Kit for detecting pancreatic cancer based on fluorescence labeling method and application of kit

Abstract

The invention relates to the technical field of biomedical detection, and discloses a kit for detecting pancreatic cancer based on a fluorescence labeling method and application of the kit, and the kit is composed of eutectic solvent precursor dispersion liquid containing an interface stabilizer and a polycarboxylate regulator. According to the method, the deep eutectic solvent is driven to be separated out from the water phase by utilizing the strong hydration capability of the polycarboxylate regulator, and the microphase enrichment region with low water content is constructed in situ. In the enrichment area, a compact hydrogen bond network limits free movement of water molecules, and a non-radiative transition channel for transferring excited state energy of fluorescent molecules to a hydroxyl vibration energy level of the water molecules is blocked. The unique microenvironment mechanism significantly reduces the fluorescence quenching effect in an aqueous system, greatly improves the quantum yield and detection sensitivity of the fluorescent probe, and effectively solves the technical problem of weak signal in traditional aqueous phase immunodetection.

Description

A reagent kit for detecting pancreatic cancer based on fluorescent labeling and its application. Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a reagent kit for detecting pancreatic cancer based on fluorescence labeling and its application. Background Technology

[0002] Fluorescent immunoassay technology holds an important position in the field of in vitro diagnostics due to its high specificity and ease of operation. However, the detection sensitivity of this technology is always limited by the inherent inhibitory effect of aqueous solutions on fluorescence signals. Commonly used organic fluorescent probes, such as fluorescein isothiocyanate, are highly sensitive to the solvent environment in the excited state. In conventional biological buffer systems, water molecules, as a highly polar solvent, can effectively absorb the energy of the excited state of fluorescent molecules through their high-frequency OH bond vibrational levels, causing energy dissipation through non-radiative transitions, resulting in significant fluorescence quenching. This solvent effect significantly reduces the quantum yield of fluorescent probes in aqueous phases, limiting their ability to detect low-abundance biomarkers.

[0003] To overcome the aqueous phase quenching effect, existing techniques typically employ the addition of an organic co-solvent to the reaction system to reduce solvent polarity. While these methods can enhance fluorescence signals to some extent, high concentrations of organic solvents can disrupt the hydration membrane on the protein surface, inducing conformational changes or even denaturation and inactivation of antigens or antibodies, thereby leading to decreased binding efficiency or loss of linearity in the immune response. Another common solid-phase carrier separation strategy, while reducing background interference, involves cumbersome procedures and fails to fundamentally address the energy dissipation of the luminescent group by solvent molecules during the liquid-phase reaction. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a reagent kit for detecting pancreatic cancer based on fluorescence labeling and its application, solving the problem of severe fluorescence quenching faced by existing fluorescence immunoassay techniques in aqueous reaction systems.

[0005] To address the aforementioned problems, the present invention provides the following technical solution: Firstly, the present invention provides a reagent kit for detecting pancreatic cancer based on fluorescence labeling, employing the following technical solution: A reagent kit for detecting pancreatic cancer based on fluorescence labeling, the reagent kit comprising independently packaged first and second components, wherein the first and second components, when mixed, form a liquid-solid competitive equilibrium system with anti-fluorescence quenching function; wherein the first component is a structured solvent dispersion, the raw materials for its preparation including: hydrogen bond acceptor, hydrogen bond donor, interface stabilizer, and first buffer; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1.8~2.2; the second component is a phase separation driving liquid, the raw materials for its preparation including: a polycarboxylate regulator, an inorganic co-salt, and a second buffer; the molar concentration of the polycarboxylate regulator in the second component is 0.6M~1.0M.

[0006] By employing the above technical solution, a microscopic heterogeneous reaction system was constructed by utilizing the thermodynamic interaction between ionotropic and stable-liquid sequence substances in aqueous solution. The mechanism is as follows: the polycarboxylate regulator, acting as a strong electrolyte with high charge density, reduces the activity of free water in the bulk solution through strong hydration during mixing. The salting-out effect then causes the eutectic solvent component, formed by the hydrogen bond acceptor and hydrogen bond donor, to precipitate from the bulk aqueous phase. Under the action of the interfacial stabilizer, the precipitated eutectic solvent component disperses to form micron- or submicron-sized enriched micelles. Due to the relative hydrophobicity of the fluorescent probe and the antigen-antibody complex, according to the principle of "like dissolves like," it is distributed and enriched within the low-water-content eutectic solvent micelles. Within the micellar microenvironment, the eutectic solvent restricts the movement of trace water molecules through a dense hydrogen bond network, inhibiting the nonradiative transition path from the excited state energy of the fluorescent molecule to the vibrational energy level of the water molecule's hydroxyl group, thereby improving the fluorescence quantum yield. Meanwhile, the interface stabilizer forms a steric hindrance layer on the micelle surface, blocking the direct contact of high-concentration salt ions with the antibody and preventing salting out precipitation; the hydrogen bond donor is complexed and fixed by the hydrogen bond acceptor, inhibiting its ability to destroy the secondary structure of the protein, so that the antibody can maintain its native activity while enhancing the signal.

[0007] Preferably, the raw materials of the first component are composed of the following proportions: the mass fraction of the eutectic solvent precursor formed by the hydrogen bond acceptor and the hydrogen bond donor in the first component is 15% to 25%; the mass fraction of the interface stabilizer in the first component is 2% to 3%; the balance is the first buffer solution; the hydrogen bond acceptor is selected from choline chloride, and the hydrogen bond donor is selected from urea or acetamide.

[0008] By adopting the above technical solution, choline chloride and urea or acetamide are controlled within a specific molar ratio and mass fraction range, a stable supramolecular cluster structure can be formed. This structure can maintain micelle morphology without irreversible macroscopic stratification when repelled by polycarboxylic acid salts. Polyvinylpyrrolidone, as an interface stabilizer, has a concentration and molecular weight that match the interfacial tension requirements of microphase separation, ensuring that the system forms a uniform microemulsion dispersion.

[0009] Preferably, the raw materials of the second component are composed of the following proportions: the polycarboxylate regulator is selected from trisodium citrate dihydrate; the inorganic salt is selected from sodium chloride or potassium chloride, and its molar concentration is 0.1M~0.2M; the pH value of the second component is 6.8~7.0.

[0010] By adopting the above technical solution, trisodium citrate dihydrate provides strong liquid stabilization ability and is the main driving force for phase separation; sodium chloride or potassium chloride is used to adjust the basic ionic strength and maintain electrolyte balance; the pH value is controlled between 6.8 and 7.0 to avoid urea decomposition due to alkaline environment during subsequent mixing, and it is also within the suitable pH window for antigen-antibody binding.

[0011] Preferably, the interface stabilizer is polyvinylpyrrolidone with a K value of 27-32.

[0012] By adopting the above technical solution and selecting polyvinylpyrrolidone with a specific K value, its molecular chain length is moderate. It can be adsorbed on the surface of eutectic solvent droplets to provide steric stabilization, and will not hinder molecular diffusion and mass transfer due to excessively high solution viscosity caused by excessively large molecular weight.

[0013] Preferably, the kit further includes a third component, which is a fluorescent probe that specifically identifies pancreatic cancer markers, wherein the pancreatic cancer markers are selected from one or more of CA19-9, CEA, or GPC1.

[0014] By adopting the above technical solution and incorporating specific fluorescent probes into the system, highly sensitive detection of specific biomarkers for pancreatic cancer was achieved.

[0015] Preferably, the system after mixing the first component and the second component at a volume ratio of 3:1 satisfies the following parameter characteristics: the molar ratio of the polycarboxylate regulator to the hydrogen bond donor is 1:6.0~8.0; and the final pH value of the mixed system is 6.8~7.2.

[0016] By employing the above technical solution and limiting the molar ratio and pH value after mixing, the reaction system is locked in a thermodynamically metastable region. At this ratio, the liquid-dissociating and liquid-stabilizing effects reach a dynamic equilibrium, ensuring sufficient microphase separation to enhance fluorescence while avoiding protein denaturation or precipitation due to excessive salt concentration, thus optimizing the signal-to-noise ratio.

[0017] Secondly, the present invention provides a method for preparing a reagent kit for detecting pancreatic cancer based on fluorescence labeling, using the following technical solution: A method for preparing a reagent kit for detecting pancreatic cancer based on fluorescence labeling, comprising the steps of preparing a first component and a second component respectively: S1, preparing the first component: A, mixing hydrogen bond acceptors and hydrogen bond donors in a molar ratio, stirring and reacting at 60℃~70℃ until solid particles disappear, forming a concentrated solution; B, cooling the concentrated solution obtained in step A to 40℃~45℃, adding it to a buffer solution containing an interface stabilizer, and performing high-speed shear mixing to obtain a metastable micelle dispersion; S2, preparing the second component: dissolving a polycarboxylate regulator and an inorganic co-salt in water, adjusting the pH value, and filtering for sterilization; S3, filling the first component obtained in S1 and the second component obtained in S2 into different containers respectively.

[0018] By employing the above technical solution and a stepwise preparation process, the structural integrity and storage stability of the components are ensured. In step S1, an anhydrous thermal eutectic reaction is used to promote the formation of a tight hydrogen bond network between hydrogen bond acceptors and hydrogen bond donors at the molecular level. This forms the structural basis for the subsequent formation of an anti-quenching microenvironment. A eutectic solvent concentrate is first prepared, then dispersed in a buffer solution containing an interface stabilizer, and subjected to high-speed shearing. This allows the eutectic solvent precursor to be encapsulated by the interface stabilizer, forming a homogeneous emulsion dispersion. High-concentration polycarboxylate regulators and structured solvents are packaged separately to avoid aggregation or macroscopic phase separation caused by prolonged contact during storage and transportation, ensuring the uniformity and effectiveness of the kit in clinical use.

[0019] Preferably, the specific process parameters for step S1 are as follows: In step A, the stirring reaction time is 30-45 minutes, and the reaction process is carried out in a closed anhydrous environment; In step B, the high-speed shear mixing speed is 300-500 rpm, the mixing time is 20-30 minutes, and the final pH value of the first component is adjusted to 7.0±0.1.

[0020] By adopting the above technical solution, the sealed anhydrous environment avoids the interference of air moisture on the formation of eutectic; the specific shear speed and time control the particle size distribution of the dispersed phase, making it at the submicron level, which is beneficial for the subsequent formation of microphases to have a large specific surface area and accelerate the enrichment process of the probe.

[0021] Preferably, in step S2, the pH value of the second component is adjusted using a 1.0M citric acid solution, and then filtered through a 0.22µm filter membrane after adjustment.

[0022] By adopting the above technical solution, citric acid is used to adjust the pH instead of a strong inorganic acid, introducing the common ion effect and avoiding the introduction of impurity anions that would disrupt the ionic strength balance of the system; filtration ensures the sterility of the reagent and prevents reagent deterioration caused by microbial growth.

[0023] Thirdly, this invention provides an application of a fluorescent labeling-based pancreatic cancer detection kit in the in vitro detection of pancreatic cancer biomarkers, comprising the following steps: mixing the biological sample to be tested, the first component, and the second component at a volume ratio of 1:3:1, allowing the polycarboxylate regulator to drive the structured solvent in the first component to form a microphase enrichment; incubating at 37°C for 15-20 minutes, allowing the fluorescent probe and antigen complex to enter the microphase enrichment region; and directly detecting the fluorescence signal of the incubated mixture.

[0024] By employing the above-described technical solution, the in-situ physicochemical changes of the reagent kit components during mixing enable the reconstruction of the reaction microenvironment. When the first component is mixed with the second component and the sample, the high concentration of citrate ions in the second component rapidly binds to free water in the mixture, leading to a decrease in the solubility of the originally dispersed eutectic solvent micelles in the first component, resulting in microphase separation and aggregation. Due to the presence of the interface stabilizer, this aggregation remains at the micrometer scale. At this point, the more hydrophobic antigen-antibody complex in the sample is driven to partition into the low-water-content eutectic solvent-enriched phase. This process enriches the analyte, increasing the local concentration of the reaction, i.e., improving the reaction kinetic rate; it also constructs a low-dielectric-constant luminescent environment, enhancing the fluorescence signal intensity, thereby achieving high-sensitivity, low-background detection of pancreatic cancer biomarkers. This application method eliminates the need for complex washing steps, simplifying the operation process.

[0025] This invention provides a reagent kit for detecting pancreatic cancer based on fluorescence labeling and its application. It has the following beneficial effects: 1. This invention utilizes the strong hydration ability of polycarboxylate regulators to drive the formation of a low-water-content microphase enrichment region in the eutectic solvent. The dense hydrogen bond network within this enrichment region restricts the free movement of water molecules and blocks the non-radiative transition channels from the excited state energy of fluorescent molecules to the vibrational energy level of water hydroxyl groups, thereby significantly reducing the fluorescence quenching effect in aqueous systems and improving the quantum yield and detection sensitivity of the fluorescent probe.

[0026] 2. This invention employs a composite formulation of an interface stabilizer and a deep eutectic solvent precursor to form a steric hindrance layer at the microphase interface. Simultaneously, it utilizes the complexation effect of hydrogen bond acceptors on hydrogen bond donors to reduce their chemical activity. This component design effectively isolates the antibody from direct contact with high-concentration salt ions and inhibits the damaging effect of urea on protein peptide bonds. This allows immunoglobulins to maintain their intact secondary structure and antigen-binding activity in an enhanced system with high ionic strength and potential denaturing agents, avoiding protein denaturation or precipitation.

[0027] 3. This invention utilizes a microphase separation mechanism initiated by a phase separation-driven liquid, based on the principle of "like dissolves like," to extract and partition hydrophobic antigen-antibody complexes from a complex serum matrix into a dispersed phase. This process achieves local concentration of the analyte, increases the antigen-antibody binding reaction rate, and reduces background interference from impurities in the serum to the detection signal through physical isolation. High signal-to-noise ratio rapid detection can be achieved without cumbersome washing and separation steps. Attached Figure Description

[0028] Figure 1 is a schematic diagram of the preparation method steps of the present invention. Detailed Implementation

[0029] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0031] Choline chloride, CAS number 67-48-1, purity ≥98%, is used as a hydrogen bond acceptor; urea, CAS number 57-13-6, purity ≥99.5%, is used as a hydrogen bond donor. Acetamide, CAS number 60-35-5, purity ≥99%, is used as an alternative hydrogen bond donor. Polyvinylpyrrolidone, CAS number 9003-39-8, specification K30, average molecular weight 40,000-50,000, is used as an interface stabilizer; trisodium citrate dihydrate, CAS number 6132-04-3, is used as a polycarboxylate regulator. Sodium chloride, chemical formula NaCl, CAS number 7647-14-5, is used as an inorganic co-salt; potassium chloride, chemical formula KCl, CAS number 7447-40-7, is used as an alternative inorganic co-salt. Citric acid monohydrate, CAS number 5949-29-1, is used for pH adjustment. Fluorescein isothiocyanate, abbreviated as FITC, CAS number 3326-32-7, is used for antibody labeling. Mouse anti-human CA19-9 monoclonal antibody, mouse anti-human CEA monoclonal antibody, and mouse anti-human GPC1 monoclonal antibody are all biological grade, with a protein concentration of 1.0 mg / mL and a purity >95% (SDS-PAGE). Other common chemical reagents such as disodium hydrogen phosphate and potassium dihydrogen phosphate are commercially available analytical grade products.

[0032] Preparation Example A1: This preparation example provides a method for preparing a structured solvent dispersion (first component), including the following steps: (1) Preparation of the eutectic solvent precursor: Weigh 13.96 g (0.1 mol) of hydrogen bond acceptor choline chloride and 12.01 g (0.2 mol) of hydrogen bond donor urea, mix them and place them in a reaction vessel; heat to 65°C under sealed and anhydrous conditions, stir at 60 rpm for 40 minutes until the solid particles completely disappear, and obtain a colorless and transparent eutectic solvent concentrate; (2) Preparation of interface stabilizer base solution: Weigh 3.25g of polyvinylpyrrolidone (K30) and dissolve it in 100mL of 20mM phosphate buffer (pH7.0), and stir until completely dissolved; (3) Shear emulsification: Cool the eutectic solvent concentrate obtained in step (1) to 42℃, and add 26.0g to the interface stabilizer base solution in step (2); perform high-speed shear mixing at 400rpm for 25 minutes, during which the pH value is finely adjusted to 7.0 using 1.0M hydrochloric acid or sodium hydroxide solution.

[0033] Preparation Example A2: This preparation example provides a method for preparing a structured solvent dispersion (first component), including the following steps: (1) Preparation of eutectic solvent precursor: Weigh 13.96g (0.1mol) of hydrogen bond acceptor choline chloride and 10.81g (0.18mol) of hydrogen bond donor urea, mix them and place them in a reaction vessel; heat to 60°C under closed and anhydrous conditions, stir at 50rpm for 45 minutes to obtain eutectic solvent concentrate; (2) Preparation of interface stabilizer base solution: Weigh 2.60g of polyvinylpyrrolidone (K30) and dissolve it in 100mL of 20mM phosphate buffer (pH 7.0); (3) Shear emulsification: Cool the eutectic solvent concentrate obtained in step (1) to 40°C, take 19.5g and add it to the interface stabilizer base solution in step (2); perform high-speed shear mixing at 300rpm for 30 minutes, and adjust the pH value to 7.0.

[0034] Preparation Example A3: This preparation example provides a method for preparing a structured solvent dispersion (first component), including the following steps: (1) Preparation of eutectic solvent precursor: Weigh 13.96g (0.1mol) of hydrogen bond acceptor choline chloride and 13.21g (0.22mol) of hydrogen bond donor urea, mix them and place them in a reaction vessel; heat to 70°C under closed and anhydrous conditions, stir at 80rpm for 30 minutes to obtain eutectic solvent concentrate; (2) Preparation of interface stabilizer base solution: Weigh 3.90g of polyvinylpyrrolidone (K30) and dissolve it in 100mL of 20mM phosphate buffer (pH 7.0); (3) Shear emulsification: Cool the eutectic solvent concentrate obtained in step (1) to 45°C, take 32.5g and add it to the interface stabilizer base solution in step (2); perform high-speed shear mixing at 500rpm for 20 minutes, and adjust the pH value to 7.0.

[0035] Preparation Example A4: This preparation example provides a method for preparing a structured solvent dispersion (first component), including the following steps: (1) Preparation of eutectic solvent precursor: Weigh 13.96g (0.1mol) of hydrogen bond acceptor choline chloride and 11.82g (0.2mol) of hydrogen bond donor acetamide, and the remaining operating conditions are the same as in step (1) of Preparation Example A1 to obtain a eutectic solvent concentrate; (2) Preparation of interface stabilizer base liquid: Same as in step (2) of Preparation Example A1; (3) Shear emulsification: Take 26.0g of the concentrate obtained in step (1), and the operating conditions are the same as in step (3) of Preparation Example A1.

[0036] Preparation Example A5: This preparation example provides a method for preparing a solvent dispersion (first component) for comparison, including the following steps: (1) Preparation of the mixture: Weigh 13.96g (0.1mol) of hydrogen bond acceptor choline chloride and 6.01g (0.1mol) of hydrogen bond donor urea (molar ratio 1:1); heat to 70°C and stir until a homogeneous liquid is barely formed (Note: the melting point is high at this ratio, and precipitation is easy when cooling); (2) Shear emulsification: Cool the liquid obtained in step (1) to 45°C (at this time it is a turbid slurry), take 20.0g and add it to a buffer solution containing polyvinylpyrrolidone, and operate as in step (3) of Preparation Example A1.

[0037] Preparation Example A6: This preparation example provides a method for preparing a solvent dispersion (first component) for comparison, including the following steps: (1) Preparation of eutectic solvent precursor: same as step (1) in Preparation Example A1; (2) Mixing: take 26.0 g of the eutectic solvent concentrate obtained in step (1) and add it directly to 100 mL of 20 mM phosphate buffer (without polyvinylpyrrolidone); shear mix at 400 rpm for 25 minutes and adjust the pH to 7.0.

[0038] Preparation Example B1: This preparation example provides a method for preparing a phase separation driving liquid (second component), comprising the following steps: weighing 23.53 g (0.08 mol) trisodium citrate dihydrate and 0.88 g (0.015 mol) sodium chloride, adding deionized water to dissolve and making up to 100 mL; adjusting the pH of the solution to 6.9 using 1.0 M citric acid solution; and finally filtering through a 0.22 µm microporous membrane for sterilization.

[0039] Preparation Example B2: This preparation example provides a method for preparing a phase separation driving liquid (second component), comprising the following steps: weighing 17.65 g (0.06 mol) trisodium citrate dihydrate and 0.75 g (0.01 mol) potassium chloride, adding deionized water to dissolve and making up to 100 mL; adjusting the pH of the solution to 6.8 using 1.0 M citric acid solution; and finally filtering through a 0.22 µm microporous membrane for sterilization.

[0040] Preparation Example B3: This preparation example provides a method for preparing a phase separation driving liquid (second component), comprising the following steps: weighing 29.41 g (0.1 mol) trisodium citrate dihydrate and 1.17 g (0.02 mol) sodium chloride, adding deionized water to dissolve and making up to 100 mL; adjusting the pH of the solution to 7.0 using 1.0 M citric acid solution; and finally filtering through a 0.22 µm microporous membrane for sterilization.

[0041] Preparation Example B4: This preparation example provides a method for preparing a comparative solution (second component), comprising the following steps: weighing 5.88 g (0.02 mol) trisodium citrate dihydrate and 0.88 g sodium chloride, adding deionized water to dissolve and making up to 100 mL, and adjusting the pH to 6.9.

[0042] Preparation Example B5: This preparation example provides a method for preparing a comparative solution (second component), comprising the following steps: weighing 23.53 g (0.08 mol) of trisodium citrate dihydrate, without adding inorganic salts, dissolving in deionized water and bringing the volume to 100 mL, and adjusting the pH to 6.9.

[0043] Example 1: This example provides a kit for detecting pancreatic cancer based on fluorescence labeling. Please refer to Figure 1. The kit includes the following steps: (1) Provision of the first component: Take the structured solvent dispersion prepared in Preparation Example A1. The molar ratio of choline chloride to urea in the dispersion is 1:2. The mass fraction of the eutectic solvent precursor is 20%. The mass fraction of polyvinylpyrrolidone (K30) is 2.5%. Fill it into a light-proof container as the first component of the kit. (2) Provision of the second component: Take the phase separation driving solution prepared in Preparation Example B1. The concentration of trisodium citrate in the solution is 0.8M. The concentration of sodium chloride is 0.15M. The pH value is 6.9. Fill it into a sealed container as the second component of the kit. (3) Provision of the third component: Provide a mouse anti-human CA19-9 monoclonal antibody solution labeled with fluorescein isothiocyanate (FITC) at a concentration of 50µg / mL as the third component of the kit. (4) Assembly: Combine and package the first, second and third components and store them at 4°C.

[0044] Example 2: This example provides a kit for detecting pancreatic cancer based on fluorescence labeling, including the following steps: (1) Provision of the first component: Take the structured solvent dispersion prepared in Preparation Example A2, in which the molar ratio of choline chloride to urea is 1:1.8, the mass fraction of the eutectic solvent precursor is 15%, and the mass fraction of polyvinylpyrrolidone (K30) is 2.0%, and fill it into a light-proof container as the first component of the kit; (2) Provision of the second component: Take the phase separation driving solution prepared in Preparation Example B2, in which the concentration of trisodium citrate is 0.6M, the concentration of potassium chloride is 0.1M, and the pH value is 6.8, and fill it into a sealed container as the second component of the kit; (3) Provision of the third component: Provide a phycoerythrin (PE) labeled mouse anti-human CEA monoclonal antibody solution with a concentration of 50µg / mL as the third component of the kit; (4) Assembly: Combine and package the first, second, and third components and store them at 4°C.

[0045] Example 3: This example provides a kit for detecting pancreatic cancer based on fluorescence labeling, including the following steps: (1) Provision of the first component: Take the structured solvent dispersion prepared in Preparation Example A3, in which the molar ratio of choline chloride to urea is 1:2.2, the mass fraction of the eutectic solvent precursor is 25%, and the mass fraction of polyvinylpyrrolidone is 3.0%, and fill it into a light-proof container as the first component of the kit; (2) Provision of the second component: Take the phase separation driving solution prepared in Preparation Example B3, in which the concentration of trisodium citrate is 1.0M, the concentration of sodium chloride is 0.2M, and the pH value is 7.0, and fill it into a sealed container as the second component of the kit; (3) Provision of the third component: Provide a mouse anti-human GPC1 monoclonal antibody solution labeled with fluorescein isothiocyanate (FITC) at a concentration of 50µg / mL as the third component of the kit; (4) Assembly: Combine and package the first, second, and third components and store them at 4°C.

[0046] Example 4: This example provides a kit for detecting pancreatic cancer based on fluorescent labeling, including the following steps: (1) Provision of the first component: Take the structured solvent dispersion prepared in Preparation Example A4, in which acetamide is used instead of urea as a hydrogen bond donor, and the molar ratio of choline chloride to acetamide is 1:2, and fill it into a light-proof container as the first component of the kit; (2) Provision of the second component: Take the phase separation driving solution prepared in Preparation Example B1, in which the concentration of trisodium citrate is 0.8M and the concentration of sodium chloride is 0.15M, and fill it into a sealed container as the second component of the kit; (3) Provision of the third component: Provide a mouse anti-human CA19-9 monoclonal antibody solution labeled with fluorescein isothiocyanate (FITC) at a concentration of 50µg / mL as the third component of the kit; (4) Assembly: Combine and package the first, second and third components and store them at 4°C.

[0047] Comparative Example 1: This comparative example provides a conventional immunofluorescence detection kit (blank control), which differs from Example 1 only in that it does not contain the eutectic solvent precursor and the polycarboxylate modifier.

[0048] (1) First component: 20mM phosphate buffer (pH 7.0), free of choline chloride, urea and polyvinylpyrrolidone; (2) Second component: 20mM phosphate buffer (pH 7.0), containing 0.15M sodium chloride, but free of trisodium citrate dihydrate; (3) Third component: same as in Example 1; (4) Assembly: same as in Example 1.

[0049] Note: This is used to simulate the detection effect of conventional aqueous systems in existing technologies.

[0050] Comparative Example 2: This comparative example provides a kit that disrupts the hydrogen bond network structure. The difference between this kit and Example 1 is that the ratio of hydrogen bond acceptor to hydrogen bond donor in the first component cannot form a stable eutectic structure.

[0051] (1) First component: Take the dispersion prepared in Preparation Example A5, wherein the molar ratio of choline chloride to urea is 1:1 (non-eutectic ratio, which cannot form a dense supramolecular hydrogen bond network); (2) Second component: Same as in Example 1 (Preparation Example B1); (3) Third component: Same as in Example 1; (4) Assembly: Same as in Example 1.

[0052] Note: This is used to verify the importance of a specific molar ratio (1:1.8~2.2) for constructing a fluorescence-resistant quenching microenvironment.

[0053] Comparative Example 3: This comparative example provides a kit that cannot drive microphase separation. The difference between this kit and Example 1 is that the concentration of the polycarboxylate regulator in the second component is too low.

[0054] (1) First component: Same as in Example 1 (Preparation Example A1); (2) Second component: Take the solution prepared in Preparation Example B4, wherein the molar concentration of trisodium citrate dihydrate is 0.2M (far lower than the critical salt concentration required for driving phase separation); (3) Third component: Same as in Example 1; (4) Assembly: Same as in Example 1.

[0055] Note: This is used to verify the necessity of high-concentration regulators in the liquid-liquid competition mechanism.

[0056] Comparative Example 4: This comparative example provides a kit lacking an interface stabilization mechanism, which differs from Example 1 in that the first component does not contain an interface stabilizer.

[0057] (1) First component: The dispersion prepared in Preparation Example A6 is free of polyvinylpyrrolidone (PVP); (2) Second component: Same as in Example 1 (Preparation Example B1); (3) Third component: Same as in Example 1; (4) Assembly: Same as in Example 1.

[0058] Note: This study is used to verify the role of interface stabilizers in preventing macroscopic precipitation and protecting antibody activity.

[0059] Comparative Example 5: This comparative example provides a kit that uses a conventional organic solvent instead of a structured solvent. The difference between this kit and Example 1 is the solvent composition of the first component.

[0060] (1) First component: phosphate buffer containing 20% ​​(v / v) dimethyl sulfoxide (DMSO) and 2.5% polyvinylpyrrolidone; (2) Second component: same as in Example 1 (Preparation Example B1); (3) Third component: same as in Example 1; (4) Assembly: same as in Example 1.

[0061] Note: This is used to compare the differences between the DES system of this invention and traditional organic solvent enhancement methods, and to verify the protective advantages of this invention for protein activity.

[0062] Comparative Example 6: This comparative example provides a single solvent system kit, which differs from Example 1 in that it does not construct a competing system and does not cause phase separation.

[0063] (1) First component: Same as in Example 1 (Preparation Example A1); (2) Second component: 20 mM phosphate buffer (pH 7.0) containing 0.15 M sodium chloride, but not trisodium citrate dihydrate; (3) Third component: Same as in Example 1; (4) Assembly: Same as in Example 1.

[0064] Note: This is used to verify the impact of the presence of DES without the formation of an enriched phase (i.e., no local concentration effect) on detection performance, thereby demonstrating the importance of the "in-situ microphase separation" step.

[0065] Test Example 1: Phase Transition Behavior and Dispersion State Characterization of the Reaction System. This test example aims to determine the transmittance changes of different ratio systems by spectrophotometry, and to verify whether the structured solvent dispersion and phase separation driving liquid described in this invention can undergo microphase separation based on the liquid-solid competition mechanism to construct a low-water-content microenvironment.

[0066] The experimental procedure is as follows: Turn on the UV-Vis spectrophotometer, set the wavelength to 600 nm, and preheat for 30 minutes. Use deionized water as a blank control for baseline calibration and adjust the transmittance to 100%.

[0067] Take the components prepared in Examples 1-4 and Comparative Examples 1, 3, and 6. Measure the first and second components (or corresponding buffer solutions) according to the proportions described in each example and comparative example, keeping the total volume to 2 mL.

[0068] Add each component to a quartz cuvette, mix by pipetting 5 times, let stand for 30 seconds, and wait for the system reaction to stabilize.

[0069] Place the cuvette containing the mixture into the sample cell and record the transmittance (T%) at 600 nm.

[0070] Each sample was measured in triplicate, and the data were recorded and the mean and standard deviation were calculated.

[0071] Table 1. Transmittance measurement data of the mixed systems of each embodiment and comparative example.

[0072] According to the data in Table 1, Comparative Example 1, as a pure aqueous buffer system, had a transmittance close to 100%, indicating that the system was homogeneous and clear. The transmittance of Examples 1 to 4 all showed a significant decreasing trend, ranging from 12% to 27%. The differences in the data indicate that when the structured solvent dispersion containing choline chloride-urea (or acetamide) was mixed with a high-concentration polycarboxylate solution, the optical properties of the system underwent a fundamental change. The significant decrease in transmittance was caused by the scattering of light by a large number of micron- or submicron-sized droplets generated within the system, confirming that the mixing process triggered instantaneous microphase separation.

[0073] Comparing the data from Example 1 (18.73%) and Comparative Example 6 (97.60%), it can be seen that in the absence of a polycarboxylate regulator, the eutectic solvent precursor dissociates and becomes infinitely miscible in water, failing to form a discontinuous phase. While Comparative Example 3 (94.17%) contains a certain amount of trisodium citrate (0.2M), its transmittance remains at a high level, indicating that this concentration has not reached the critical salt concentration for initiating phase separation and cannot generate sufficient salting-out driving force to repel hydrogen bond acceptors and donors from the aqueous phase.

[0074] Based on the experimental results of the combined examples and comparative examples, it can be confirmed that the present invention successfully induced the aggregation of eutectic solvent components by controlling the concentration of polycarboxylate to create a strong electrolyte environment. This aggregation behavior leads to an increase in system turbidity, which macroscopically manifests as a decrease in transmittance, and microscopically corresponds to the formation of high-density, low-water-content enriched phase droplets. This physical phase transition provides the necessary hydrophobic microenvironment for subsequent antigen-antibody immunoreaction and fluorescent probe enrichment. Example 3, due to the use of higher concentrations of salt and DES components, formed droplet densities that were the lowest, resulting in the lowest transmittance; while Example 2, at the lower limit of the ratio, had relatively high transmittance, but was still significantly different from the homogeneous system, proving that the process parameters within the scope of protection of the present invention can effectively achieve microphase separation.

[0075] Test Example 2: Characterization of Fluorescent Probe Microenvironment Enrichment Effect and Signal Enhancement Performance. This test example aims to determine the fluorescence intensity of FITC-labeled monoclonal antibodies under different solvent environments by fluorescence spectroscopy, and to verify the enhancement effect and anti-quenching performance of the structured solvent microphase system constructed in this invention on fluorescence signals.

[0076] The experimental steps are as follows: Start the fluorescence spectrophotometer, set the excitation wavelength (Ex) to 492nm, the emission spectrum scanning range to 500nm to 600nm, the width of both the excitation slit and the emission slit to 5nm, and the voltage to 600V.

[0077] According to the grouping of Examples 1 to 4 and Comparative Examples 1, 2, and 6, the first and second components (or corresponding buffer solutions) in the corresponding kits were taken and mixed according to the proportions specified in their respective instructions. The mixture was vortexed for 30 seconds to ensure the formation of a stable dispersion or homogeneous solution.

[0078] Add an equal amount of the third component (FITC-labeled mouse anti-human CA19-9 monoclonal antibody solution) to each of the above mixtures to ensure that the final antibody concentration in the system is consistent (approximately 2 µg / mL), and then incubate at 25°C in the dark for 10 minutes.

[0079] The sample to be tested was transferred to a four-sided transparent quartz cuvette, placed in the sample cell for spectral scanning, and the fluorescence intensity value (RFU) at 518 nm (the maximum emission wavelength of FITC) was recorded.

[0080] Three samples were prepared in parallel for each experimental group and measured independently. The raw data were recorded and the fluorescence enhancement factor relative to Comparative Example 1 was calculated.

[0081] Table 2. Fluorescence intensity measurement data of each example and comparative example system.

[0082] According to the data in Table 2, the average fluorescence intensity in the conventional PBS buffer environment (Comparative Example 1) was 167.2 RFU, which served as a baseline. The fluorescence intensity distribution of Examples 1 to 4 ranged from 1155.5 to 1590.8 RFU, representing a fluorescence enhancement factor of 6.91 to 9.51 times compared to Comparative Example 1. This significant signal amplification effect confirms that the microenvironment constructed in this invention effectively overcomes the fluorescence quenching effect commonly found in aqueous solutions.

[0083] Comparing the data of Example 1 (1428.7 RFU) and Comparative Example 6 (215.2 RFU), it can be seen that although both contain the same amount of eutectic solvent component (choline chloride-urea), Comparative Example 6 lacks a polycarboxylate-driven phase separation process. The DES component is dispersed at the molecular level in the aqueous phase, failing to form an independent microphase region. In this case, the fluorescent probe is still mainly exposed to a highly polar aqueous environment, and water molecules, acting as strong fluorescence quenchers, dissipate the excited-state energy through non-radiative transitions. Conversely, Example 1, driven by the salting-out effect, forms DES-rich microdroplets. The hydrophobic interaction selectively extracts and enriches the FITC-labeled antibody in this low-aqueous microphase, thereby blocking the contact quenching of water molecules and achieving physical enhancement of the fluorescence signal.

[0084] Furthermore, Comparative Example 2 (452.0 RFU) used a 1:1 molar ratio of raw materials, which deviated from the eutectic point, resulting in a less dense solvent network structure and weakened hydrogen bond strength. Data showed that its fluorescence enhancement effect was far lower than that of Example 1. This indicates that phase separation alone is insufficient; a specific hydrogen bond acceptor / donor stoichiometry is also required to construct a supramolecular structure with high viscosity and a rigid hydrogen bond network. This rigid structure restricts rotational and vibrational relaxation within the fluorescent molecules, further reducing non-radiative energy loss. In summary, this invention achieves a significant improvement in detection sensitivity through a dual mechanism of phase separation enrichment and a rigid, low-water microenvironment.

[0085] Test Example 3: Comparison and Evaluation of Detection Sensitivity and Linearity Range. This test example aims to construct a standard curve to compare the lower limit of detection (LOD) and linear response range of different reaction systems for the tumor marker CA19-9, thereby verifying the technical effect of the present invention in improving the detection sensitivity of low-abundance samples through the microphase separation enrichment effect.

[0086] The experimental steps are as follows: Prepare a series of CA19-9 antigen standard solutions. Use phosphate buffer containing 1% bovine serum albumin (BSA) to serially dilute the high-concentration antigen stock solution to obtain a series of standard solutions with concentrations of 0, 5, 10, 50, 100, and 200 U / mL.

[0087] Example 1, Example 4, Comparative Example 1, Comparative Example 5 (organic solvent system containing 20% ​​DMSO) and Comparative Example 6 (system without phase separation) were selected as test objects.

[0088] In a 96-well black ELISA plate, first add 50 µL of the antigen standards of each concentration mentioned above, and then add the first component, the second component, and the third component (FITC-labeled antibody) according to the proportions specified in each example, bringing the total volume of each well to 200 µL.

[0089] Place the ELISA plate on a microplate shaker and mix at 400 rpm for 2 minutes, then let it stand at room temperature in the dark for 15 minutes.

[0090] The fluorescence intensity of each well was measured using a fluorescence microplate reader at an excitation wavelength of 492 nm and an emission wavelength of 518 nm.

[0091] Three replicates were set for each concentration point, and the average value was taken. A standard curve was plotted with antigen concentration (U / mL) on the x-axis and fluorescence intensity (RFU) on the y-axis. Linear regression analysis was performed, and the linear correlation coefficient (R²) was calculated. 2 ).

[0092] The standard deviation (SD) of background noise was calculated based on the measurement results of the blank sample (0 U / mL), and the lower limit of detection for each system was calculated according to the formula LOD=3×SD / Slope.

[0093] Table 3. Detection performance data of each system against CA19-9 standard.

[0094] According to the data in Table 3, the detection system of Example 1 exhibited the highest response sensitivity. Its standard curve slope was 34.15, significantly higher than that of Comparative Example 1 (1.41). This means that, under the same antigen concentration change, the signal increment generated by the system of this invention is more than 20 times that of the conventional aqueous system. The calculated limit of detection (LOD) was 0.16 U / mL, significantly better than that of Comparative Example 1 (2.55 U / mL), achieving effective detection of trace antigens.

[0095] Analysis of the data differences between Example 1 and Comparative Example 6 (LOD 0.89 U / mL) reveals that although both contain eutectic solvent components, Example 1 initiated phase separation through salting-out. Since the volume of the eutectic solvent phase in the reaction system is much smaller than the volume of the aqueous phase (the volume ratio is typically less than 1:10), a significant "volume concentration effect" occurs when the antigen-antibody complex is extracted into the eutectic solvent microdroplets based on the principle of "like dissolves like." This physical enrichment directly increases the local concentration of luminescent groups within the microenvironment, and, combined with the aforementioned anti-quenching mechanism, achieves cascaded signal amplification.

[0096] Although Comparative Example 5 improved the fluorescence signal to some extent by adding organic solvents (slope of 10.22), its blank background value was high (112.4 RFU) and its standard deviation was large (±8.5), resulting in no significant improvement in LOD (2.49 U / mL). Furthermore, Comparative Example 5 had a low linear correlation coefficient (0.9542), which is attributed to the destructive effect of organic solvents on protein structure. With increasing antigen concentration or prolonged reaction time, some antibodies denatured and became inactive, leading to a decrease in antigen binding efficiency and disrupting the linearity of the dose-response relationship. In contrast, Example 1 of the present invention achieved high sensitivity while maintaining a good linear relationship (R0). 2 =0.9982), demonstrating that the structured solvent system has excellent biocompatibility and can maintain the native conformation and activity of proteins while enriching signals.

[0097] Test Example 4: Bioactivity Protection and Thermal Stability Assessment. This test example investigates the effects of different solvent systems on the bioactivity of proteins (antibodies) through accelerated aging experiments, and verifies the stabilizing effect of the structured solvent microenvironment on the conformation of fluorescently labeled antibodies under high temperature conditions.

[0098] The experimental steps are as follows: Select the components corresponding to Example 1, Comparative Example 1 (PBS control), Comparative Example 4 (PVP-free system) and Comparative Example 5 (DMSO organic solvent system).

[0099] The first and second components of each experimental group were mixed in proportion, and then the third component (FITC-labeled antibody) with a final concentration of 5 µg / mL was added. The mixture was then vortexed to prepare the base solution for testing.

[0100] The above-mentioned base solution was dispensed into several microcentrifuge tubes and placed in a 42°C constant temperature water bath for heat stress treatment.

[0101] Samples were removed at four time points: 0 hours, 2 hours, 6 hours, and 12 hours after heating, and quickly placed in an ice bath to cool to room temperature.

[0102] Add an equal and sufficient amount of CA19-9 antigen standard (100 U / mL) to the samples at each time point and incubate at 25°C for 15 minutes to carry out the immune response.

[0103] The fluorescence intensity at 518 nm was measured, and the activity retention rate at each time point was calculated with the measurement value at 0 hours as the baseline (100%).

[0104] Table 4. Antibody activity retention rate data for each system under heat stress conditions.

[0105] According to the data in Table 4, Comparative Example 5 showed the most severe activity degradation under heat stress at 42℃, with an activity retention rate of only 11.4% after 12 hours. This is because dimethyl sulfoxide, as a highly polar organic solvent, disrupts the hydration membrane on the protein surface and competitively forms hydrogen bonds with the peptide chain, leading to irreversible curling and denaturation of the antibody, thus losing its ability to recognize antigens. Comparative Example 1 showed a retention rate of 62.8% after 12 hours, indicating that in a pure aqueous environment, heating accelerated the thermal motion and hydrolysis of the protein, resulting in partial antibody inactivation.

[0106] Example 1 retained a high activity of 91.7% after 12 hours of high-temperature treatment. Its stability mechanism lies in the fact that the eutectic solvent component constructs a high-viscosity, high-ionic-strength pseudo-intracellular environment within the microdroplets. Choline chloride and urea replace unstable free water molecules through a hydrogen bond network, forming a more stable "structured solvent shell" with the polar groups on the protein surface. This rigid shell restricts the thermal vibration of the peptide chain, significantly increasing the protein's thermal denaturation temperature, allowing it to maintain its native tertiary structure even at high temperatures.

[0107] Comparing the data differences between Example 1 and Comparative Example 4 (retention rate 45.9%) reveals the crucial role of the interface stabilizer polyvinylpyrrolidone (PVP). In Comparative Example 4, which lacks PVP, although DES droplets were formed, the droplets possessed high surface energy. Antibody molecules tended to migrate to the phase boundary at the droplet-water interface and, under the influence of interfacial tension, unfolded to expose the hydrophobic core (interfacial denaturation), or aggregated and precipitated due to interfacial adsorption. In Example 1, the introduction of PVP molecules formed a steric hindrance protective layer on the droplet surface, effectively preventing direct contact between the antibody and the phase interface, thereby avoiding interface-induced protein inactivation.

[0108] Test Example 5: Determination of Anti-interference Ability and Recovery Rate in Complex Biological Matrix. This test example simulates a real clinical testing environment and performs an antigen spike recovery experiment in a human serum matrix to examine the anti-interference ability of the reaction system against complex components such as serum proteins and lipids, and to verify whether the microphase separation system can eliminate matrix effects through physical isolation mechanisms.

[0109] The experimental procedure is as follows: Collect mixed serum from healthy volunteers, which was confirmed to be negative for CA19-9 antigen (concentration <2U / mL) and used as the biological matrix background.

[0110] Add a high concentration of CA19-9 standard to the blank serum to prepare a spiked serum sample with a theoretical concentration of 50.0 U / mL.

[0111] Example 1, Comparative Example 1 (conventional PBS system), and Comparative Example 4 (PVP-free system) were selected as test subjects. The first and second components of each group were mixed in proportion.

[0112] Add 20 µL of the spiked serum sample and a quantitative amount of FITC-labeled antibody to each mixture, vortex to mix, and incubate at 25°C for 15 minutes.

[0113] The fluorescence intensity of each well was measured using a fluorescence microplate reader, and the concentration was calculated by substituting the measured concentration into the standard curve established in Test Example 3.

[0114] Each system was measured in parallel 5 times, and the average measured concentration, recovery rate (measured concentration / theoretical concentration × 100%) and coefficient of variation (CV) were calculated.

[0115] Table 5. Spike recovery and precision data in human serum matrix.

[0116] According to the data in Table 5, the detection accuracy of each system differed significantly in serum matrices containing a large number of non-target proteins (such as albumin and globulin). Comparative Example 1 showed an average recovery rate of only 72.8%, exhibiting a significant negative bias. This is because large protein molecules in serum undergo non-specific adsorption in the aqueous phase, covering some antigen sites. Simultaneously, colored substances such as hemoglobin or bilirubin absorb some of the excitation or emission light (internal filtration effect), leading to signal suppression in the conventional aqueous system and increasing the risk of false negatives.

[0117] Example 1 showed a recovery rate of 99.8% and a coefficient of variation of only 2.15%, demonstrating extremely high accuracy and precision. Its anti-interference mechanism lies in the salting-out effect generated by the strong electrolyte environment formed by the high concentration of polycarboxylate, which retains most of the hydrophilic serum proteins in the high-water-content continuous phase (aqueous phase); while the microdroplet phase formed by the eutectic solvent (DES) selectively enriches the relatively hydrophobic and highly affinity antigen-antibody complexes. This "liquid-liquid microextraction" process achieves physical spatial separation of the analyte and interfering substances at the microscale, effectively avoiding matrix effects.

[0118] Comparing the data from Example 1 and Comparative Example 4, although the latter had a relatively acceptable average recovery rate (93.8%), its coefficient of variation was as high as 15.24%, indicating extremely high data dispersion. This suggests that in the absence of polyvinylpyrrolidone (PVP), the surface of DES microdroplets has a high interfacial energy, easily adsorbing proteins from serum to form an interfacial film, leading to unstable droplet aggregation or emulsification disruption. In contrast, the PVP segments in Example 1 formed a hydrophilic steric hindrance layer on the droplet surface.

[0119] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A reagent kit for detecting pancreatic cancer based on fluorescent labeling, characterized in that: The kit comprises an independently packaged first component and a second component. The first component is a structured solvent dispersion, and its preparation materials include: hydrogen bond acceptor, hydrogen bond donor, interface stabilizer, and first buffer; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1.8~2.

2. The second component is a phase separation driving liquid, and its preparation materials include: polycarboxylate regulator, inorganic co-salt, and second buffer; the molar concentration of the polycarboxylate regulator in the second component is 0.6M~1.0M.

2. The reagent kit for detecting pancreatic cancer based on fluorescent labeling according to claim 1, characterized in that: The raw materials of the first component are composed of the following proportions: the mass fraction of the eutectic solvent precursor formed by the hydrogen bond acceptor and the hydrogen bond donor in the first component is 15% to 25%; the mass fraction of the interface stabilizer in the first component is 2% to 3%; the balance of the first buffer solution; the hydrogen bond acceptor is selected from choline chloride, and the hydrogen bond donor is selected from urea or acetamide.

3. The reagent kit for detecting pancreatic cancer based on fluorescent labeling according to claim 1, characterized in that: The raw materials of the second component are composed of the following proportions: the polycarboxylic acid salt regulator is selected from trisodium citrate dihydrate; the inorganic salt is selected from sodium chloride or potassium chloride, and its molar concentration is 0.1M~0.2M; the pH value of the second component is 6.8~7.

0.

4. The reagent kit for detecting pancreatic cancer based on fluorescent labeling according to claim 1, characterized in that: The interface stabilizer is polyvinylpyrrolidone, with a K value of 27-32.

5. The reagent kit for detecting pancreatic cancer based on fluorescent labeling according to claim 1, characterized in that: It also includes a third component, which is a fluorescent probe that specifically recognizes pancreatic cancer markers, wherein the pancreatic cancer markers are selected from one or more of CA19-9, CEA, or GPC1.

6. The reagent kit for detecting pancreatic cancer based on fluorescent labeling according to claim 1, characterized in that: The system obtained by mixing the first component and the second component at a volume ratio of 3:1 satisfies the following parameter characteristics: The molar ratio of the polycarboxylate regulator to the hydrogen bond donor is 1:6.0~8.0; the final pH of the mixed system is 6.8~7.

2.

7. A method for preparing a reagent kit for detecting pancreatic cancer based on fluorescent labeling, as described in any one of claims 1-6, characterized in that: The process includes the following steps for preparing the first and second components: S1. Preparation of the first component: A. Mix hydrogen bond acceptors and hydrogen bond donors in a molar ratio and stir at 60℃~70℃ until the solid particles disappear, forming a concentrated solution; B. Cool the concentrated solution obtained in step A to 40℃~45℃, add it to a buffer solution containing an interface stabilizer, and perform high-speed shear mixing to obtain a metastable micelle dispersion; S2. Preparation of the second component: Dissolve the polycarboxylate regulator and inorganic co-salt in water, adjust the pH value, and filter to remove bacteria; S3. Fill the first component obtained in S1 and the second component obtained in S2 into different containers.

8. The method for preparing a reagent kit for detecting pancreatic cancer based on fluorescent labeling according to claim 7, characterized in that: The specific process parameters for step S1 are as follows: In step A, the stirring reaction time is 30-45 minutes, and the reaction process is carried out in a closed anhydrous environment; In step B, the high-speed shear mixing speed is 300-500 rpm, the mixing time is 20-30 minutes, and the final pH value of the first component is adjusted to 7.0±0.

1.

9. The method for preparing a reagent kit for detecting pancreatic cancer based on fluorescent labeling according to claim 7, characterized in that: In step S2, the pH value of the second component is adjusted using a 1.0M citric acid solution, and then filtered through a 0.22µm filter membrane.

10. The application of the pancreatic cancer detection kit based on fluorescent labeling as described in any one of claims 1-6 in the in vitro detection of pancreatic cancer biomarkers.

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