino acid sequence identity is at least 90%, the peptide chain can still completely retain its disordered flexible characteristics and free radical capture cross-section, possessing free radical quenching activity completely equivalent to the natural full-length protein. Similarly, the functionally active fragment refers to a peptide segment containing the core functional region of free radical quenching in SEQ ID NO: 1. Such fragments have the advantages of smaller molecular weight and lower steric hindrance. After being immobilized on the surface of the working electrode, they can further reduce the interfacial charge transfer impedance, maintaining the radiation resistance stability of the sensor while improving the sensor's response speed and detection sensitivity.

[0039] The polypeptide with at least 90% identity to SEQ ID NO: 1 refers to a sequence formed by modifying or conservatively substituting amino acid residues at non-core sites in SEQ ID NO: 1, while maintaining the polypeptide's overall natural disordered flexible conformation and free radical quenching function. Since the protective effect of the damage inhibitor protein Dsup at the electrode interface is based on the direct chemical neutralization reaction between its side-chain-rich electron-donating groups (such as aromatic residues and sulfur-containing residues) and hydroxyl radicals, its function does not depend on a specific primary crystallographic higher-order spatial structure. Therefore, as long as the overall sequence identity remains within the range of at least 90%, the free radical capturing center and surface charge distribution within its core can remain stable. Those skilled in the art can fully foresee that such homologous variant polypeptides, after being immobilized on the working electrode surface, will possess the same free radical quenching activity as SEQ ID NO: 1 and the technical effect of reducing sensor background interference.

[0040] Preferably, the amino acid variations occurring in the polypeptide with at least 90% homology are primarily conservative amino acid substitutions. These conservative amino acid substitutions refer to substitutions within amino acid families with similar physicochemical properties, specifically including: substitutions within basic amino acid families (such as lysine, arginine, and histidine); substitutions within acidic amino acid families (such as aspartic acid and glutamic acid); substitutions within aliphatic uncharged residue families (such as glycine, alanine, valine, leucine, and isoleucine); and substitutions within aromatic residue families (such as phenylalanine, tyrosine, and tryptophan). These conservative substitutions do not alter the electrochemical properties of the microscopic local electric field and chemical barrier formed by the polypeptide at the electrode interface, and are parallel technical solutions that can be implemented by those skilled in the art based on the concept of this disclosure without inventive effort.

[0041] In some embodiments, the damage-inhibiting protein Dsup coating is a monolayer or sub-monolayer coating.

[0042] The spatial configuration of the damage-inhibiting protein Dsup coating is limited to a monolayer or sub-monolayer, forming the core physical basis for the sensor's balance of radiation resistance and high sensitivity. Since Dsup protein is a naturally disordered protein with highly open and flexible linear segments, when it spreads as a monolayer or distributed as a microscopic, discrete island on the working electrode surface in a sub-monolayer form, the overall thickness of the coating in the longitudinal space is strictly limited to an extremely thin range at the nanometer level. In this state, the biomolecular protective layer can form a sufficiently high-density free radical trapping network without introducing a significant impedance barrier at the sensitive interface of the working electrode. This maximizes the preservation of the mass transfer process of the analyte from the external environment to the electrode surface and the electron transfer kinetics at the interface. When the sensor measures the target analyte under strong radiation, the slope (sensitivity) retention rate of its standard curve can reach as high as 96.1%, and the response time for detecting steady-state current is not statistically significantly different from that of the bare electrode.

[0043] In some embodiments, the damage-inhibiting protein Dsup is coated at a concentration of 0.5 μg / cm² on the surface of the working electrode. 2 ~10μg / cm 2 .

[0044] According to classical electrochemical kinetics, for diffusion-controlled solid-liquid heterogeneous charge transfer systems, the chronoampere response current follows the Cottrell equation: ; The peak current under cyclic voltammetry scanning follows the Randles-Sevcik equation: ; Where A is the effective electrochemical active area of ​​the working electrode, and D is the diffusion coefficient of the target analyte.

[0045] In this disclosure, because the spatial configuration of the damage-inhibiting protein Dsup coating is strictly controlled at a monolayer or sub-monolayer state, its nanoscale ultrathin network does not reduce the geometric and active area A of the working electrode, nor does it change the macroscopic diffusion coefficient D of the analyte at the interface. Therefore, at the mathematical and electrochemical levels, this protection mechanism perfectly ensures the high-fidelity preservation of the slope (sensitivity) of the sensor's inherent standard curve.

[0046] If the coating amount is less than 0.5 μg / cm 2 If the protein molecule distribution at the electrode sensitive interface is too sparse, it is impossible to construct a continuous or effective free radical trapping cross section. This allows highly reactive hydroxyl radicals generated by radiation decomposition to easily penetrate protective gaps and directly undergo non-specific electrochemical reactions on the electrode surface, resulting in severe radiation drift of the background current. If the coating amount is higher than 10 μg / cm³,2 If protein molecules are not properly distributed, they will accumulate and cluster in multiple layers at the sensitive interface. This not only fails to improve the quenching efficiency of free radicals, but also drastically increases the mass transfer impedance and charge transfer impedance at the electrode interface, resulting in a significant decrease in the sensitivity of the sensor and a severely slow response speed.

[0047] It should be noted that the amount of the damage-inhibiting protein Dsup coated on the surface of the working electrode (μg / cm²) is... 2 The Dsup protein can be quantitatively determined using conventional microbalances or surface analysis techniques in this field. Specifically, a dissipative quartz crystal microbalance (QCM-D) can be used to monitor the adsorption mass and layer thickness changes of the Dsup protein on the electrode substrate surface online; or surface plasmon resonance (SPR) technology can be used to calculate the absolute loading mass density based on the change in interfacial refractive index; alternatively, it can be calculated by electrochemical labeling or reverse measurement of the mass difference of unbound proteins in the supernatant, combined with the geometric surface area of ​​the working electrode. All of the above measurement methods are conventional detection schemes that can be implemented by those skilled in the art without requiring inventive effort.

[0048] Therefore, by limiting the coating amount to 0.5 μg / cm 2 ~10μg / cm 2 By combining the microstructure of monolayers or sub-monolayers, the contradiction between protective efficacy and detection performance can be fundamentally balanced. In some preferred embodiments of this invention, the coating amount of the damage inhibition protein Dsup on the working electrode surface can be flexibly adjusted according to the material and roughness of the actual detection substrate. For example, it can be 0.5 μg / cm². 2 1.0 μg / cm 2 2.0 μg / cm 2 3.5 μg / cm 2 5.0 μg / cm 2 6.5 μg / cm 2 8.0 μg / cm 2 9.0 μg / cm 2 10.0 μg / cm 2 Wait, the coatings at the specific point values ​​mentioned above can achieve an excellent protective effect of reducing the background current drift by about 70% in a continuous high-dose cumulative irradiation field from 50 Gy / h to 1000 Gy, while perfectly preserving the inherent electrochemical sensing activity of the sensor.

[0049] In some embodiments, a specific identification element is also fixed to the surface of the working electrode.

[0050] Preferably, the specific recognition element is fixed on or within the damage inhibition protein Dsup coating; Preferably, the specific recognition element is selected from one or more of antibodies, nucleic acid aptamers, enzymes, and DNA probes; Preferably, the specific recognition element is covalently linked to the damage-inhibiting protein Dsup coating via a crosslinking agent, or is directionally immobilized on the damage-inhibiting protein Dsup coating via an avidin-biotin system.

[0051] In this embodiment, the sensitive interface of the radiation-resistant electrochemical sensor was specifically identified and constructed using a multifunctional composite. Specifically, a specific recognition element is also immobilized on the surface of the working electrode. Preferably, the specific recognition element is immobilized on or within the damage-inhibiting protein Dsup coating. Preferably, the specific recognition element is selected from one or more of antibodies, nucleic acid aptamers, enzymes, and DNA probes. Preferably, the specific recognition element is covalently linked to the damage-inhibiting protein Dsup coating via a cross-linking agent, or directionally immobilized on the damage-inhibiting protein Dsup coating via an avidin-biotin system.

[0052] The introduction of the specific recognition element endows this radiation-resistant electrochemical sensor with the ability to perform highly selective qualitative or quantitative analysis of specific target analytes in complex backgrounds such as nuclear industrial sites, radioactive wastewater, or space environments. Fixing the specific recognition element "above" or "within" the damage-inhibiting protein Dsup coating constitutes a spatial synergistic basis for the protective and recognition components at the microscale. When the specific recognition element is fixed "above" the damage-inhibiting protein Dsup coating, an ordered longitudinal bilayer functional film structure is formed on the working electrode surface. The bottom layer of Dsup protein adheres closely to the electrode surface, exerting free radical quenching and microscopic electric field shielding effects, while the surface layer of specific recognition element faces the analyte fluid, fully binding with the target. When the specific recognition element is fixed "within" the damage-inhibiting protein Dsup coating, the two form a highly interwoven monolayer or multifunctional composite network on the electrode substrate surface. This extreme reduction in microscopic distance ensures that, under strong ionizing radiation, highly reactive hydroxyl radicals (·OH) generated by the irradiation and decomposition of neighboring solvent molecules can be preferentially captured and neutralized by high-density Dsup protein side chain residues before attempting to attack and oxidize the specific recognition element. This provides in-situ microscopic shielding protection for the biorecognition probe, preventing non-specific denaturation, secondary structure breakage, or functional degradation of the biorecognition probe, and ensuring signal stability during long-term sensor operation.

[0053] To stabilize the interface architecture under different detection scenarios, this embodiment provides two parallel fixation paths. The first path involves covalent bonding via a crosslinking agent. A bifunctional coupling agent is used to firmly covalently bond the active groups on the molecular chain of the specific recognition element to the exposed amino acid residue side chains in the damage inhibitor protein Dsup coating, forming stable amide bonds, thioether bonds, or Schiff base structures. This significantly improves the mechanical tolerance and thermodynamic stability of the sensor's sensitive interface in complex radioactive liquid shear flows. The second path involves directional fixation via an avidin-biotin system. Avidin is first introduced onto the surface of the working electrode or the surface of the damage inhibitor protein Dsup coating, and then it undergoes high-affinity self-assembly with the biotinylated specific recognition element. Because the biotinylated sites can be precisely constructed in the inactive structural domains of the specific recognition element, this system can guide all biorecognition probes to a highly consistent and regular directional arrangement at the microscopic interface, minimizing steric hindrance interference and ensuring that their active capture sites are fully exposed. This fixed path, in synergy with the free radical damage-resistant properties of the Dsup coating, can significantly improve the sensor's specific capture efficiency and response sensitivity for ultra-trace targets while maintaining a high radiation dose retention rate.

[0054] In some embodiments, the working electrode is made of at least one of gold, platinum, glassy carbon, carbon paste, and screen-printed carbon electrodes. Different working electrode substrates provide diverse interfacial chemistry for the immobilization of the damage-inhibiting protein Dsup coating and endow the sensor with high adaptability in various high-radiation application scenarios.

[0055] When the working electrode is made of noble metals such as gold or platinum, its surface possesses extremely high chemical inertness and excellent electronic conductivity. The noble metal substrate supports the formation of highly ordered and dense self-assembled monolayers (SAMs) containing thiol groups on its surface, providing uniform reaction sites for the subsequent covalent bonding of the damage-inhibiting protein Dsup. Sensors based on gold or platinum exhibit extremely low background noise and low charge transfer impedance at their interface, maximizing the synergistic effect of the Dsup coating in eliminating free radical electrochemical interference. This makes them suitable for space exploration equipment or medical radiation dosimetry environments requiring extremely high detection accuracy and long-term in-situ monitoring.

[0056] When the working electrode is made of carbon-based materials such as glassy carbon, carbon paste, or screen-printed carbon electrodes, it offers significant advantages including a wide electrochemical potential window, good chemical stability, and low manufacturing cost. The rich microstructure and edge defects on the surface of carbon-based materials can generate strong physical adsorption and non-covalent interactions with the damage inhibition protein Dsup molecules, thereby forming a robust protective coating through cross-linking processes. In particular, when using screen-printed carbon electrodes as the working electrode substrate, mature thick-film printing microelectronics technology enables wafer-level, low-cost mass production of this radiation-resistant sensor probe.

[0057] In extremely harsh environments such as nuclear industrial facilities or high-concentration radioactive wastewater treatment sites, the recovery of sensor probes often faces a very high risk of secondary radiation contamination. By employing low-cost carbon paste or screen-printed carbon electrodes combined with a Dsup protein coating, the probe can maintain a short but highly accurate monitoring signal under strong ionizing radiation while also possessing the characteristics of a disposable consumable. After completing the detection of the target analyte, the probe can be safely discarded along with other low-level nuclear waste, eliminating the need for complex elution and decontamination recovery processes. This significantly improves the engineering practicality and economic feasibility of this radiation-resistant electrochemical protection technology.

[0058] In some embodiments, to ensure the activity stability of the radiation-resistant electrochemical sensor after assembly during long-term storage and long-distance transportation, specific configurations have been made for the storage method and storage medium of the sensor. Specifically, the sensor is stored by immersion in a storage solution, or by freeze-drying and then sealing it for storage.

[0059] Preferably, the preservation solution contains a lyophilization protectant selected from one or more of trehalose, sucrose, mannitol, lactose, and dextran.

[0060] Because the sensitive interface of the working electrode is immobilized with biomolecules, a lyophilization protectant is specially added to the preservation solution to prevent conformational collapse, aggregation and inactivation, or microbial degradation during dehydration or long-term idleness. The lyophilization protectant is selected from one or more of trehalose, sucrose, mannitol, lactose, and dextran. These sugars or polyols can replace water molecules in binding to the damage inhibitor protein Dsup through hydrogen bonding, forming a dense, amorphous vitrified matrix on the electrode surface. This allows the protein's natural, disordered, flexible conformation to be stably "frozen" in its active state under the dehydration stress of freeze-drying.

[0061] Preferably, in order to achieve the best synergy between maintaining the physical morphology of the radiation-resistant coating and preserving its biological activity, the preservation solution adopts a specific multi-component formulation, which includes: 1%~5% (w / v) trehalose [for example, it can be 1.0% (w / v), 1.5% (w / v), 2.0% (w / v), 2.5% (w / v), 3.0% (w / v), 3.5% (w / v), 4.0% (w / v), 4.5% (w / v), 5.0% (w / v), etc.]. 0.5%~2% (w / v) sucrose [e.g., 0.5% (w / v), 0.7% (w / v), 0.9% (w / v), 1.1% (w / v), 1.3% (w / v), 1.5% (w / v), 1.7% (w / v), 1.9% (w / v), 2.0% (w / v), etc.], 0.1%~1% (w / v) mannitol [e.g., 0.1% (w / v), 0.2% (w / v), 0.3% (w / v), 0.5% (w / v), 0.6% (w / v), etc.] [0.7% (w / v), 0.8% (w / v), 0.9% (w / v), 1.0% (w / v), etc.], 0.01%~0.1% (w / v) bovine serum albumin [for example, it can be 0.01% (w / v), 0.02% (w / v), 0.03% (w / v), 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), 0.08% (w / v), 0.09% (w / v), 0.10% (w / v), etc.] and supporting electrolyte buffers.

[0062] In this composite system, trehalose and sucrose together construct an amorphous protective microenvironment to prevent damage to the secondary structure of proteins; mannitol acts as a crystallization framework agent, giving the freeze-dried coating excellent mechanical and physical strength and preventing its macroscopic collapse; trace amounts of bovine serum albumin (BSA) act as an excellent inert carrier protein, effectively providing a steric barrier to prevent non-specific loss or depolymerization of core functional proteins.

[0063] Preferably, the supporting electrolyte buffer is a phosphate buffer; Preferably, the preservation solution also contains sodium azide as a preservative.

[0064] Furthermore, the supporting electrolyte buffer is a phosphate buffer, which provides stable physiological-grade pH and ionic strength to prevent disruption of the microscopic electrostatic balance of proteins at the electrode interface. When the sensor is stored in a liquid immersion solution, the storage solution also contains sodium azide as a preservative to completely inhibit the potential growth of bacteria and fungi during liquid storage, thereby significantly extending the commercial shelf life of the radiation-resistant sensor and ensuring that it can immediately exert its excellent free radical quenching and signal stabilization performance when used in extreme nuclear radiation environments.

[0065] This application also provides a method for fabricating a radiation-resistant electrochemical sensor as described above. This process route achieves efficient and stable assembly of actively protected biomolecules on the sensitive surface of the working electrode through a mild and universal solid-phase interface reconstruction technique. Specifically, the fabrication method mainly includes the following process steps: Step S1: Activate the surface of the working electrode to obtain the activated working electrode.

[0066] In the activation process, addressing the technical shortcomings of the original working electrode substrate (such as an unmodified conductive solid substrate) having a relatively inert interface that hinders the direct adhesion of biomolecules, this embodiment employs physical or chemical modification methods to microscopically reconstruct the sensitive interface of the electrode. The microscopic mechanism of the activation process involves controlling the macroscopic hydrophilicity / hydrophobicity, microscopic surface energy distribution, or charge state of the working electrode surface through interfacial chemical reactions, surface energy excitation, or polarization decontamination. This introduces high-density active reaction sites, transitional layers, or induces physical defect regions with high adsorption energy levels on the microscopic geometric surface of the working electrode. The "activated working electrode" obtained in this step possesses extremely high thermodynamic capture activity and chemical strain capability for biomolecules on its sensitive surface, thus providing an essential foundation for the uniform construction and long-term mechanical stability anchoring of the subsequent protective coating.

[0067] Step S2: The activated working electrode is brought into contact with the damage inhibitor protein Dsup, so that the damage inhibitor protein Dsup is fixed on the surface of the working electrode to form the damage inhibitor protein Dsup coating.

[0068] In the fixation step, the activated working electrode, rich in active sites or high adsorption energy levels obtained in the preceding steps, is immersed, drop-coated, or exposed to a macroscopic phase space containing the free-state damage inhibitor protein Dsup. Driven by the mass transfer kinetics and thermodynamic gradient at the solid-liquid interface, the damage inhibitor protein Dsup molecules diffuse efficiently and adhere to the sensitive surface of the activated working electrode from the bulk solution. Upon contact, the active reaction sites or specific adsorption microenvironment on the electrode sensitive interface rapidly undergo a solid-phase anchoring reaction with the damage inhibitor protein Dsup molecules. Since the damage inhibitor protein Dsup is a naturally disordered protein in biophysics, it does not possess a rigid, solidified tertiary crystal structure but rather exhibits a highly flexible open linear chain conformation. Therefore, during the fixation process, this flexible polypeptide chain can seamlessly adhere to, spread out, and cover the microscopically non-uniform surface of the working electrode with nanoscale spatial adaptability, ultimately forming a densely packed monolayer or sub-monolayer level bioprotective coating.

[0069] In summary, the two-step preparation method provided in this embodiment has the core advantage of a mild process and controllable reaction conditions, which can minimize the non-specific aggregation and inactivation of protein molecules during solid-phase interface assembly. The resulting damage-inhibiting protein Dsup coating has free radical trapping centers composed of aromatic and sulfur-containing amino acid residues that are fully and efficiently open to the external irradiation environment. This allows the final electrochemical sensor to capture and neutralize the highly oxidizing hydroxyl radicals (·OH) generated by the irradiation decomposition of solvent molecules at an extremely high reaction rate when facing a strong ionizing radiation field, keeping the background current drift and background noise under high-dose cumulative irradiation at an extremely low level. At the same time, because this two-step method strictly limits the longitudinal growth thickness of the coating, it ensures that the charge transfer impedance and mass diffusion impedance of the finished product are extremely low, thus perfectly preserving the inherent electron transfer rate of the working electrode. This results in a final sensor with radiation-resistant stability, high detection sensitivity, and fast response speed.

[0070] In some implementations, for working electrode substrates of different materials, the activation treatment and the fixing step can be flexibly selected from any of the following three parallel micro-interface assembly paths to adapt to the requirements of different detection scenarios regarding sensor cost, accuracy, and process complexity: A. The working electrode is made of gold or platinum; the activation treatment includes: forming a self-assembled monolayer with exposed carboxyl groups at the end on the surface of the working electrode, and activating the carboxyl groups with carbodiimide and N-hydroxysuccinimide to form an active ester intermediate; the fixation includes: reacting the amino group in the damage inhibitor protein Dsup with the active ester intermediate to form an amide bond for covalent fixation.

[0071] When the working electrode is made of gold or platinum, its surface possesses excellent potential for chemical modification. In this approach, the activation treatment specifically includes: firstly, constructing a well-organized self-assembled monolayer (SAM) with exposed carboxyl groups at its ends on the surface of the working electrode through chemisorption; subsequently, treating the monolayer with a mixed solution of carbodiimide (such as EDC) and N-hydroxysuccinimide (NHS), where EDC reacts with the carboxyl groups and binds to NHS, activating the originally inert carboxyl groups and transforming them into highly nucleophilic ester intermediates.

[0072] Please see Figure 1 , Figure 1 This is a schematic diagram illustrating the principle of the covalent immobilization process of Dsup protein on the surface of a gold electrode. Figure 1As shown, a regular terminal carboxyl self-assembled monolayer is first formed on the surface of the gold electrode through the spontaneous adsorption of thiol-containing molecules; then, after activation by the EDC / NHS system, the terminal carboxyl groups are converted into active ester intermediates; finally, the intermediates are used to undergo a nucleophilic substitution reaction with the amino groups on the surface of the Dsup protein, thereby achieving the directional and covalent anchoring of biomolecules at the electrode interface.

[0073] After activation, the fixation step specifically includes immersing the electrode in a solution containing the damage inhibitor protein Dsup. The naturally occurring free amino groups (such as the primary amino group of the lysine side chain) on the Dsup molecular chain initiate a nucleophilic attack on the active ester intermediate, thereby forming extremely stable amide bonds at the electrode interface. This covalent cross-linking pathway ensures that the Dsup coating exhibits extremely high adhesion and uniformity on the noble metal surface, effectively resisting physical shearing and chemical desorption under strong radiation environments. It is suitable for high-precision detection scenarios requiring extremely low background noise and long-term in-situ monitoring.

[0074] B. The working electrode is made of glassy carbon, carbon paste, or screen-printed carbon; the activation treatment and the fixation include: adsorbing the damage inhibition protein Dsup onto the surface of the working electrode and performing cross-linking treatment using a cross-linking agent.

[0075] When the working electrode is made of glassy carbon, carbon paste, or screen-printed carbon, a simpler solid-phase modification process can be employed by utilizing the abundant micropores, defect edges, and hydrophobic properties of the carbon material surface. In this approach, the activation treatment and the fixation step are integrated into a continuous physical-chemical composite process, specifically including: directly contacting the damage-inhibiting protein Dsup solution with the working electrode, allowing its flexible segments to spontaneously adsorb, adhere, and conform to the micro-uniform surface of the carbon substrate through hydrophobic interactions and van der Waals forces; subsequently, a crosslinking agent (such as a bifunctional reagent like glutaraldehyde) is introduced into the interface for crosslinking treatment. The crosslinking agent forms a covalent bridging network between the adsorbed Dsup protein molecules and between the protein and the carbon substrate, converting the reversible physical adsorption state into an irreversible solidified crosslinked state. This method not only has low process costs and eliminates the need for complex pre-chemical derivatization steps, but also perfectly matches the mass production characteristics of screen-printed carbon electrodes, greatly facilitating the large-scale production of disposable radiation-resistant electrochemical sensing chips.

[0076] C. The working electrode is a nickel ion modified electrode, and the damage inhibition protein Dsup is tagged with histidine; the immobilization includes: immobilization through the affinity between the histidine tag and the nickel ion modified electrode.

[0077] To further optimize the spatial orientation of the Dsup protein on the electrode surface and maximize the exposure of its free radical quenching active sites, the activation and immobilization process can also employ a supramolecular affinity binding strategy. In this approach, a surface pre-modified with transition metal nickel ions (Ni...) is selected. 2+ The electrode is used as the working electrode, and a recombinant damage inhibitor protein Dsup with a histidine tag is simultaneously configured. The immobilization step specifically includes: after contacting the two, utilizing the strong coordination complexation between the lone pair electrons on the imidazole ring of the histidine side chain and the empty orbitals of nickel ions on the working electrode surface, the protein is immobilized through the specific affinity between the histidine tag and the nickel ion modified electrode. Because the histidine tag is located at a specific end of the protein, this directional coordination process allows the Dsup protein chain segments to be anchored orderly on the electrode surface like "vegetation," and its main chain segments rich in aromatic and sulfur-containing amino acids can be fully extended into the liquid phase space. This not only eliminates the adverse effects of steric hindrance on free radical capture efficiency, but also gives the protective layer the potential ability to reversibly dissociate and regenerate under competition from specific reagents (such as imidazole).

[0078] In this embodiment, the interface modification process parameters and antifouling and functionalization extension steps of the radiation-resistant electrochemical sensor during the preparation process are defined in detail.

[0079] Specifically, the conditions for contact include at least one of the following: A. The reaction temperature is 4℃~25℃; for example, it can be 4℃, 6℃, 8℃, 10℃, 15℃, 18℃, 20℃, 22℃, 24℃, 25℃, etc.

[0080] B. The reaction time is 1h to 12h; for example, it can be 1h, 2h, 3h, 4h, 6h, 8h, 9h, 10h, 11h, 12h, etc.

[0081] C. The concentration of the damage inhibitory protein Dsup is 0.1 mg / mL to 2.0 mg / mL. For example, it can be 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, etc.

[0082] In the process of immobilizing the activated working electrode with the damage inhibitor protein Dsup at a heterogeneous interface, the thermodynamic and kinetic environment of the reaction has a decisive influence on the morphology and activity of the final coating. Controlling the reaction temperature between 4℃ and 25℃ ensures that the damage inhibitor protein Dsup molecules maintain a stable physiological conformation at the solution bulk and electrode interface, avoiding protein thermal denaturation or irreversible aggregation and precipitation caused by excessively high temperatures. Simultaneously, this temperature range provides the basic thermodynamic kinetic energy required to overcome the diffusion resistance at the solid-liquid interface and the activation energy of the reaction. Setting the reaction time to 1h to 12h aims to provide a sufficient kinetic evolution window for mass transfer, diffusion, conformational rearrangement, and covalent bonding or physical anchoring of protein molecules to the micro-electrode surface, ensuring that the adsorption-desorption or chemical bonding at the solid-phase interface reaches a stable equilibrium state. Meanwhile, the concentration of the damage-inhibiting protein Dsup was controlled at 0.1 mg / mL to 2.0 mg / mL, thus constructing a suitable concentration gradient driving force. This ensures that the electrode sensitive surface receives enough protein molecules to form a continuous free radical quenching shield, while avoiding the disordered multilayer accumulation of protein on the electrode surface caused by liquid phase supersaturation, thereby preventing an abnormal increase in interfacial mass transfer impedance.

[0083] In some embodiments, after forming the damage inhibition protein Dsup coating, the method further includes a step of sealing the remaining active sites on the electrode surface with a sealing agent; more preferably, the sealing agent includes at least one of bovine serum albumin, ethanolamine, and polyvinyl alcohol.

[0084] After constructing the Dsup damage inhibition protein coating, the microscopic surface of the working electrode inevitably retains some residual active sites that failed to bind to the Dsup protein. If these exposed high-energy physical adsorption regions or unreacted activated functional groups are not treated before detection, they are highly susceptible to non-specific adsorption of non-target impurity molecules in subsequent actual detection environments, thus generating background electronic noise. By introducing a sealing treatment step, an inert sealing agent is used to passivate the electrode surface. More preferably, the sealing agent includes at least one of bovine serum albumin, ethanolamine, and polyvinyl alcohol. Small-molecule ethanolamine can react with residual active esters and other active groups on the interface through its amino group, achieving microscopic chemical sealing; while large-molecule bovine serum albumin or polyvinyl alcohol utilizes its steric hindrance effect to firmly cover the exposed high surface energy defect regions. The electrode interface after sealing treatment has extremely strong resistance to non-specific adsorption, significantly improving the sensor signal-to-noise ratio in complex detection environments.

[0085] In some embodiments, after forming the damage-inhibiting protein Dsup coating, the method further includes the step of attaching a specific recognition element to the surface of the damage-inhibiting protein Dsup coating via a chemical crosslinking or affinity system.

[0086] To endow the radiation-resistant substrate platform with targeted capture capabilities for specific targets, after forming the damage-inhibiting protein Dsup coating, specific recognition elements can be firmly attached to the surface or network of the Dsup coating via covalent bridging with a bifunctional chemical crosslinking agent or by utilizing a non-covalent affinity system between a specific ligand and receptor. This step results in a composite architecture at the electrode-sensitive interface that combines underlying radiation protection with surface-specific capture, significantly expanding the sensor's application range in various high-radiation scenarios.

[0087] This application also provides an application of the radiation-resistant electrochemical sensor described above in the detection of target analytes under radiation conditions.

[0088] The application fully utilizes the free radical quenching and electric field shielding effects of the damage inhibition protein Dsup coating fixed to the sensitive interface of the working electrode at the microscale, enabling the sensor to achieve high-precision, high signal-to-noise ratio, and distortion-free real-time stable measurement of a variety of chemical, biological, or ionic target analytes in extreme and harsh backgrounds of strong ionizing radiation.

[0089] Furthermore, the radiation environment includes at least one of nuclear industrial facilities, nuclear waste disposal sites, space exploration equipment, and medical radiotherapy areas.

[0090] In these high-intensity radiation environments, ionizing radiation (including but not limited to gamma rays, X-rays, and high-energy particle beams) undergoes a strong irradiation decomposition reaction with solvent water molecules around the sensor's sensitive interface, generating highly reactive, random, and continuous strong oxidizing hydroxyl radicals (·OH) at high frequencies. In this embodiment, by placing the sensor in the aforementioned radiation environment, the damage-inhibiting protein Dsup coating on the sensitive interface of the working electrode exhibits an extremely high-density free radical trapping cross-section. Utilizing the aromatic and sulfur-containing amino acid side chains rich in its flexible linear conformation as efficient electron donors, it rapidly neutralizes and eliminates diffused hydroxyl radicals at the microscopic and in-situ scale, causing the free radical concentration at the interface to drop sharply by approximately 82%. This active chemical quenching process cuts off the non-specific interference of radiation products on the electrode surface and superimposed on the detection signal at the source, laying a hardware-level steady-state foundation for the sensor's applications in online monitoring of nuclear industrial wastewater, deep space exploration payload analysis, or real-time dose control for radiotherapy.

[0091] Furthermore, the detection mode used in the detection is selected from at least one of the amperometric method, the voltammetric method, and the impedance method.

[0092] In a strong ionizing radiation field, due to the introduction of the damage inhibition protein Dsup coating, the sensor exhibits excellent signal fidelity and bias correction effects under different detection methodologies: When using amperometric methods (such as chronoamperometric methods) for continuous fluid concentration monitoring, conventional electrodes are prone to large-scale upward baseline drift and severe random noise caused by continuous free radical discharge. In this embodiment, the Dsup coating continuously captures and quenches hydroxyl radicals, significantly reducing the background current drift under high dose rate radiation by about 70%, and outputs an extremely stable, low-noise current-time (It) response curve, ensuring the baseline stability of long-term online monitoring. When using voltammetry (such as cyclic voltammetry, square wave voltammetry, or differential pulse voltammetry) for quantitative analysis of target analytes, the active protection mechanism of the Dsup coating suppresses the large-scale radiation-additive background current to an extremely low level, preventing the characteristic redox response peaks of the target analyte from being swallowed up or masked by the radiation background current. This allows the electrochemical characteristic peaks of the target analyte to be clearly and sharply presented on the output current-potential (IV) scan curve, and the slope (detection sensitivity) retention rate of the standard curve is as high as 96.1% or more, achieving high-precision trace analysis under radiation background. When impedance methods (such as electrochemical impedance spectroscopy, EIS) are used for specific bioaffinity binding detection, the positively charged microscopic shielding electric field constructed by the Dsup coating at the interface works synergistically with the chemical trapping mechanism to block the non-specific oxidative attack of strong oxidizing free radicals on biorecognition elements such as antibodies, enzymes, or nucleic acid aptamers that may be co-modified on the electrode surface. This prevents the biological probe from undergoing spatial conformational collapse, denaturation, inactivation, or detachment. As a result, even under a cumulative irradiation dose of up to 1000 Gy, the cyclic voltammetric oxidation peak current signal of the sensor can still be clearly and completely presented, and the signal retention rate can still be maintained at an excellent level of over 76%. This demonstrates the sensor's excellent electron transfer fidelity and hardware tolerance limits under a wide radiation dose background.

[0093] Based on the aforementioned radiation-resistant electrochemical sensor, this embodiment also provides a detection device. The detection device not only includes a front-end sensing element but also integrates a back-end signal processing system, thus forming a complete set of equipment capable of independently performing in-situ monitoring of target analytes in a strong radiation environment.

[0094] Specifically, it includes: an electrochemical sensor resistant to radiation interference as described above; and a signal acquisition and processing module electrically connected to the electrochemical sensor, the signal acquisition and processing module being used to output a detection signal of the target analyte.

[0095] The radiation-resistant electrochemical sensor serves as the front-end probe of the detection device. The damage-inhibiting protein Dsup coating on the surface of its working electrode plays an active protective role in the radiation field, quenching the strong oxidizing hydroxyl radicals generated by the decomposition of environmental solvents under irradiation in real time, thereby avoiding non-specific electrochemical redox reactions at the electrode interface.

[0096] The signal acquisition and processing module, serving as the back-end processing hub, may include a potentiostat circuit, a micro-current amplifier, an analog-to-digital converter (A / D converter), and a microcontroller (MCU) in its hardware structure. The electrode system of the electrochemical sensor establishes a stable electrical connection with the input terminal of the signal acquisition and processing module via conductive contacts or cables. During operation, the signal acquisition and processing module applies a specific excitation potential (such as a step voltage or cyclic scanning voltage) to the working electrode of the sensor. The weak response current or impedance change generated by the sensor after detecting the target analyte is transmitted to the signal acquisition and processing module in real time via wires. This module amplifies, filters, and denoises the received raw analog signal, and then performs digitization. Combined with a built-in calibration curve algorithm, it calculates and outputs the quantitative detection signal of the target analyte.

[0097] In the overall architecture of this detection equipment, the front-end sensor and the back-end processing module form a significant system-level synergy. Because the Dsup coating on the sensor front end significantly eliminates radiation-induced background noise and current drift at the microscopic physicochemical source, the electrical signal transmitted to the back end inherently possesses an extremely high signal-to-noise ratio. This active noise cancellation at the front-end hardware level greatly reduces the algorithmic burden on the back-end signal acquisition and processing module, eliminating the need for complex digital filtering or baseline compensation chips. This not only helps reduce the overall hardware cost and power consumption of the detection equipment but also effectively avoids signal processing delays that may be caused by complex algorithms, ensuring the agility of the equipment's response and the authenticity of the output data.

[0098] Furthermore, in specific industrial and scientific research applications, the detection equipment is a nuclear industry radiation monitor, a nuclear waste online analyzer, a medical radiotherapy dose calibrator, or a space exploration payload analyzer.

[0099] For example, when the detection device is used as a space exploration payload analyzer, it does not require a thick physical shielding layer of lead, tungsten, or other materials to resist cosmic rays. The overall device structure is highly lightweight, making it suitable for long-term stable analysis of chemical substances in the extraterrestrial environment on microsatellites or deep space probes. When the detection device is used as an online nuclear waste analyzer, the sensor part can be extended by cable and placed inside the high-radioactive waste liquid storage tank, while the signal acquisition and processing module is placed in a safe area. The sensor can not only maintain an ultra-long working life under high cumulative doses, but its stable output signal can also help staff accurately control the waste liquid treatment process.

[0100] Based on the aforementioned requirements for radiation-resistant electrochemical sensors and their applications in mass production and extreme environments, this embodiment also provides a test kit, including: the radiation-resistant electrochemical sensor as described above. This kit integrates the core sensor element with a dedicated protective medium, giving the product excellent long-term storage stability and "ready-to-use" convenience.

[0101] Specifically, the kit includes at least one radiation-resistant electrochemical sensor as described in any of the foregoing embodiments. To ensure that the sensor maintains the high free radical quenching activity of the Dsup protein coating and the high sensitivity of the electrode interface after being removed from the laboratory preparation environment and undergoing long-term logistics transportation and shelf-life storage, the kit employs a specific protective and encapsulation structure.

[0102] Furthermore, the kit also includes the aforementioned preservation solution; the electrochemical sensor is immersed in the preservation solution for sealed packaging; or, the surface of the electrochemical sensor is coated with a lyophilized product of the aforementioned preservation solution and then vacuum-sealed.

[0103] In one liquid-encapsulated embodiment, the kit further includes the aforementioned preservation solution. The electrochemical sensor (particularly its working electrode sensitive end modified with a protective coating) is immersed in the preservation solution and entirely housed in a sealed packaging container (such as an aluminum-plastic composite packaging bag or a sealed reservoir tube). In this state, the sealed physical barrier, combined with the buffering and antibacterial effects of the preservation solution, maintains the dynamic balance of microscopic electrostatic charges at the electrode interface. Furthermore, by utilizing the steric hindrance effect of the macromolecular carrier protein in the liquid phase, the desorption or conformational decay of Dsup molecules immobilized on the electrode surface during long-term static storage is effectively suppressed.

[0104] In another solid-state freeze-dried packaging embodiment, the surface of the electrochemical sensor is coated with the aforementioned freeze-dried preservation solution and encapsulated in a vacuum-sealed package. The preparation process involves: pre-coating the electrode surface with the preservation solution, removing moisture through freeze-drying, allowing the multi-component protective agents in the preservation solution to form a dense solid protective matrix in situ at the electrode sensitive interface; subsequently, heat-sealing of the outer layer material is performed under vacuum. In this architecture, the amorphous glassy framework formed by the protective agents (such as trehalose, sucrose, and mannitol) replaces the lost water molecules through a hydrogen bond network, stably "freezing" and locking the naturally disordered flexible active conformation of the Dsup protein at the microscopic scale. The outer vacuum-sealed package completely isolates the sensor from oxygen and humidity in the external environment, effectively preventing moisture absorption and deliquescence of the freeze-dried matrix and oxidative deactivation of the bioactive coating.

[0105] The modular design of the aforementioned reagent kit not only significantly extends the commercial shelf life of radiation-resistant sensors (e.g., maintaining a signal response rate of over 90% after months of storage), but also perfectly meets the needs for instrument consumables in nuclear waste monitoring or field emergency radiation accident detection. In high-radiation-risk or space-constrained environments, operators only need to unpack the reagent kit (for lyophilized packaging, it can be quickly rehydrated and activated upon contact with the detection fluid) to directly connect the probe to the back-end instrument for accurate testing. After testing, it can be safely disposed of as a single-use consumable, fundamentally avoiding the risk of secondary radiation contamination and the cumbersome washing and decontamination processes associated with probe recycling.

[0106] The sensor disclosed in this embodiment, when applied in the above detection mode, fundamentally eliminates the source of analytical interference at the electrode interface, thus greatly reducing the reliance of the sensor's back-end data processing system on complex digital filtering and baseline subtraction algorithms. Furthermore, the single-molecule-layer-level Dsup protective layer does not increase the mass transfer and diffusion barrier, resulting in a slight increase in the detection response time of the finished product of only 0.3 seconds. While achieving high radiation resistance and stability, it perfectly preserves the original agile physical response activity of the sensor, possessing extremely high industrial engineering application value.

[0107] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0108] Example 1: Fabrication and basic performance testing of a Dsup protein-modified gold electrode sensor 1. Experimental Objective: This embodiment provides a method for preparing a Dsup protein-modified gold electrode sensor based on covalent bonding, and conducts preliminary tests on its basic anti-radiation interference performance.

[0109] 2. Experimental methods: (1) Electrode pretreatment and activation: The gold disk electrode (3 mm in diameter) was polished sequentially with 0.3 μm and 0.05 μm alumina slurries, and then ultrasonically cleaned in ethanol and ultrapure water to obtain a smooth surface. The cleaned gold electrode was immersed in an ethanol solution of 10 mmol / L mercaptoundecanoic acid and left at room temperature for 16 hours to allow the formation of a self-assembled monolayer on the electrode surface. After removal, it was thoroughly washed with ethanol and ultrapure water. Subsequently, the electrode was immersed in an aqueous solution containing 100 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 50 mmol / L N-hydroxysuccinimide (NHS) to activate the carboxyl groups for 30 minutes, and then washed with phosphate buffer (pH 7.4).

[0110] (2) Immobilization and blocking of Dsup protein: The activated electrode was immersed in phosphate buffer containing 0.5 mg / mL Dsup protein and reacted overnight at 4°C. After removal, it was washed with buffer and then blocked with 1% bovine serum albumin (BSA) for 30 minutes.

[0111] (3) Sensor assembly: Using the modified electrode as the working electrode, a three-electrode system is formed with the platinum wire counter electrode and the silver / silver chloride (Ag / AgCl) reference electrode to assemble a complete electrochemical sensor.

[0112] 3. Experimental Results and Analysis: The assembled sensor was placed... 60 The sensor was continuously irradiated for 2 hours at a dose rate of 50 Gy / h in a Co-γ-ray field. Background current was measured using cyclic voltammetry (CV) in phosphate buffer before and after irradiation. The results showed that the increase in background current after irradiation was 68% lower than that of the uncoated bare gold control electrode, and the shape of the cyclic voltammetric curve of the sensor did not change significantly after irradiation. This indicates that the Dsup coating effectively suppresses the interference of radiation-induced free radicals on the electrode interface in a strong radiation field.

[0113] Example 2: Preparation and Accuracy Testing of Dsup Protein-Modified Glassy Carbon Electrode Sensor 1. Experimental Objective: This embodiment provides a method for preparing a Dsup protein-modified glassy carbon electrode sensor based on a combination of physical adsorption and chemical cross-linking, and verifies its accuracy in detecting target objects under irradiation.

[0114] 2. Experimental Methods: A glassy carbon electrode was used as the working electrode. Its pretreatment involved polishing with 1.0 μm, 0.3 μm, and 0.05 μm alumina slurries sequentially, followed by ultrasonic cleaning. Dsup protein was immobilized using a combination of physical adsorption and cross-linking: 5 μL of a 1.0 mg / mL Dsup protein solution was drop-coated onto the glassy carbon electrode surface. After drying at room temperature, it was subjected to cross-linking treatment in glutaraldehyde saturated vapor for 1 hour, followed by washing with buffer solution. The remaining sensor assembly steps were the same as in Example 1.

[0115] 3. Experimental Results and Analysis: The same irradiation conditions as in Example 1 were used for testing. The results showed that the increase in background current after irradiation was reduced by 65% ​​compared to the uncoated control glassy carbon electrode. Simultaneously, the sensor could still detect a 1 mmol / L potassium ferrocyanide standard solution normally after irradiation, and the detection signal error was less than 5%, proving that the coating also has excellent protective and detection fidelity effects on carbon-based materials.

[0116] Example 3: Directional Immobilization of Dsup Protein and Sensor Performance Testing 1. Experimental Objective: This embodiment provides a Dsup protein directional fixation method based on histidine tag coordination, aiming to further optimize the spatial orientation of the protein and improve the radiation resistance of the sensor.

[0117] 2. Experimental Methods: The Dsup protein was pre-expressed as a recombinant protein with a 6×histidine tag via genetic engineering. A nickel-modified gold electrode was used as the working electrode (prepared by immersing the gold electrode in an ethanol solution containing thiol-modified triacetic acid to form a self-assembled monolayer, followed by complexation with nickel ions). The modified working electrode was immersed in a buffer solution containing 0.5 mg / mL of the recombinant Dsup protein with a 6×histidine tag for 2 hours at room temperature to complete directional immobilization. The remaining sensor assembly steps were the same as in Example 1.

[0118] 3. Experimental Results and Analysis: The same irradiation conditions as in Example 1 were used for testing. The results showed that the increase in background current after irradiation was reduced by 72% compared to the uncoated control electrode. This indicates that the directional fixation method resulted in better spatial exposure of the radiation-resistant active region on the Dsup protein backbone, further improving the free radical capture efficiency and significantly optimizing the stability of the sensor.

[0119] Example 4: Verification of the quenching effect of hydroxyl radicals (·OH) at the electrode interface 1. Experimental objective: This experiment is a comparative experiment, aiming to directly and intuitively demonstrate, through a specific fluorescent probe method, that the Dsup coating has an active capture and quenching effect on radiation-induced strong oxidizing hydroxyl radicals.

[0120] 2. Experimental Methods: Two sets of test electrodes were set up: a bare gold electrode for the comparative example and a Dsup-modified gold electrode prepared according to the method in Example 1 for the practical example. Both sets of electrodes were immersed in phosphate buffer (pH 7.4) containing 10 mM coumarin-3-carboxylic acid (3-CCA, a specific ·OH fluorescent probe). The electrode system was placed in… 60 Irradiation was performed for 30 minutes at a dose rate of 50 Gy / h using a Co γ-ray source. After irradiation, the electrodes were removed and the reaction solution was collected. The fluorescence intensity of the reaction product 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA) was measured using a fluorescence spectrophotometer at an excitation wavelength of 395 nm and an emission wavelength of 450 nm (Note: This fluorescence intensity is proportional to the concentration of ·OH in the solution).

[0121] 3. Experimental Results and Analysis: The experimental results showed that the fluorescence intensity of the comparative example (bare electrode group) solution was 1250 a.u., while the fluorescence intensity of the example group (Dsup-modified electrode group) solution was only 225 a.u. Compared with the comparative example, the concentration of hydroxyl radicals near the electrode interface was reduced by approximately 82% after Dsup modification. This result directly confirms that the Dsup protein immobilized on the electrode surface can efficiently quench hydroxyl radicals generated by irradiation, which is also the fundamental physicochemical mechanism by which this sensor possesses excellent anti-radiation interference performance.

[0122] Example 5: Evaluation of the impact of Dsup coating on sensor sensitivity and response speed 1. Experimental objective: This experiment aims to verify, through comparison of electrochemical kinetic data, whether the introduction of the Dsup protective coating will sacrifice the original detection sensitivity and response speed of the sensor.

[0123] 2. Experimental Methods: A bare gold electrode (comparative example) and a Dsup-modified gold electrode (example, prepared according to Example 1) were set up. Under the same three-electrode system, phosphate buffer containing 0.1 mM KCl was used as the supporting electrolyte, and the current-time (It) curve was recorded by chronoamperometry. A series of concentrations of potassium ferricyanide / potassium ferrocyanide standard solutions (concentration range 0.1 mM to 10 mM) were tested, and the working potential was kept constant at +0.25 V (vs. Ag / AgCl).

[0124] 3. Experimental Results and Analysis: (1) Sensitivity comparison (reference) Figure 2 The current-concentration (IC) standard curve plotted based on the test data shows that the standard curve equation for the comparative example (bare electrode) is as follows: I(μA) = 12.8 C(mM) + 0.5 (R) 2 =0.998); The standard curve equation for the example group (Dsup modified electrode) is I(μA) = 12.3C(mM) + 0.3(R) 2 =0.996). Calculations showed that the sensitivity retention rate of the Dsup-modified electrode was as high as 96.1% of the comparative example, and there was no statistically significant difference in the slope between the two.

[0125] (2) Response time comparison (reference) Figure 3 The time required for the current response to reach 95% of its steady-state value was recorded after a 2 mM potassium ferricyanide standard solution was instantaneously added to a blank buffer. The response time of the comparative example (bare electrode) was 2.8 seconds, and the response time of the example group (Dsup-modified electrode) was 3.1 seconds. These results demonstrate that the Dsup coating, controlled at the monolayer level, is extremely thin and has minimal impact on the electron transfer kinetics at the electrode interface and the diffusion mass transfer process of the target molecules, allowing the sensor to perfectly retain its high sensitivity and extremely fast response speed.

[0126] Example 6: Limiting determination of sensor signal retention rate under different cumulative irradiation doses 1. Experimental Objective: This experiment is a destructive environment test designed to evaluate the performance retention capability of Dsup-modified sensors under extreme high-dose cumulative irradiation, so as to clarify the upper limit of their radiation-resistant applications in extreme scenarios such as the nuclear industry.

[0127] 2. Experimental Methods: The Dsup-modified gold electrode sensor prepared according to Example 1 was used as the experimental group, and the bare gold electrode sensor was used as the control group. Multiple sensor groups were placed... 60 In a Co-γ-ray irradiation field, continuous irradiation for different durations was performed at a dose rate of 50 Gy / h to achieve cumulative irradiation doses of 100 Gy, 500 Gy, and 1000 Gy, respectively. After each irradiation phase, the sensor was placed in a phosphate buffer containing 2 mM K3 [Fe(CN)6] for cyclic voltammetry scanning. The oxidation peak current value was recorded and compared with the initial peak current value recorded before irradiation to calculate the signal retention rate (%).

[0128] 3. Experimental Results and Analysis: The test results are shown in Table 1.

[0129] Table 1. Comparison of signal retention rates between bare gold electrodes and Dsup-modified gold electrodes under different cumulative irradiation doses.

[0130] This set of extreme damage data fully demonstrates that, even when faced with extreme radiation doses of up to 1000 Gy, the Dsup coating can still maintain a robust protective barrier, significantly improving the stability and lifespan of the sensor across a wide dose range.

[0131] To further explore the microscopic mechanism of the Dsup protective coating, this application further verified the quantitative relationship between interface coating density and electrochemical performance through the following examples.

[0132] Example 7: Quantitative study on the relationship between Dsup protein coating amount and solution concentration To establish a quantitative correlation between the macroscopic concentration of the solution and the microscopic coating amount on the electrode surface, a dissipative quartz crystal microbalance (QCM-D) was used to monitor the adsorption mass of sup protein on the gold electrode surface in real time.

[0133] QCM-D calculates the mass change on the electrode surface by detecting the change in the oscillation frequency (ΔF) of the quartz crystal. The relationship between the frequency change and the mass change is given by the Sauerbrey equation: Δm= C Δf / nΔm= C Δf / n; Where C is the mass sensitivity constant (for a 5MHz quartz crystal, C ≈ 17.7 ng·Hz). -1 ·cm -2 ), where n is the overtone factor. Simultaneously, electrochemical impedance spectroscopy (EIS) was used to cross-validate the protein coverage at the electrode interface. The test results are shown in Table 2.

[0134] Table 2. Correspondence between Dsup protein solution concentration and actual coating amount on gold electrode surface (QCM-D determination)

[0135] Measurement conditions: Gold electrode (QCM-D chip, area 0.196 cm²) 2 Dsup protein was dissolved in PBS buffer (pH 7.4) and statically adsorbed at room temperature (25°C) for 2 hours. The data are the mean ± standard deviation of three independent experiments.

[0136] As shown in Table 2, the actual coating amount on the electrode surface corresponding to the 0.5 mg / mL Dsup protein solution concentration used in the examples is approximately 1.70 μg / cm². 2 .

[0137] In practice, those skilled in the art can flexibly achieve a coating concentration of 0.5–10 μg / cm by adjusting the concentration of the Dsup protein solution (0.1–10 mg / mL) and the fixed time (1–12 h) according to the target coating amount. 2 Any amount of coating within the range.

[0138] Example 8: Verification of the critical point between radiation resistance and charge transfer impedance under different coating amounts To further prove 0.5~10 μg / cm 2 This study aimed to achieve the optimal balance between radiation protection efficacy and highly sensitive electrochemical response, using five groups of Dsup-modified gold electrode sensors with different coating amounts for comparative testing. The coating amount was determined by adjusting the Dsup protein solution concentration (0.05, 0.1, 0.5, 2.0, 10.0 mg / mL) combined with real-time monitoring using QCM-D, corresponding to the five characteristic coating amounts of 0.15, 0.44, 1.70, 3.86, and 8.00 μg / cm² in Table 1. An additional coating amount of 15.0 μg / cm² was also included. 2 (Obtained by extending the fixation time to 12 hours with 10 mg / mL Dsup solution).

[0139] Test method: Place each group of sensors in 60 Irradiation was performed for 2 hours in a Co gamma-ray irradiation field at a dose rate of 50 Gy / h (cumulative dose 100 Gy). Measurements were taken before and after irradiation. (1) Cyclic voltammetry (CV) background current drift (reflects the ability to resist radiation interference); (2) Retention rate of CV oxidation peak current in 2 mM potassium ferricyanide solution (reflecting the retention of sensitivity); (3) Changes in charge transfer impedance (R_ct) in electrochemical impedance spectroscopy (EIS) (reflecting interfacial mass transfer resistance).

[0140] The test results are shown in Table 3.

[0141] Table 3. Comparison of sensor performance before and after irradiation with different Dsup coating amounts (cumulative dose of 100 Gy)

[0142] Data Analysis: (1) Coating amount is too low (<0.5 μg / cm) 2 When the coating amount is only 0.15 μg / cm 2 At this stage, the Dsup protein is distributed as a discrete "island-like" submonolayer on the electrode surface, failing to form a continuous free radical trapping network. Irradiated hydroxyl radicals can easily penetrate the protective gaps and reach the electrode surface, resulting in a background current drift of up to 3.95 μA and an oxidation peak current retention rate of only 71.5%, showing very limited improvement compared to the bare electrode (68.2%). While a coating amount of 0.44 μg / cm² showed some improvement, the background drift still reached 2.18 μA, and the radiation resistance effect remained unsatisfactory.

[0143] (2) The coating amount is within the optimal range (0.5~10 μg / cm). 2When the coating amount reaches 1.70 μg / cm 2 At that time, the Dsup protein formed a continuous and dense monolayer on the electrode surface, with an intact and unblemished free radical capture network. The background current drift decreased sharply to 0.85 μA (a reduction of 82.4%), and the oxidation peak current retention rate reached as high as 95.1%. The coating amount was increased to 3.86 μg / cm² and 8.00 μg / cm². 2 Meanwhile, the radiation resistance continued to improve (background drift decreased to 0.72 and 0.58 μA, respectively), and the sensitivity retention rate remained above 92%. Notably, even with a coating amount of 8.00 μg / cm², the charge transfer impedance R_ct was only 485 Ω, still far lower than that of 15.0 μg / cm². 2 The Ω of 820 Ω indicates that the coating has not substantially hindered interfacial electron transfer within this range.

[0144] (3) Excessive coating amount (>10 μg / cm) 2 When the coating amount reaches 15.0 μg / cm 2 At this time, protein molecules undergo severe multilayer disordered stacking and spatial aggregation on the electrode surface. Although the radiation resistance is further improved (background drift of only 0.51 μA), the charge transfer impedance R_ct increases sharply to 820 Ω, causing the oxidation peak current retention rate to plummet to 71.3%—a value even lower than that of a coating amount of only 0.15 μg / cm³. 2 The results indicate that excessively thick protein multilayer films create severe mass transfer and electron transfer barriers at the electrode interface, completely contradicting the core design principle of this invention: "balancing radiation resistance and high sensitivity."

[0145] III. Conclusion: Based on the above data, when the Dsup coating amount is less than 0.5 μg / cm 2 When the coating amount exceeds 10 μg / cm³, a continuous free radical trapping network cannot be formed on the electrode surface, resulting in severely insufficient radiation protection. 2 At this time, the multilayer accumulation of proteins leads to a sharp increase in interfacial impedance, severely degrading the sensor's sensitivity and response speed. Only by strictly controlling the coating amount within 0.5~10 μg / cm³ can this be effectively addressed. 2 Within this critical range, the following can be achieved simultaneously: (1) a dense and continuous monolayer free radical trapping network that controls the background current drift below 1.0 μA; (2) an interfacial charge transfer impedance R_ct maintained below 500 Ω to ensure an oxidation peak current retention rate ≥92%; and (3) the sensor possessing excellent anti-interference capability and high sensitivity and fast response characteristics in a strong radiation environment. The determination of this critical range constitutes the key technical means for achieving the synergistic electrochemical effect of "anti-radiation and high sensitivity" in this invention.

[0146] Example 9: Construction of a Dsup / glucose oxidase complex enzyme sensor and its detection of glucose under irradiation. 1. Experimental objective: This embodiment provides a method for preparing an enzyme sensor based on Dsup coating and glucose oxidase (GOx) co-modification, and verifies the detection performance of the composite sensor on glucose substrate under ionizing radiation environment, to demonstrate the compatibility of Dsup coating with macromolecular biorecognition element in spatial configuration, and that Dsup coating does not interfere with the electron transfer process of enzymatic reaction.

[0147] 2. Sensor fabrication: (1) Pretreatment of glassy carbon electrode and fixation of Dsup: Following the method in Example 2, after polishing and cleaning the glassy carbon electrode, Dsup protein was fixed by a combination of physical adsorption and cross-linking: 5 μL of Dsup protein solution with a concentration of 1.0 mg / mL was drop-coated onto the electrode surface, dried at room temperature, and then cross-linked in glutaraldehyde saturated vapor for 1 hour.

[0148] (2) Glucose oxidase fixation: The Dsup-modified glassy carbon electrode was immersed in a PBS solution containing 2.0 mg / mL glucose oxidase (GOx, derived from Aspergillus niger) and 0.5% glutaraldehyde and crosslinked for 4 hours at 4°C. After washing, it was blocked with 1% BSA for 30 minutes.

[0149] (3) Preparation of control group: A bare glassy carbon electrode with GOx directly fixed (without Dsup coating) was set up as a control.

[0150] 3. Irradiation experiments and electrochemical detection: Place the two sets of sensors 60 In a Co γ-ray irradiation field, irradiation was carried out at a dose rate of 50 Gy / h for 0, 2, 4, 8, and 16 hours (corresponding to cumulative doses of 0, 50, 100, 200, 400, and 800 Gy). Before and after irradiation, steady-state response currents after the addition of different concentrations of glucose (0, 0.5, 1, 2, 5, 10, and 20 mM) were recorded using chronoamperometry at a working potential of +0.3 V (vs. Ag / AgCl) in PBS buffer containing 0.1 M KCl (with 0.5 mM p-benzoquinone as an electron mediator).

[0151] 4. Experimental Results and Analysis: (1) Standard curves before and after irradiation: The current response of the two sets of sensors to different concentrations of glucose was measured before irradiation and after 100 Gy irradiation (reference). Figure 4 ).

[0152] The results show that the standard curve equation for the Dsup / GOx composite sensor before irradiation is: I(μA) = 0.52C(mM) + 0.08(R) 2 =0.998); The standard curve equation for the Dsup / GOx composite sensor after 100 Gy irradiation is: I(μA) = 0.49C(mM) + 0.06(R) 2 =0.996), with a sensitivity retention rate of 94.2%; The standard curve equation for the GOx sensor before irradiation is: I(μA) = 0.55C(mM) + 0.05 (R² = 0.997); The standard curve equation for the GOx sensor after 100 Gy irradiation is: I(μA) = 0.28C(mM) + 0.15(R) 2 =0.982), with a sensitivity retention rate of only 50.9%.

[0153] (2) Signal retention rate under different cumulative doses: The Dsup / GOx composite sensor was tested and found to have sensitivity retention rates of 97.6%, 94.2%, 88.5%, 79.3%, and 68.1% after cumulative irradiation of 50, 100, 200, 400, and 800 Gy, respectively, while the control GOx sensor had retention rates of 86.4%, 50.9%, 31.8%, 18.2%, and 9.1%, respectively.

[0154] (3) Response time: The response times of the Dsup / GOx composite sensor before and after irradiation were 3.2 seconds and 3.5 seconds, respectively, which were not significantly different from the 2.9 seconds and 3.8 seconds of the bare GOx sensor.

[0155] (4) Mechanism Analysis: Dsup protein is an intrinsically disordered protein (IDP). Its highly flexible linear conformation allows it to adhere tightly to the electrode surface at a nanometer thickness, while providing ample interfacial space for the GOx macromolecule (molecular weight approximately 160 kDa). Rigid steric hindrance does not obscure the free radical quenching active site of Dsup, nor does it hinder electron transfer between the active site (FAD cofactor) of GOx and the electrode. GOx catalyzes the oxidation of glucose to H2O2, and H2O2 is oxidized on the electrode surface, releasing electrons. The Dsup monolayer coating does not constitute an impedance barrier for electron transfer. Furthermore, the Dsup coating provides in-situ microscopic radiation protection for GOx by actively quenching hydroxyl radicals generated by irradiation, allowing the enzyme protein to maintain its native conformation and catalytic activity even after 100 Gy of cumulative irradiation.

[0156] The above results fully demonstrate that the Dsup coating and GOx macromolecules form a spatially compatible composite interface on the electrode surface: on the one hand, the flexible and disordered conformation of Dsup is not masked by the co-immobilization of GOx, thus preserving its free radical quenching activity; on the other hand, the electron transfer in the enzymatic reaction of GOx is not hindered by the presence of Dsup. Conversely, the Dsup coating provides in-situ microscopic radiation protection for GOx by actively quenching radiation-induced hydroxyl radicals, significantly extending the effective working life of the enzyme sensor under radiation conditions.

[0157] Example 8: Long-term storage stability test of Dsup modified electrode sensor under different storage conditions 1. Experimental objective: This embodiment aims to verify the effect of the composite lyophilization protection formulation on the activity retention of the Dsup modified electrode sensor during long-term storage (liquid immersion and lyophilized sealing), and to prove that the preservation solution formulation can effectively maintain the sensor's radiation resistance and electrochemical detection sensitivity after the storage period.

[0158] 2. Sensor fabrication: Dsup-modified gold electrode sensors were prepared according to the method in Example 1. The prepared sensors were randomly divided into the following groups: (1) Preservation solution formulation group (experimental group): The sensor was immersed in a preservation solution containing the following components: 3% (w / v) trehalose, 1% (w / v) sucrose, 0.5% (w / v) mannitol, 0.05% (w / v) bovine serum albumin, and 0.02% (w / v) sodium azide, with phosphate buffered saline (PBS, pH 7.4) as the solvent. Some sensors were directly immersed in the preservation solution and stored at 4°C in the dark (liquid preservation group); some sensors were freeze-dried and then vacuum-sealed for preservation (lyophilized preservation group).

[0159] (2) Control group setup: Control group 1 (no protectant): The sensor was immersed in PBS buffer without any lyophilization protectant and stored at 4°C in the dark.

[0160] Control group 2 (single protectant): The sensor was immersed in PBS buffer containing only 3% trehalose (excluding sucrose, mannitol, and BSA) and stored at 4°C in the dark.

[0161] Control group 3 (without Dsup coating): Bare gold electrodes (without Dsup coating) were placed in the same preservation solution as the experimental group and stored at 4°C in the dark.

[0162] 3. Storage conditions and time points: Each group of sensors was stored for 0 days (freshly prepared, immediately tested), 7 days, 30 days, and 90 days, respectively. Three sensors from each group were used for parallel testing at each time point. The lyophilized group was rehydrated with PBS buffer for 30 minutes before testing.

[0163] 4. Testing Method: At each storage time point, the following tests were performed on the sensors after they were removed (or rehydrated): (1) Cyclic voltammetry (CV) background current test: Record the CV curve (potential range -0.2V to +0.6V, scan rate 50mV / s) in PBS buffer (pH 7.4), record the background current value, and evaluate whether non-specific degradation of the electrode interface occurs during storage.

[0164] (2) Detection with potassium ferricyanide standard solution: The CV oxidation peak current value was recorded in PBS buffer containing 2 mM K3[Fe(CN)6], and the oxidation peak current retention rate (%) relative to the freshly prepared sensor (0 days) was calculated to evaluate the retention of the electrode's electrochemical activity.

[0165] (3) Post-irradiation performance test: Place the stored sensor in 60 In a Co γ-ray irradiation field, the coating was irradiated for 2 hours at a dose rate of 50 Gy / h (cumulative dose 100 Gy). After irradiation, the CV oxidation peak current in 2 mM K3[Fe(CN)6] was measured again, and the signal retention rate (%) after irradiation was calculated to evaluate the retention of the radiation resistance activity of the Dsup coating after storage.

[0166] (4) Electrochemical impedance spectroscopy (EIS): EIS spectra (frequency range 100kHz~0.01Hz, amplitude 5mV) were recorded in PBS buffer containing 2mM K3[Fe(CN)6], and the charge transfer impedance R_ct was obtained by fitting to evaluate the impedance change of the electrode interface during storage.

[0167] 5. Experimental results: The test results are shown in Tables 4 and 5.

[0168] Table 4. Oxidation peak current retention rate (%) of Dsup modified electrode sensor after storage under different storage conditions, relative to fresh sensor at 0 days.

[0169] Table 5. Charge transfer impedance R_ct (Ω) of Dsup modified electrode sensor after storage under different storage conditions.

[0170] 6. Results Analysis and Discussion Based on the experimental data analysis in Tables 4 and 5, we can see that: (1) In the absence of irradiation, as the storage time increased, the oxidation peak current retention rate of control group 1, which did not contain any protective agent, decreased sharply, reaching only 78.6% and 52.4% after 30 days and 90 days of storage, respectively, and its charge transfer impedance R ct It surged from an initial 312 Ω to 1120 Ω in 90 days.

[0171] This indicates that under unprotected conditions, protein molecules at the sensitive interface of the working electrode are highly susceptible to non-specific conformational collapse, aggregation, or detachment from the interface, leading to a significant increase in interfacial mass transfer and electron transfer barriers. However, the experimental groups using the composite formulation of this application (including liquid preservation and lyophilization preservation) still exhibited unirradiated oxidation peak current retention rates of 93.1% and 96.5%, respectively, after 90 days of storage, and the charge transfer impedance R0 was also low. ct The Ω values ​​increased only slightly to 402 Ω and 358 Ω, respectively. This fully demonstrates that the multi-component synergistic protection system of trehalose, sucrose, mannitol, and bovine serum albumin can effectively replace water molecules and Dsup protein and biomacromolecules at the electrode interface to form a stable hydrogen bond network, thus stabilizing the natural active conformation of protein molecules at both the macroscopic and microscopic scales.

[0172] (2) After 100 Gy cumulative dose irradiation, due to protein degradation during storage, the irradiation signal retention rate of control group 1 was extremely low, only 28.7%, after 90 days of storage. Control group 2, protected by single trehalose, also showed significant performance degradation, with a signal retention rate of only 52.3% after 90 days of irradiation. In contrast, the liquid experimental group and the freeze-dried experimental group using the composite preservation solution formulation of this application maintained extremely high signal retention rates of 90.6% and 94.2%, respectively, after 90 days of storage and 100 Gy irradiation.

[0173] The results show that the composite freeze-drying protection formulation of this application can not only maintain the basic conductivity of the sensor surface, but more importantly, it can completely preserve the core protective element: the free radical quenching active center and spatial trapping cross section of the damage inhibitor protein Dsup, ensuring that it can still remove interfacial hydroxyl radicals in a timely and efficient manner when used in a strong radiation environment after a long shelf life.

[0174] (3) Comparison of synergistic advantages between freeze-drying and liquid preservation: Comparison of experimental data shows that the performance of the freeze-dried group at each time point (including current retention rate and impedance robustness) is slightly better than that of the liquid preservation group. After 90 days of storage and 100 Gy irradiation, the signal retention rate of the freeze-dried group (94.2%) is 3.6 percentage points higher than that of the liquid group (90.6%), and its R at 90 days is also higher. ct (358 Ω) is significantly lower than that of the liquid group (402 Ω).

[0175] This indicates that the freeze-drying process, by physically "freezing" the flexible linear conformation of proteins in a dry state, can more thoroughly isolate the potential damage of moisture to the secondary structure of proteins and the chemical strain in a long-term liquid environment. Combined with the mannitol crystal framework and BSA steric hindrance shielding in the specific ratio of this application, the optimal synergy between the physical morphology and biological activity of the radiation-resistant coating under storage and transportation conditions is achieved.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An electrochemical sensor resistant to radiation interference, characterized in that, Including the electrode system; The electrode system includes at least a working electrode; The surface of the working electrode is coated with a damage-inhibiting protein Dsup coating for quenching radiation-induced free radicals.

2. The radiation-resistant electrochemical sensor as described in claim 1, characterized in that, The damage-inhibiting protein Dsup is derived from tardigrades, and its amino acid sequence is shown in SEQ ID NO:1, or it is a polypeptide or its functional active fragment that has at least 90% identity with SEQ ID NO:1 and retains hydroxyl radical quenching activity.

3. The radiation-resistant electrochemical sensor as described in claim 1, characterized in that, The damage inhibition protein Dsup coating is a monolayer or sub-monolayer coating; and / or, The damage inhibition protein Dsup is coated at a concentration of 0.5 μg / cm² on the surface of the working electrode. 2 ~10μg / cm 2 ; and / or, The surface of the working electrode is further fixed with a specific recognition element; preferably, the specific recognition element is fixed on or within the damage inhibitory protein Dsup coating; preferably, the specific recognition element is selected from one or more of antibodies, nucleic acid aptamers, enzymes, and DNA probes; preferably, the specific recognition element is covalently linked to the damage inhibitory protein Dsup coating by a cross-linking agent, or is directionally fixed to the damage inhibitory protein Dsup coating by an avidin-biotin system; and / or, The working electrode is made of at least one of gold, platinum, glassy carbon, carbon paste, and screen-printed carbon electrodes; and / or, The sensor is preserved by immersion in a preservation solution or by freeze-drying and then sealing it for preservation. Preferably, the preservation solution contains a freeze-drying protectant selected from one or more of trehalose, sucrose, mannitol, lactose, and dextran. Preferably, the preservation solution comprises: 1%~5% (w / v) trehalose, 0.5%~2% (w / v) sucrose, 0.1%~1% (w / v) mannitol, 0.01%~0.1% (w / v) bovine serum albumin, and a supporting electrolyte buffer. Preferably, the supporting electrolyte buffer is a phosphate buffer. Preferably, the preservation solution also contains sodium azide as a preservative.

4. A method for preparing a radiation-resistant electrochemical sensor as described in any one of claims 1-3, characterized in that, include: The surface of the working electrode is activated to obtain an activated working electrode; The activated working electrode is brought into contact with the damage inhibitor protein Dsup, thereby fixing the damage inhibitor protein Dsup onto the surface of the working electrode and forming the damage inhibitor protein Dsup coating.

5. The method for preparing the radiation-resistant electrochemical sensor as described in claim 4, characterized in that, The activation treatment of the working electrode and the fixation are selected from any one of the following methods: A. The working electrode is made of gold or platinum; the activation treatment includes: forming a self-assembled monolayer with exposed carboxyl groups at the ends on the surface of the working electrode, and activating the carboxyl groups with carbodiimide and N-hydroxysuccinimide to form an active ester intermediate; the fixation includes: reacting the amino group in the damage inhibitor protein Dsup with the active ester intermediate to form an amide bond for covalent fixation; B. The working electrode is made of glassy carbon, carbon paste, or screen-printed carbon; the activation treatment and the fixation include: adsorbing the damage inhibition protein Dsup onto the surface of the working electrode and performing cross-linking treatment using a cross-linking agent; C. The working electrode is a nickel ion modified electrode, and the damage inhibition protein Dsup is tagged with histidine; the immobilization includes: immobilization through the affinity between the histidine tag and the nickel ion modified electrode.

6. The method for preparing the radiation-resistant electrochemical sensor as described in claim 4, characterized in that, The conditions for contact include at least one of the following: A. The reaction temperature is 4℃~25℃; B. The reaction time is 1 hour to 12 hours; C. The concentration of the damage-inhibiting protein Dsup is 0.1 mg / mL to 2.0 mg / mL; and / or, After forming the damage-inhibiting protein Dsup coating, the method further includes a step of sealing the remaining active sites on the electrode surface with a sealing agent; more preferably, the sealing agent includes at least one of bovine serum albumin, ethanolamine, and polyvinyl alcohol; and / or, After forming the damage-inhibiting protein Dsup coating, the method further includes the step of attaching a specific recognition element to the surface of the damage-inhibiting protein Dsup coating through a chemical crosslinking or affinity system.

7. A testing device, characterized in that, include: The radiation-resistant electrochemical sensor as described in any one of claims 1-3; and a signal acquisition and processing module electrically connected to the electrochemical sensor, the signal acquisition and processing module being used to output the detection signal of the target analyte. Preferably, the detection equipment is a nuclear industry radiation monitor, a nuclear waste online analyzer, a medical radiotherapy dose calibrator, or a space exploration payload analyzer.

8. A test kit, characterized in that, include: The radiation-resistant electrochemical sensor as described in any one of claims 1-3.

9. The radiation protection detection kit as described in claim 8, characterized in that, The kit further includes the preservation solution of claim 3; the electrochemical sensor is immersed in the preservation solution for sealed packaging; or, the surface of the electrochemical sensor is coated with a lyophilized product of the preservation solution of claim 3 and vacuum-sealed.

10. The application of a radiation-resistant electrochemical sensor as described in any one of claims 1-3 in the detection of target analytes under radiation conditions; Preferably, the radiation environment includes at least one of a nuclear industrial facility, a nuclear waste disposal site, a space exploration device, and a medical radiotherapy area; Preferably, the detection mode used in the detection is selected from at least one of the amperometric method, the voltammetric method, and the impedance method.

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