Method for cellulose acetate film zone separation and ratio fluorescence quantitative detection of lactoferrin based on electric field driving

Abstract

The invention relates to a method for quantitatively detecting lactoferrin based on electric field driving-cellulose acetate film zone separation and ratio fluorescence, which takes a cellulose acetate film as a separation platform and a fluorescence platform, and utilizes potential difference to drive lactoferrin to generate zone separation. Meanwhile, lactoferrin is coordinated with carbon dot-lanthanide metal ions of the fluorescent probe, lanthanide ions are sensitized through an antenna effect, strong specific fluorescence is emitted, and the lactoferrin is quantitatively detected according to the ratio of the fluorescence intensity of the lanthanide ions to the fluorescence intensity of the carbon dots. The method does not need expensive instruments, has the advantages of high sensitivity, low detection cost, easiness in operation and the like, and is suitable for accurately detecting the content of lactoferrin in complex dairy products.

Description

Technical Field

[0001] This invention belongs to the field of food testing and relates to a method for detecting lactoferrin content, particularly a method for detecting lactoferrin based on electric field-driven cellulose acetate membrane band separation and ratio fluorescence quantitative detection. Background Technology

[0002] Lactoferrin is an iron-binding glycoprotein with a molecular weight of 80 kDa and approximately 700 amino acid residues. It is mainly found in mammalian tissues and exocrine secretions, particularly milk. The lactoferrin content in cow's milk is typically 0.02–0.75 mg / mL. Lactoferrin possesses various biological activities, such as antibacterial, immunomodulatory, and antiviral activity. The national standard GB 14880-2012, "Applicable Standard for Food Fortifiers," allows lactoferrin to be used as a nutrient fortifier, specifying a maximum addition level of 1.0 g / kg for infant formula. Therefore, accurately detecting the lactoferrin content in raw milk and formula milk powder is of great significance. Currently, the main methods for detecting lactoferrin fall into three categories: immunoassay, chromatography, and biological methods. Immunoassay and biological methods suffer from poor reproducibility and unstable results, and require highly skilled operators. For example, antigens, antibodies, and nucleic acid aptamers are expensive, and experimental conditions are demanding. Chromatographic methods are costly, time-consuming, require sophisticated equipment, involve complex pretreatment, and cannot definitively determine the biological activity of lactoferrin. Furthermore, milk contains lactose, vitamins, minerals, and other complex matrices besides protein, which interfere with detection and affect specificity. While the national standard method uses heparin affinity chromatography to purify lactoferrin, heparin is expensive and not reusable, resulting in high costs. Cellulose acetate membranes offer advantages such as minimal resistance to molecular movement and no protein adsorption. This material is environmentally friendly in production and use and is relatively inexpensive. Cellulose acetate membranes are widely used in the separation of biomolecules such as serum proteins, glycoproteins, and lipoproteins. Lanthanide metal ions such as Tb... 3+ and Eu 3+ with Fe 3+Lanthanides, exhibiting similarities in charge and ionic radius, have been found to specifically bind to the two Fe binding sites of iron-free lactoferrin, forming lanthanide-lactoferrin complexes. This "antenna effect" sensitizes lanthanides, significantly enhancing their fluorescence intensity. Carbon dots are fluorescent nanomaterials with excellent optical properties, good biocompatibility, and high water solubility. Their preparation also aligns with green chemistry and sustainable development requirements. Combining carbon dots with lanthanides to construct ratiometric fluorescent probes can greatly improve accuracy and sensitivity. Cellulose acetate films are chemically stable and do not denature lactoferrin, thus preserving its conformation. They can form complexes with carbon dots@lanthanide fluorescent probes loaded on cellulose acetate films, creating a fluorescent band on the film. The fluorescence intensity ratio of this band reflects the content of the target analyte, lactoferrin, enabling rapid and accurate detection of the concentration of active lactoferrin in dairy products. This method, which uses a cellulose acetate membrane as a separation platform and a ratio fluorescence sensing platform, to separate and enrich lactoferrin, then coordinate it with lanthanide metal ions for luminescence, and quantitatively detect lactoferrin based on the ratio of the two fluorescence signals, is unprecedented in reported lactoferrin detection methods and is therefore highly significant. Summary of the Invention

[0003] This invention relates to a method for zone separation and ratio fluorescence quantitative detection of lactoferrin using cellulose acetate membranes driven by an electric field. A method for detecting lactoferrin based on zonal separation and ratiometric fluorescence quantification, involving the preparation method of a carbon dot@lanthanide metal-supported cellulose acetate membrane detection platform, comprises the following steps: Preparation of the carbon dots: Carbon dots are prepared by chemical synthesis. The carbon source, precursor, passivating agent urea (or none) and etchant (formic acid and H2O2) are heated in a high-pressure reactor at 80-600 °C for 120-200 min. After the reaction is completed, the mixture is cooled to room temperature, filtered through a filter membrane, dialyzed through a dialysis bag (300-700 MWCO) for 48-62 h, and then freeze-dried to obtain carbon dot powder. Preparation of the carbon dot@lanthanide metal ion fluorescent probe: The solid powder carbon dots obtained by freeze drying are dissolved in distilled water, and then 200~300 mg of lanthanide metal salt is added. The mixture is magnetically stirred at room temperature for 2~3 h, and then transferred to a dialysis bag for dialysis for 24~36 h to remove free lanthanide ions. The dialysis solution is then freeze-dried to obtain the carbon dot@lanthanide metal ion fluorescent probe. Preparation of the carbon dot@lanthanide metal ion fluorescent probe loaded with cellulose acetate film: Dissolve 10-25 mg of carbon dot@lanthanide metal ion fluorescent probe in 5-20 mL of separation and enrichment buffer, then immerse the dried, uncontaminated, streaked and numbered cellulose acetate film in the solution for 5-20 min. If necessary, sonicate for 1-5 min to ensure complete saturation of the cellulose acetate film, resulting in a uniform color and no spots along the entire film, thus obtaining the prepared carbon dot@lanthanide metal ion fluorescent probe loaded with cellulose acetate film. Preparation of dairy product samples for separation and enrichment using carbon dot@lanthanide metal ion fluorescent probe loaded with cellulose acetate membrane: Accurately transfer 10~50 mL of dairy product sample into a centrifuge at 4~6 ℃ and centrifuge at 10000~12000 r / min for 10~20 min to remove the upper layer of milk fat. Adjust the pH of the lower layer of dilute liquid to 4.6 with hydrochloric acid solution, allow it to stand and precipitate, filter with filter paper to remove casein, and collect the lower clear liquid to obtain the sample solution for separation and enrichment. Preparation of the sample enrichment by carbon dot@lanthanide metal ion fluorescent probe loaded with cellulose acetate membrane: Take out the prepared carbon dot@lanthanide metal ion fluorescent probe loaded with cellulose acetate membrane and place it on a smooth and dry filter paper. Gently dab off the excess liquid on the surface with the smooth and dry filter paper. Then, use a micropipette to transfer 5~10 µL of the sample solution to be tested and evenly spread it on the membrane using the streak method to obtain the carbon dot@lanthanide metal ion fluorescent probe loaded with cellulose acetate membrane after spotting. The procedure for separating and enriching the carbon dot@lanthanide metal ion fluorescent probe-loaded cellulose acetate membrane is as follows: Buffer solutions are poured into two separate buffer tanks. Then, the two ends of the spotted cellulose acetate membrane are placed over the two buffer tanks to form a "bridge," as shown in the attached diagram. Figure 1 As shown, equilibrate for 5-10 min. Set a DC power supply with a voltage of 20-150 V and a time of 20-60 min to begin zone separation. Obtain the separated carbon dots@lanthanide metal ion fluorescent probe-supported cellulose acetate film; The data acquisition and analysis method based on ratiometric fluorescence quantification involves placing a carbon dot@lanthanide metal ion-loaded cellulose acetate membrane after spotting into a fluorescence spectrometer to collect fluorescence intensity and calculate the fluorescence ratio. The lactoferrin content in the dairy sample is then calculated based on a standard curve of lactoferrin content versus fluorescence ratio. This results in a carbon dot@lanthanide metal ion fluorescent probe-loaded cellulose acetate membrane detection platform for lactoferrin content detection, based on separation, enrichment, purification, and ratiometric fluorescence quantification.

[0004] The carbon dots are prepared by one or more of the following methods: hydrothermal method, solvothermal method, biomass method, microwave-assisted method, etc.

[0005] The carbon source is one or more of the following: citric acid, glucose, natural biomass, etc.

[0006] The lanthanide metals mentioned are one or more of europium and terbium.

[0007] The lanthanide metal salts mentioned are one or more of Eu(NO3)3·6H2O, EuCl3·6H2O, Tb(NO3)3·6H2O or TbCl3·6H2O.

[0008] The cellulose acetate film is one or more of the following: acetic acid content of 40-100%.

[0009] The buffer solution is one or more of Tris-HCl buffer solution (pH 7.5-8.5) and Tris-glycine buffer solution (pH 7.5-8.5).

[0010] In the detection platform of this invention, a carbon dot@lanthanide metal-supported cellulose acetate membrane is used as a separation and enrichment support, and also as a ratiometric fluorescence detection platform. Compared with other separation, enrichment and fluorescence methods used for lactoferrin content detection, the prepared carbon dot@lanthanide metal-supported cellulose acetate membrane detection platform has the advantages of effectively eliminating the complex matrix background of dairy products, high sensitivity, good repeatability and high accuracy. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the electric field-driven zone separation device of the present invention.

[0012] 1-Cellulose acetate membrane, 2-Buffer bath, 3-Power supply, 4-Wire Detailed Implementation

[0013] The present invention will now be described with reference to the accompanying drawings and specific embodiments: Example

[0014] The specific steps are as follows: (1) First, lignite carbon dots@terbium ion fluorescent probes were prepared by hydrothermal method. 10 mL of H2O2 and 40 mL of formic acid were mixed and ultrasonicated for 20 min. Then, 0.9 g of lignite was added, and the mixture was shaken in a constant temperature shaker at 80 ℃ for 200 min. After the reaction was completed, the mixture was cooled to room temperature and then filtered with filter paper. The product was transferred to a 500 MWCO dialysis bag and dialyzed at room temperature for 48 @ 62 h. After dialysis, the product was taken out and freeze-dried to obtain solid powder carbon dots. (2) Dissolve the prepared solid powder carbon dots in a small amount of distilled water, then add 200 mg of crystal TbCl3·6H2O, sonicate for 10 min until mixed, then stir magnetically at room temperature for 2-3 h, then transfer to a 500 MWCO dialysis bag and dialyze at room temperature for 24-36 h to remove free terbium ions, and freeze-dry the dialysate to obtain lignite carbon dots@terbium ion fluorescent probe; (3) Then, prepare a cellulose acetate film loaded with lignite carbon dots@terbium ion fluorescent probe. Weigh 25 mg of lignite carbon dots@terbium ion fluorescent probe and dissolve it in 10 mL of Tris-glycine buffer solution (pH=8.3) for separation and enrichment. Sonicate for 5 min until dissolved and mixed. Then, immerse the dried, uncontaminated, streaked and numbered cellulose acetate film in the solution for 5 min and sonicate for 2 min to ensure that the cellulose acetate film is completely saturated and that the entire film has a uniform color and no spots. This is the prepared lignite carbon dots@lanthanide metal ion fluorescent probe loaded cellulose acetate film. (4) After centrifuging fresh milk to remove fat, adjust the pH to 4.6 with 1 mol / L hydrochloric acid solution, allow it to stand at room temperature to separate into layers, and then filter it with filter paper to collect the filtrate. Take 5 µL of the filtrate and the lactoferrin standard solution and spot them onto the prepared lignite carbon dots@lanthanide metal ion fluorescent probe-supported cellulose acetate membrane. Then pour the Tris-glycine buffer solution (pH=8.3) into the two buffer tanks respectively, set the DC power supply to 110 V and separate for 30 min. Then collect the fluorescence intensity on the membrane, calculate the fluorescence ratio, and fit a standard curve based on the concentration of lactoferrin standard solution and the fluorescence ratio. The lactoferrin content in fresh milk can be calculated based on the standard curve.

[0015] The prepared detection platform exhibits high accuracy and a wide linear range (1×10⁻⁶) for the detection of lactoferrin. -5 ~ 4×10 -3 mol / L), low detection limit (1×10⁻⁶ mol / L). -5 The sensor exhibits characteristics of (mol / L). Furthermore, detection results on actual samples (such as fresh milk and formula milk powder) demonstrate that the prepared sensor has excellent practical application value.

[0016] The above embodiments are merely illustrative of the present invention and not intended to limit it. Many improvements and modifications can be made to the present invention based on the above description. Within the scope of the appended claims, the present invention can be implemented in other ways different from those described above; the use of other reagents or materials, or other lanthanide luminescent metal ions, are all within the scope of the claims of this invention.

Claims

1. A method for zone separation and ratiometric fluorescence quantitative detection of lactoferrin using cellulose acetate membranes driven by an electric field, characterized in that, The detection method includes the following steps: (1) Sample pretreatment: Accurately transfer 10~50 mL of dairy product sample into a centrifuge at 4~6 ℃ and centrifuge at 10000~12000 r / min for 10~20 min to remove the upper layer of milk fat. Adjust the pH of the lower layer of dilute liquid to 4.6 with hydrochloric acid solution, let it stand to precipitate, filter with filter paper to remove casein, collect the lower layer of clear liquid, seal and refrigerate for later use. (2) Synthesis of carbon dots: Carbon dots were prepared by hydrothermal method. The carbon source (such as citric acid, glucose, biomass), passivating agent urea (or none), etching agent formic acid and H2O2 were heated in a high-pressure reactor at 80 ℃~200 ℃ for 120~200 min. After the reaction was completed, the mixture was cooled to room temperature, filtered with a filter membrane, and then dialyzed with a dialysis bag (300~700MWCO) for 48~62 h. After freeze drying, carbon dot powder was obtained. (3) Synthesis of carbon dot@lanthanide metal ion fluorescent probe: The solid powder carbon dots obtained by freeze drying are dissolved in distilled water, and then 200~300 mg of lanthanide metal salt is added. The mixture is magnetically stirred at room temperature for 2~3 h, and then transferred to a dialysis bag for dialysis for 24~36 h to remove free lanthanide ions. The dialysis solution is freeze-dried to obtain carbon dot@lanthanide metal ion fluorescent probe. (4) Preparation of carbon dot@lanthanide metal ion fluorescent probe loaded with cellulose acetate film: Dissolve 10~25 mg of carbon dot@lanthanide metal ion fluorescent probe in 5~20 mL of separation and enrichment buffer, and then immerse the dried and uncontaminated cellulose acetate film that has been streaked and numbered in the solution for 5~20 min. If necessary, it can be sonicated for 1~5 min to make the cellulose acetate film completely saturated, and the entire film has a uniform color and no spots. (5) Spotting: Take out the prepared carbon dot@lanthanide metal ion fluorescent probe loaded cellulose acetate film and place it on a smooth and dry filter paper. Use the smooth and dry filter paper to gently remove the excess liquid on the surface. Then, use a micropipette to transfer 5~10 µL of the sample solution to be tested and apply it evenly to the film using the streak method. (5) Electric field driven cellulose acetate membrane zone separation: Pour the buffer solution into two buffer tanks respectively, and then place the two ends of the sample-treated cellulose acetate membrane over the two buffer tanks to form a "bridge". The electric field driven device is shown in Figure 1. Equilibrate for 5~10 min. Turn on the power to start separation; (6) Data acquisition and analysis: After separation, the carbon dot@lanthanide metal ion loaded cellulose acetate film was taken out and immediately placed in a fluorescence spectrometer to collect fluorescence intensity and calculate fluorescence ratio. The content of lactoferrin in the dairy product sample was calculated according to the standard curve of lactoferrin content-fluorescence ratio value.

2. The method for zone separation and ratiometric fluorescence quantitative detection of lactoferrin using cellulose acetate membrane driven by an electric field, as described in claim 1, is characterized in that... The cellulose acetate film described in step (1) has an acetic acid content of 40-100%.

3. The method for zone separation and ratiometric fluorescence quantitative detection of lactoferrin using cellulose acetate membranes driven by an electric field, as described in claim 1, is characterized in that... The lanthanide metal mentioned in step (1) is either europium or terbium.

4. The method for zone separation and ratiometric fluorescence quantitative detection of lactoferrin using cellulose acetate membrane driven by an electric field, as described in claim 1, is characterized in that... The lanthanide metal salt mentioned in step (1) is one or more of Eu(NO3)3·6H2O, EuCl3·6H2O, Tb(NO3)3·6H2O or TbCl3·6H2O.

5. The method for zone separation and ratiometric fluorescence quantitative detection of lactoferrin using cellulose acetate membrane driven by an electric field, as described in claim 1, is characterized in that... The separation and enrichment buffer solution is one or more of Tris-HCl buffer solution and Tris-glycine buffer solution, with a pH of 7.5-8.

5.

6. The method for zone separation and ratiometric fluorescence quantitative detection of lactoferrin using cellulose acetate membrane driven by an electric field, as described in claim 1, is characterized in that... The power supply is a DC power supply with a voltage range of 20~150 V and a duration of 20~60 min.

7. The method for zone separation and ratiometric fluorescence quantitative detection of lactoferrin using cellulose acetate membranes driven by an electric field, as described in claim 1, is characterized in that... The method for establishing the standard curve of fluorescence ratio of carbon dot@lanthanide metal ion-lactoferrin complex includes the following steps: taking lactoferrin standard solutions of different concentration gradients for separation, immediately placing the membrane on a fluorescence spectrometer to collect fluorescence intensity after separation, calculating the ratio of lanthanide metal ion fluorescence intensity to carbon dot fluorescence intensity, and then plotting the standard curve of lactoferrin content-fluorescence ratio.

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