CN113155744B - Method for quantitatively detecting target protein in serum by micro-nano optical fiber coupler label-free biosensing - Google Patents
Method for quantitatively detecting target protein in serum by micro-nano optical fiber coupler label-free biosensing Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
The invention relates to the technical field of biosensing of optical fiber sensors, and discloses a method for quantitatively detecting target protein in serum by using a micro-nano optical fiber coupler without standard biosensing. The invention pre-adsorbs standard serum to the sensor modified by the antibody to be used as an antifouling interface, and takes a linear dynamic detection result under the standard serum environment as a calibration straight line. The effect of reducing the individual differential effect of human serum with standard serum on the non-standard quantitative assay is then proposed. And then taking the offset of the standard serum as reference, obtaining the deviation of the offsets of different human serum and standard human serum, and then carrying out quantitative detection on the target protein in the human serum according to a calibration curve. The quantitative detection result of the OMC is in good agreement with the clinical detection result. The strategy fills the blank of the non-standard quantitative detection of the immunosensor, and further promotes the practicability of the micro-nano optical fiber coupler biosensor.
Description
Technical Field
The invention relates to the technical field of biosensing of optical fiber sensors, in particular to a method for quantitatively detecting target protein in serum by using a micro-nano optical fiber coupler without standard biosensing.
Background
The optical fiber micro-nano coupler (OMC) sensor is widely researched and applied in the field of biosensing due to low cost, simple structure, small electromagnetic interference and high sensitivity. However, when detecting a target protein in serum, nonspecific adsorption often occurs, which increases a background noise signal, hinders detection, and degrades sensor performance. When defects occur in the process of modifying the functions of the surface of the immunosensor or antibodies do not completely cover the surface of the sensor, complex proteins in serum can be adsorbed to blank sites on the surface of the sensor due to hydrophobicity, static electricity and intermolecular exchange interaction and can be combined with groups modified on the surface of the sensor. In these cases, a label-free immunosensor is required to distinguish the response caused by non-specific adsorption from the response caused by binding of the analyte to a particular receptor. In addition, practical application of the biological immunosensor requires accurate quantitative detection of target proteins to be detected in clinical serum samples. However, in the case of non-standard quantity detection, it is necessary to solve the problem of individual difference in human serum. Due to individual differences, different human serum samples are injected into the OMC to reflect different wavelength expressions, the baseline is highly uncertain, and it is difficult to use a certain serum transmission spectrum as a reference, which directly restricts the non-standard accurate quantitative detection of the biosensor on clinical samples.
Gold nanoparticles, magnetic nanoparticles, silica particles, quantum dots, and the like are used to label on the secondary antibody to enhance specific binding signals and improve signal-to-noise ratio, thereby ignoring the effect of non-specific adsorption. However, the preparation, functionalization and sandwich immunoassay of these nanomaterials require additional time, cost and handling procedures. At the same time, the performance reliability of the sensor is also affected by the uncertainty of nanoparticle labeling. The use of anti-non-specific protein adsorption materials as surface coatings is the main strategy for the application of the sensor label-free immunoassay in serum. Polyethylene glycol and oligo-ethylene glycol (PEG and OEG) materials are common choices, but they are easily oxidized in the presence of transition metal ions and oxygen, limiting the long-term application of biosensors. In addition, zwitterionic polymers which have proven to have good antifouling properties are also good candidates. However, the synthesis of such materials suffers from high cost, complex polymerization steps, and the convenience and high control of the synthetic derivative films remains a challenge.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for quantitatively detecting a target protein in serum by a micro-nano fiber coupler without standard biological sensing, so that the method can reduce the influence of individual differences of human serum on the quantitative detection without standard, and ensure more accurate, high-sensitivity and high-specificity subsequent quantitative detection results of the target protein OMC.
In order to achieve the above purpose, the invention provides the following technical scheme:
the preposing method for quantitatively detecting the target protein in the serum by the micro-nano optical fiber coupler without the standard biological sensing comprises the following steps:
selecting one of the measured standard serums with one concentration to carry out OMC sensor detection, recording the offset of the wavelength signal, and recording the offset as A, wherein the offset is the difference between the wavelength signal when the measured standard serum starts to be detected and the wavelength signal when the detection is finished (the detection is carried out twice); selecting the measured healthy serum with one concentration according to the same method to detect the wavelength signal offset, and calculating the offset as B1-BXX is the number of healthy serum sources; comparing offset A and offset B1-BXThe mean and standard deviation of the relative wavelength shift difference of (a);
through the detection in the step, the average value of the relative wavelength deviation difference and a group of detected standard serum concentration a and detected healthy serum concentration b with the minimum standard deviation are selected as the concentrations adopted in the actual detection;
and 4, dissociating and cleaning the surface of the OMC sensor for later use.
In a specific embodiment of the invention, the invention takes sigma human serum as standard serum;
preferably, the active group is selected from carboxyl or aldehyde group; in a specific embodiment of the invention, the invention selects the carboxyl group provided by PAA, to which an antibody to a protein of interest is attached after activation by NHS and EDC; meanwhile, the present invention uses ethanolamine to quench the excessive carboxyl or aldehyde groups, specifically 1M ethanolamine-HCl (pH 8.5).
Preferably, the active groups of step 1 are immobilized on OMC by electrostatic adsorption or covalent bond. In the specific implementation mode of the invention, the invention adopts an electrostatic adsorption mode for immobilization, namely, the OMC surface is negatively charged through potassium hydroxide, then the OMC surface is adsorbed through a positive electrolyte solution of PDDA, and finally the active group-carboxyl is electrostatically adsorbed through a negative electrolyte solution of PAA and provided by the negative electrolyte solution of PAA.
The step 2 of the invention is individual difference correction of human serum, and the offset of standard serum with a certain concentration covers the offset of most clinical serum through the step, so that the individual difference is corrected, and the detection is more accurate. In a specific embodiment of the present invention, the standard serum with 25% and 30% concentration is selected and compared with the wavelength shift of 23 parts of the sample of the serum with 25% concentration, and the result shows that the comparison result of the wavelength shift of 25% standard serum and the wavelength shift of 23 parts of the sample of the serum with 25% concentration in the vicinity of the same wavelength is as follows: the average value of the relative wavelength deviation difference is 0.64nm, and the standard deviation is 0.42 nm; the comparison of the wavelength shift of 30% standard serum with that of 23 samples of healthy human serum with a concentration of 25% showed the following results: the mean value of the relative wavelength shift difference was 0.08nm, and the standard deviation was 0.27 nm. The experimental results show that 30% standard serum is more suitable than 25% standard serum for the detection of clinical serum samples with 25% concentration as a reference to reduce the influence of individual serum difference on the non-standard quantitative detection. Therefore, the invention determines that the sigma human serum is taken as standard serum, the concentration is 30 percent, and the concentration in clinical serum detection is 25 percent. The serum concentration was adjusted by PBS dilution.
In a specific embodiment of the invention, the standard serum concentration of the blocked OMC sensor surface is 50%.
Since one sensor often cannot cover the entire measurement range, OMC immunosensors with different linear dynamic ranges are used in embodiments of the invention to detect target proteins at different concentrations in standard serum to obtain calibration curves. For example, the standard serum provided by the embodiment of the invention has a linear reaction of the target protein and the antibody of the OMC surface-immobilized target protein within the range of 200fg/mL-1pg/mL, within the range of 200pg/mL-800pg/mL, and within the concentration range of 2ng/mL-8 ng/mL; target proteins at different concentrations in standard serum were detected by three OMC immunosensors of different linear dynamic ranges.
By the preposed method, the invention corrects possible nonspecific adsorption and individual difference in the detection process, and can ensure more accurate subsequent detection, high sensitivity and high specificity.
Meanwhile, the invention also provides a method for quantitatively detecting the target protein in the serum by the micro-nano optical fiber coupler without the standard biological sensor, which continuously performs the following steps on the basis of the preposition method:
the detected standard serum with the concentration of a is detected by an OMC sensor, and a wavelength signal f at the beginning of detection is recordeds1And the wavelength signal f at the time of completion of detections2The offset is counted as fs2-fs1；
Adjusting the concentration of the clinical serum to be detected to the concentration b in claim 1, carrying out OMC sensor detection, and recording the wavelength signal f at the beginning of detectionp1And the wavelength signal f at the time of completion of detectionp2；
According to the formula (f)p2-fp1)-(fs2-fs1) Obtaining the wavelength signal of clinical serum to be tested, substitutingAnd obtaining the content of the target protein in the clinical serum to be detected in the calibration curve.
In the specific implementation mode of the invention, CEA is used as an experimental object to perform methodological verification, and excellent consistency is achieved by comparing with a clinical detection result; the pre-method and the subsequent detection method can be used for target protein with non-diagnostic purposes, namely, the quantitative detection of the target protein cannot realize the expected diagnosis and needs to be combined with other detection results for determination.
According to the technical scheme, the invention provides a method for realizing high-sensitivity and high-specificity non-standard quantitative detection of target protein in serum by using a micro-nano optical fiber coupler (OMC) biosensor. The invention pre-adsorbs serum to the sensor modified by the antibody to be used as an antifouling interface, extracts a specific binding signal caused by curve deviation relative to an overlapped blank serum baseline so as to realize linear dynamic detection of the OMC immunosensor with different detection ranges in a standard human serum environment, and takes a linear dynamic detection result in the standard human serum environment as a calibration straight line. It was then proposed to reduce the effect of individual differences in human serum on the non-standard quantitative test with standard human serum. And finally, taking the offset of the standard human serum as a reference, obtaining the deviation of the offset of different clinical human serum to be detected and the standard human serum, and then carrying out quantitative detection on the target protein in the human serum according to a calibration curve. The quantitative detection result of the OMC is in good agreement with the clinical detection result. The strategy fills the blank of the non-standard quantitative detection of the immunosensor, and further promotes the practicability of the micro-nano optical fiber coupler biosensor.
Drawings
FIG. 1 is a diagram of an experimental setup provided in an embodiment of the present invention;
FIG. 2 is a schematic diagram of the immobilization of an antibody on an OMC surface according to an embodiment of the present invention;
FIG. 3 is a diagram of an immunosensor blocking strategy provided in accordance with an embodiment of the present invention; the remaining activated carboxyl groups were quenched by 1M ethanolamine-HCl (pH 8.5) to avoid more non-specific proteins on the modified sensor from binding to the remaining carboxyl groups, and serum pre-adsorbed on functionalized OMCs as an antifouling interface;
FIG. 4 is a comparison of an antigen detection curve (200fg/mL) with a baseline overlapping curve of blank serum provided in an embodiment of the present invention, wherein the signal of the detected sample is much greater than the baseline signal, and the difference in the signals is considered as an antigen-antibody specific binding signal;
FIG. 5 is a linear reaction of 25% sigma human serum CEA with OMC surface immobilized CEA antibody in the range of 200fg/mL-1pg/mL provided by the present invention;
FIG. 6 is a linear reaction of 25% sigma human serum CEA with OMC surface immobilized CEA antibody in the range of 200pg/mL-800pg/mL provided by the present invention;
FIG. 7 is a linear reaction of 25% sigma human serum CEA and OMC surface immobilized CEA antibody in a concentration range of 2ng/mL-8ng/mL provided by an embodiment of the present invention;
FIG. 8 is a diagram showing a specific signal extraction strategy for label-free quantitative detection, where Δ represents the signal value of the binding of the target protein to the antibody, and fs1And fs2The signals represent the wavelength signals of sigma human serum at the start of detection (start of addition of reaction) and at the end of detection (end of reaction). f. ofp1And fp0Respectively represent the wavelength signals of a clinical serum (serum in a healthy state) sample not containing the target protein at the beginning of detection (the beginning of addition of the reaction) and at the end of detection (the end of the reaction), and f is the difference of individual human serump0Has uncertainty; f. ofp2Is a wavelength signal when the detection of a clinical serum sample to be detected (serum in a current state) is finished (after the reaction is finished); f. ofp1Since the signal is a wavelength signal measured at the time of starting addition, it can be regarded as a wavelength signal at the time of starting detection of clinical serum to be tested (or serum in a healthy state) containing no target protein regardless of the state from which the sample is taken, and fp2The wavelength signal value under the current state can be measured by a clinical serum sample to be measured; due to fp0Uncertainty of (c), fail to pass fp2Obtaining the signal value of the combination of the target protein and the antibody, so the invention needs to determine the infinite capacity of the human serum individual difference correction link (step 2)Is close to fp0-fp1＝fs2-fs1The measured standard serum concentration a of the situation and the corresponding clinical measured serum concentration b;
the upper panel of figure 9 is a comparison of the wavelength shift of a second 25% sigma human serum addition to 23 samples of 25% different healthy human serum after blocking with 50% sigma human serum. FIG. 9 lower panel shows a comparison of the wavelength shift of a second 30% sigma human serum addition after blocking with 50% sigma human serum, with 23 samples of 25% different healthy human serum;
FIG. 10 is a graph showing the detection results of 4 serum CEA by OMC immunosensor and clinical controls.
Detailed Description
The embodiment of the invention discloses a method for quantitatively detecting target protein in serum by a micro-nano optical fiber coupler through label-free biosensing, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations of parts, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
The method for quantitative detection of target protein in serum by the micro-nano optical fiber coupler without standard biosensing provided by the invention is further explained below.
Example 1: the method of the invention
(1) Experimental device
A halogen lamp was used as the light source and a microscope objective with a numerical aperture of 0.65 was used to focus the light into the OMC. An optical microfiber coupler is secured in the fluid cell to deliver the sample solution. A spectrometer with a resolution of 0.3nm was used as the detector and experimental data were acquired by a laptop. In order to eliminate the influence of temperature, a biosensing experiment is carried out in a clean room environment, and the temperature of the whole experiment is kept to be 23 +/-0.2 ℃; the experimental setup is schematically shown in FIG. 1.
(2) OMC surface functionalization and detection
1. Washing OMC with ionized water;
2. immersing OMC in 0.1M KOH solution for 10 minutes, and then washing with ionized water;
3. adding a positive electrolyte solution of PDDA (polymer dispersed DA) of 2mg/mL to soak OMC for 30 minutes, and then washing with deionized water to remove excess reagent in the sample cell;
4. adding 2mg/mL PAA negative electrolyte solution to soak OMC for 30 minutes, and then washing with deionized water to remove excess reagent in the sample cell;
5. 1: 1 the mixture soaked the OMC for 30 minutes to activate the carboxyl groups of PAA. Then washing with PBS;
6. and determining the concentration, the fixing time and the fixing times of the antibody through the dynamic response time and the process of the antibody fixing. The first fixation is generally carried out for 2 hours, the second fixation is carried out for 10 minutes to increase the adhesion of the antibody on the surface of the optical fiber, and then the optical fiber is washed by PBS, and the flow chart is shown in FIG. 2;
7. OMC was exposed to 1M ethanolamine-HCl (pH 8.5) for 30 min, then washed with PBS; the schematic view is shown in FIG. 3;
(3) human serum individual difference correction comparison experiment
When the wavelength shift of sigma human serum is taken as a reference to reduce the influence of individual differences of human serum, the shift of sigma human serum from healthy human serum of corresponding concentration should be as small as possible. The effect of different concentrations of sigma human serum on correcting individual differences in 23 25% concentration healthy human serum was therefore examined. After blocking with 50% sigma human serum, sigma human serum was added twice, then the offset of the second sigma human serum was recorded, then 25% concentration healthy human serum was added and its offset was recorded. The wavelength shifts of the added second 25% and 30% sigma human serum were compared to the wavelength shifts of 23 25% concentration healthy human serum samples.
On the same micro-nano fiber coupler, the wavelength shift of 23 parts of a 25% concentration healthy human serum sample was compared with the wavelength shift of a second addition of 25% sigma human serum. As a result, as shown in the upper graph of FIG. 9, the average value of the relative wavelength shift difference was 0.64nm and the standard deviation was 0.42nm around the wavelength of 710 nm. The lower graph of fig. 9 shows the results of comparing the wavelength shift of 30% sigma human serum added a second time with the wavelength shift of 23 samples of healthy human serum at a concentration of 25%, the relative wavelength shift difference around a wavelength of 710nm having an average value of 0.08nm and a standard deviation of 0.27 nm. The experimental result shows that for the detection of clinical serum samples with 25% concentration, 30% sigma human serum is more suitable to be used as reference than 25% sigma human serum so as to reduce the influence of individual difference of serum on the non-standard quantitative detection.
After the concentration is determined, 50% sigma human serum is adopted for sealing for 30 minutes, and then PBS is used for washing the surface of the sensor;
(4) calibration curve
Adding 25% sigma human serum as blank serum, adding CEA with different concentrations in 25% sigma human serum after the blank serum curves are overlapped, and making a calibration curve;
when the wavelength shift of the signal of the test sample from the blank serum signal is greater than 0.6nm, as shown in fig. 4, the signal difference is considered to be an antigen-antibody specific binding signal. In this case, it is considered that the sensor device has little response to any molecule other than the target protein CEA.
Since one sensor often cannot cover the whole measurement range, OMC immunosensors with different linear dynamic ranges are required to detect different concentrations of target protein CEA in 25% sigma human serum, as shown in fig. 5, 6, and 7, to obtain a calibration curve.
(5) Quantitative diagnosis of target protein CEA in serum
1. Dissociation was performed using 0.1M glycine-HCl (pH 2.3).
2. 2 additions of 30% sigma human serum and the wavelength shift of the second 30% sigma human serum, f in FIG. 8, was recordeds2-fs1。
3. Adding the clinical human serum to be tested, and recording the wavelength offset, i.e. f in FIG. 8p2-fp1(ii) a Subtracting fs2-fs1And substituting the signal difference intoThe corresponding concentration is obtained from the calibration curve, and the quantitative detection of the target protein in the serum of different 25% of people is realized.
(6) The detection result of the method is compared with the clinical detection result
The results of the detection of CEA in 4 sera by using the OMC immunosensor of the present invention were compared with the clinical test results, and the results are shown in FIG. 10, which are in good agreement.
The foregoing is only for the purpose of understanding the method of the present invention and the core concept thereof, and it will be understood by those skilled in the art that various changes and modifications may be made without departing from the principle of the invention, and the invention also falls within the scope of the appended claims.
Claims (6)
1. The preposing method for quantitatively detecting the target protein in the serum by the micro-nano optical fiber coupler without the standard biological sensing is characterized by comprising the following steps:
step 1, performing surface functionalization on OMC, fixing an active group capable of being coupled with an antibody of a target protein on the OMC, fixing the antibody on the OMC, and then quenching the residual active group on the surface of the surface functionalized OMC sensor by using a chemical reagent;
step 2, setting measured standard serum with different concentrations, and obtaining the measured health human serum from different human bodies, wherein the measured health human serum from each source is also set with different concentrations; sealing the surface of the OMC sensor by adopting standard serum with the concentration exceeding the concentration of the standard serum to be detected and the concentration of the serum of the healthy person to be detected, and then cleaning the surface of the OMC sensor for later use;
selecting one of the measured standard serum with one concentration to carry out OMC sensor detection, recording the offset of the wavelength signal, and calculating the offset as A, wherein the offset is the difference value of the wavelength signal when the measured standard serum starts to detect and the wavelength signal when the detection is finished; selecting the detected human serum of all the sources with one concentration according to the same method, and calculating the deviation as B1～BXX is the quantity of the source of the human serum of the health; comparing offset A and offset B1～BXRelative to each otherThe mean and standard deviation of the wavelength shift difference;
through the detection in the step, the average value of the relative wavelength deviation difference and a group of detected standard serum concentration a and detected healthy human serum concentration b with the minimum standard deviation are selected as the concentrations adopted in the actual detection;
step 3, cleaning the surface of the OMC sensor, then using the measured standard serum with the concentration b as blank serum to carry out OMC sensor detection, adding target proteins with different concentrations into the measured standard serum with the concentration b after the blank serum curves are superposed, preparing target protein standard detection liquid with gradient concentration to carry out OMC sensor detection, and obtaining a calibration curve;
and 4, dissociating and cleaning the surface of the OMC sensor for later use.
2. The method for preamble according to claim 1, wherein the standard serum is sigma human serum.
3. The method as recited in claim 1, wherein the reactive group in step 1 is a carboxyl group or an aldehyde group.
4. The method as set forth in claim 1, wherein the chemical agent in step 1 is ethanolamine.
5. The method of claim 1, wherein the reactive groups of step 1 are immobilized on the OMC by electrostatic adsorption or covalent bonding.
6. The method for quantitatively detecting the target protein in the serum by the micro-nano optical fiber coupler without the label biosensing is characterized by further comprising the following steps on the basis of the preposing method of any one of claims 1 to 5:
subjecting the standard serum to be measured at the concentration of a according to claim 1 to OMC sensor detection, and recording the wavelength signal f at the beginning of detections1And the wavelength signal f at the time of completion of detections2Deviation fromThe displacement meter is fs2-fs1；
Adjusting the concentration of the clinical serum to be detected to the concentration b in claim 1, carrying out OMC sensor detection, and recording the wavelength signal f at the beginning of detectionp1And the wavelength signal f at the time of completion of detectionp2；
According to the formula (f)p2-fp1)-(fs2-fs1) And obtaining a wavelength signal of the clinical serum to be tested, and substituting the wavelength signal into the calibration curve in claim 1 to obtain the content of the target protein in the clinical serum to be tested.
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Denomination of invention: A Method for Quantitative Detection of Target Proteins in Serum Using Micro-Nano Fiber Optic Coupler without Label Biosensing Effective date of registration: 20230620 Granted publication date: 20220311 Pledgee: Bank of China Limited Dongshan Branch of Guangzhou Pledgor: Guangdong Changguang Zhongke Biotechnology Co.,Ltd. Registration number: Y2023980044836 |