CN113899896A

CN113899896A - Microchip for identifying selenium sugar, qualitative analysis method of selenium sugar and application

Info

Publication number: CN113899896A
Application number: CN202110935655.8A
Authority: CN
Inventors: 邓盛元; 吴希; 徐誉延; 穆瑶; 郭睿; 宋美燕; 任婷蓉; 李臻怡
Original assignee: Nanjing University of Science and Technology
Current assignee: Nanjing University of Science and Technology
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2022-01-07

Abstract

The invention discloses a microchip for identifying selenosaccharides and a qualitative analysis method and application of selenium sugars, and the scientific name of the selenosaccharides is 1β-methylselenide-N-acetyl-D-galactosamine". The invention discloses a method for manufacturing a microchip capable of identifying the monosaccharide molecule: firstly, silanizing a reaction area reserved on a clean glass slide to make the surface load an amine group; The base-N'-(3-dimethylaminopropyl) carbodiimide salt and N-hydroxysuccinimide are activated, and an amide condensation reaction occurs with the aminated solid-phase carrier to obtain a base covered by phenylboronic acid. The sheet is connected by the covalent bond between the boronic acid group and the cis-adjacent diol in the sugar structure to capture the free selenosaccharide in the solution. The invention also discloses a qualitative analysis method of the monosaccharide molecule, which uses lectin and acetylgalactosamine. The affinity recognition of the ligand realizes the firm and specific entrapment of the selenosaccharide in a specific situation, and further labels the fluorescent dye on the lectin to indicate whether the selenosaccharide is contained by microscopic imaging.

Description

Microchip for identifying selenium sugar, qualitative analysis method of selenium sugar and application

Technical Field

The invention belongs to the field of biological detection, and particularly relates to a microchip for identifying selenium sugar, a qualitative analysis method of selenium sugar and application of the microchip.

Background

Point-of-care testing (POCT) is a new method for instantly analyzing and rapidly obtaining results at a sampling site, is the most rapid field of testing medical development at present, and has wide application in the fields of blood sugar testing, blood coagulation testing, pregnancy testing, cardiac marker testing, blood gas electrolytes, infectious disease testing, drug testing, tumor markers and the like.

Selenoglycose (selenosugar) is used as a main urine selenium metabolite in a human body, and a traditional analysis method thereof usually mainly comprises large detection equipment such as High Performance Liquid Chromatography (HPLC), but because the traditional detection has a complex operation flow and high equipment cost, a patient sample is often required to be collected for centralized processing, and the requirement of rapid detection cannot be met. Compared with the prior art, the POCT system has the advantages of being simple in operation, free of site limitation and the like, capable of meeting the diversity of medical requirements and playing a role in primary medical treatment.

Disclosure of Invention

The invention aims to provide a microchip for identifying selenium sugar, a qualitative analysis method of selenium sugar and application, aiming at the defects of the prior art.

The purpose of the invention is realized by the following technical scheme: a microchip for identifying selenium sugar molecules takes a glass slide as a solid phase carrier, and 3-aminopropyl triethoxysilane is used for silanization treatment, so that the surface of the microchip is loaded with amino groups, and a solid-borne environment is provided for phenylboronic acid. EDC and NHS are used as reaction media, so that p-carboxyphenylboronic acid and an aminated solid phase carrier are subjected to an amide condensation reaction, and the phenylboronic acid is modified on a substrate to obtain the microchip for identifying selenium sugar molecules.

Further, the preparation method comprises the following steps:

(1) activating a cleaned glass slide, and silanizing a reserved reaction area of the activated glass slide by using 3-aminopropyltriethoxysilane as a reaction medium and taking absolute ethyl alcohol as the reaction medium to load amino on the surface of the glass slide.

(2) 2- (N-morpholino) ethanesulfonic acid is taken as a buffer solution, N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are sequentially added into a carboxyphenylboronic acid solution, and the mixture is uniformly mixed to activate carboxyl on the carboxyphenylboronic acid.

(3) And (3) taking an amino substrate as a solid phase carrier, dropwise adding the activated p-carboxyphenylboronic acid solution in the step (2) to perform an amide condensation reaction, washing after the reaction is not less than 24 hours, and removing the unreacted reactant completely to obtain the phenylboronic acid-covered substrate.

Further, the selenium sugar molecule is a sugar molecule containing acetylgalactosamine ligand, and the structural formula is as follows:

a qualitative analysis method of selenium sugar comprises the following steps:

the boric acid group is used for being connected with the covalent bond of cis-vicinal diol in a sugar structure, so that free selenium sugar in a solution is captured.

The lectin is used for affinity recognition of the acetylgalactosamine ligand to realize firm and specific entrapment on the selenose in specific situations, a fluorescent dye is further marked on the lectin, and microscopic imaging is used for indicating whether the selenose is contained or not.

Further, the microchip of claim 1 as a boric acid based substrate for the identification of selenium sugars.

A fluorescence immunochromatographic test paper for detecting selenium sugar comprises a sample pad, a combination pad, an NC membrane, a T line, a C line and a water absorption pad. Wherein the sample pad, the combination pad, the NC membrane and the water absorption pad are connected in sequence; the NC film is provided with a T line and a C line, the T line is close to the combination pad, and the C line is close to the water absorption pad; fixing a boric acid group on a T line by adopting bovine serum albumin, fixing D-galactose on a C line by adopting bovine serum albumin, and stacking rhodamine or a fluorescence-labeled lectin on a bonding pad; . Wherein the sample pad is a cellulose membrane.

Further, the using method comprises the following steps:

(1) and dropwise adding the solution to be detected on the sample pad, uniformly soaking the sample pad with the solution to be detected, filtering, laterally flowing onto the combination pad through capillary action, and dissolving the lectin fixed on the combination pad to ensure that the lectin performs specific recognition on the N-acetylgalactosamine.

(2) The solution on the conjugate pad also flows laterally into the NC membrane by capillary action; and the boric acid group on the T line is used for covalently bonding the vicinal diol on the monosaccharide in the solution to be detected through the capillary action of the NC film, so that the target molecule is captured.

(3) The surplus lectin that flows through the T-line is adsorbed on the C-line by affinity recognition of D-galactose, and the surplus lectin that flows through the C-line is absorbed by the absorbent pad.

(4) And detecting the fluorescence intensity of the T line under different selenium sugar molecule concentrations by using a fluorescence detector, and realizing quantitative analysis of the selenium sugar according to a relation between the calibrated selenium sugar concentration x and the fluorescence peak area y of the T line. Wherein, the relation formula for calibrating x and y is specifically as follows: and detecting the fluorescence intensity of the T line under different selenium sugar molecular concentrations by using a fluorescence detector, obtaining the fluorescence peak area y of the T line under different selenium sugar concentrations x by adopting baseline integration, and fitting to obtain an x-y relational expression.

Further, the preparation method comprises the following steps:

(1) the preparation method comprises the following steps of taking 2- (N-morpholino) ethanesulfonic acid as a buffer solution, sequentially adding N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide into a carboxyphenylboronic acid (PBA) solution, uniformly mixing to activate carboxyl on the carboxyphenylboronic acid, adjusting the pH to be neutral, adding Bovine Serum Albumin (BSA) for overnight reaction, and preparing the BSA-PBA solution.

(2) And (2) taking PBS as a buffer solution, sequentially adding N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide into the lactobionic acid (Gal) solution, uniformly mixing to activate carboxyl on the lactobionic acid, adding Bovine Serum Albumin (BSA) to perform overnight reaction, and preparing the BSA-Gal solution.

(3) Placing the NC membrane in a thermostat, pretreating at 45-65% humidity, and carrying out sample application by adopting the BSA-PBA solution prepared in the step (1) to form a T line on the NC membrane; then spotting is carried out by using the BSA-Gal solution prepared in the step (2) above, a C line is formed on the NC membrane, and then drying is carried out in an incubator at 37 ℃.

(4) The conjugate pad was cut to the appropriate width, soaked with rhodamine-labeled GSL I, BSL I lectin probes, removed and placed in a petri dish for drying in a 37 ℃ incubator.

(5) And sequentially assembling the sample pad, the combination pad, the NC membrane and the water absorption pad which are cut into a proper size and processed.

Further, the relation between the selenoglycose concentration x and the fluorescence peak area y of the T-line is-2.693 × (lgx)²+4.252×lgx+14.257。

Further, the device also comprises a bottom plate; the sample pad, the combination pad, the NC membrane and the water absorption pad are all arranged on the bottom plate. The contact surface width of the sample pad and the combination pad is about 1-2 mm; the contact surface width of the combination pad and the NC membrane is about 1-2 mm; the contact surface width of the NC film and the water absorption pad is about 1-2 mm.

The invention has the beneficial effects that:

1. according to the invention, a substrate capable of rapidly and specifically capturing selenium sugar in a complex system is developed according to a bioorthogonal principle, and then the conversion from the abundance of a target object to a visible fluorescent signal is realized by utilizing the immobilization, capture and detection of a probe, so that a microchip model for identifying the selenium sugar is constructed, the detection application of the selenium sugar is expanded, and the simple and accurate analysis of selenium nutrients is realized.

2. The invention can detect trace element selenium in urine metabolite, the method is established on the reaction of sugar and phenylboronic acid, the combination of agglutinin and sugar, the covalent bond connection of boric acid group and cis-vicinal diol in sugar structure and the affinity recognition of agglutinin and acetylgalactosamine ligand, form the sandwich structure of 'phenylboronic acid/selenium sugar/agglutinin'; wherein the lectin is fluorescently labeled, and the concentration of the selenoglycose is converted into a signal of the lectin according to the stoichiometry; the selenium sugar molecule is integrated in a test strip to realize the instant detection of the selenium sugar molecule. Compared with the traditional detection method, the lectin probe adopted by the invention can recognize the acetylgalactosamine ligand in an affinity manner, has higher sensitivity and specificity to the selenium sugar, and can realize firm and specific entrapment to the selenium sugar in specific situations.

3. The invention provides a method for realizing immobilization of boric acid groups by silanization treatment and amide reaction, and compared with other recognition units, the phenylboronic acid has the advantages of low price, good stability, easy reversible regeneration and the like.

Drawings

FIG. 1 is a schematic diagram of a qualitative analysis method for detecting selenium sugar molecules;

FIG. 2 is a schematic structural diagram of an immunochromatographic test strip for detecting selenoglycose molecules;

FIG. 3 is a schematic view of an embodiment of the immunochromatographic test strip of the present invention;

FIG. 4 is a graph of fluorescence intensity of immunochromatographic test paper at different concentrations of selenoglycose molecules; wherein a is a fluorescence micrograph of a C line on the immunochromatographic test paper at a selenium sugar concentration of 0mM, b is a fluorescence micrograph of a T line on the immunochromatographic test paper at a selenium sugar concentration of 0mM, C is a fluorescence micrograph of a C line on the immunochromatographic test paper at a selenium sugar concentration of 0.1mM, d is a fluorescence micrograph of a T line on the immunochromatographic test paper at a selenium sugar concentration of 0.1mM, e is a fluorescence micrograph of a C line on the immunochromatographic test paper at a selenium sugar concentration of 1mM, f is a fluorescence micrograph of a T line on the immunochromatographic test paper at a selenium sugar concentration of 1mM, g is a fluorescence micrograph of a C line on the immunochromatographic test paper at a selenium sugar concentration of 10mM, and h is a fluorescence micrograph of a T line on the immunochromatographic test paper at a selenium sugar concentration of 10 mM;

FIG. 5 is a graph of the fit of the selenoglycose concentration x to the fluorescence peak area y of the T-line;

in the figure: the device comprises a sample pad 1, a combination pad 2, a nitrocellulose membrane 3, a detection line 4, a control line 5, a water absorption pad 6 and a bottom plate 7.

Detailed Description

The invention relates to a preparation method of a microchip for identifying selenium sugar, which comprises the following specific steps:

(1) the slide glass is taken as a solid phase carrier, and silanization treatment is firstly carried out on a reaction area reserved on the clean slide glass, so that the surface of the slide glass is loaded with amidogen.

Taking a cleaned glass slide, soaking the glass slide in Piranha solution at 90 ℃ for 2 hours to load hydroxyl on the surface of the glass slide, and silanizing a reserved reaction area of the activated glass slide by using 3-aminopropyltriethoxysilane (3-aminopropyltriethoxysilane, APTES) by using absolute ethyl alcohol as a reaction medium to load amino on the surface of the glass slide to provide a solid loading environment for phenylboronic acid. The reaction process is as follows:

(2) the carboxyl group of the carboxylated phenylboronic acid is then activated with N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide salt and N-hydroxysuccinimide.

2- (N-morpholino) ethanesulfonic acid (4-morpholino sulfonic acid hydrochloride, MES) is used as a buffer solution, N-Ethyl-N '- (3-Dimethylaminopropyl) Carbodiimide hydrochloride (N-Ethyl-N' - (3-dimethylamino propyl) Carbodiimide hydrochloride, EDC) and N-HydroxySuccinimide (N-HydroxySuccinimide, NHS) are sequentially added into a carboxyphenylboronic acid solution, the mixture is uniformly mixed, and carboxyl on the carboxyphenylboronic acid is activated, wherein the molar ratio of EDC to NHS is 1: 4. the reaction process is as follows:

(3) the activated p-carboxyl phenylboronic acid solution and the aminated solid phase carrier are subjected to amide condensation reaction to obtain the substrate covered by the phenylboronic acid.

And (3) taking an amino substrate as a solid phase carrier, dropwise adding the activated p-carboxyphenylboronic acid solution in the step (2) to ensure that amino on the aminated solid phase carrier and carboxyl on the p-carboxyphenylboronic acid are subjected to an amide condensation reaction, washing for many times after the reaction is carried out for 24 hours, and removing the unreacted reactant completely to obtain the phenylboronic acid-covered substrate which is the microchip for identifying the selenium sugar. The reaction process is as follows:

the chemical name of the selenose is 1 beta-methyl selenium-N-acetyl-D-galactosamine, which is a selenium metabolism marker with predominant abundance in human urine and has the chemical formula C₉H₁₈O₅NSe, is a sugar molecule containing acetylgalactosamine ligand, and has the following structural formula:

the invention relates to a qualitative analysis method of selenium sugar, which specifically comprises the following steps:

(1) the microchip for identifying selenoglycose is linked to the sugar structure by covalent bond of a boronic acid group and a cis-vicinal diol, and captures free selenoglycose in a solution. The method specifically comprises the following steps:

the phenylboronic acid substrate (microchip for identifying selenose) is used as a substrate, the characteristic that boric acid groups can form reversible covalent bonds with 1, 2-and 1, 3-diols in a sugar structure is utilized, cyclic borate ester is formed by combining, washing is carried out for multiple times after reaction for 1h, sugar molecules which are not completely reacted are removed, and the phenylboronic acid substrate is used as a molecular probe of a biosensor to identify carbohydrate compounds, so that free selenose in a solution is captured, and the selenium sugar molecules are identified and captured on the substrate. The reaction process is as follows:

(2) the strong and specific entrapment of selenium sugar in a urine matrix is realized by using the affinity recognition of rhodamine or fluorescence labeled lectin and acetylgalactosamine ligand (acetylgalactosamine structural fragment), washing is carried out for a plurality of times after the reaction is carried out for 1h, the lectin probe which is not completely reacted is removed, and whether the selenium sugar is contained or not is indicated by the fluorescence intensity of a fluorescent substance of microscopic imaging. As can be seen from FIG. 1, the boronic acid substrate realizes the capture of the target molecule, but since the urine environment contains a plurality of interfering sugar molecules, such as glucose, etc., the qualitative analysis of the target molecule is realized through the affinity recognition of the fluorescence-labeled lectin to acetylgalactosamine. The lectin labeled with rhodamine or fluorescence may be rhodamine-labeled GSL I, BSL I, rhodamine-labeled DBA (lentil lectin), or fluorescein-labeled SBA (soybean lectin).

As shown in FIG. 2, the fluorescence immunochromatographic test paper for detecting selenoglycose of the present invention mainly comprises a sample pad 1 (cellulose membrane), a binding pad 2, a nitrocellulose membrane 3(NC membrane), a detection line 4(T line), a control line 5(C line), a water absorption pad 6 and a bottom plate 7. Wherein, the sample pad 1, the combination pad 2, the NC membrane and the water absorption pad 6 are connected in sequence and are all arranged on the bottom plate 7; the contact surfaces of the sample pad 1 and the combination pad 2, the combination pad 2 and the NC membrane, and the NC membrane and the water absorption pad 6 are about 1-2 mm wide; the NC film is provided with a T line and a C line, the T line is close to the combining pad 2, and the C line is close to the water absorption pad 6; the bovine serum albumin is used to fix the boric acid group in the microchip on the T line, the bovine serum albumin is used to fix the D-galactose on the C line, and the excess rhodamine or the fluorescence labeled lectin is deposited on the binding pad 2.

The invention relates to a method for using a fluorescence immunochromatographic test paper for detecting selenoglycose, which comprises the following steps:

(1) and dropwise adding the solution to be detected on the sample pad 1, uniformly soaking the sample pad 1 with the solution to be detected, filtering, laterally flowing onto the binding pad 2 through capillary action, and dissolving the lectin fixed on the binding pad 2, so that the lectin performs specific recognition on the N-acetylgalactosamine (the part of the 1 beta-methylselenium-N-acetyl-D-galactosamine bound with the lectin), and the conversion of the abundance of the target molecule to the visible fluorescent signal is realized.

(2) The solution on the conjugate pad 2 also flows laterally into the NC membrane by capillary action; and the boric acid group on the T line is used for covalently bonding the vicinal diol on the monosaccharide in the solution to be detected through the capillary action of the NC film, so that the target molecule is captured.

(3) The surplus lectin that flows through the T-line is adsorbed on the C-line by affinity recognition of D-galactose, and the surplus lectin that flows through the C-line is absorbed by the absorbent pad 6.

(4) The fluorescence intensity of the T line under different selenium sugar molecule concentrations is detected by a fluorescence detector, the fluorescence peak area (y) of the T line under different selenium sugar concentrations (x) is obtained by adopting baseline integration, and an x-y relational expression is obtained by fitting, so that the quantitative analysis of the target molecule is realized.

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments.

Example 1

(1) To 10ml of 100mM 2- (N-morpholino) ethanesulfonic acid buffer solution (pH6.0), 16.63mg of p-carboxyphenylboronic acid (PBA), 807.2mg of N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride and 117.4mg of N-hydroxysuccinimide were sequentially added, reacted at room temperature for 2 hours, and after adjusting the pH to 7.4, 70mg of Bovine Serum Albumin (BSA) was added for overnight reaction to prepare a BSA-PBA solution.

(2) 37.0mg of lactobionic acid (Gal), 807.2mg of N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride and 117.4mg of N-hydroxysuccinimide are sequentially added to 5ml of 0.01M PBS buffer solution (pH 7.2-7.4), and after reacting for 2 hours at room temperature, 70mg of Bovine Serum Albumin (BSA) is added for overnight reaction to prepare a BSA-Gal solution.

(3) Pre-treating the NC membrane in a 37 ℃ thermostat at 45% -65% humidity for 10min, carrying out spotting by adopting 10 microliter 10mM BSA-PBA solution prepared in the step (1), and forming a T line on the NC membrane at the speed of 1.5 microliter/cm; then, spotting was performed using 10. mu.l of 20mM BSA-Gal solution prepared in the above step (2) to form a C line on an NC membrane at a rate of 1.0. mu.l/cm, followed by drying in an incubator at 37 ℃ for 2 hours.

(4) The conjugate pad was cut to the appropriate width and probed with 100. mu.g/ml rhodamine-labeled GSL I, BSL I lectin at 50. mu.l/cm²The amount of the composition is soaked for 10min, taken out and placed in a culture dish, and dried in a thermostat at 37 ℃ for 2 h.

(5) The sample pad, the conjugate pad, the NC film, and the absorbent pad cut to an appropriate size and processed were sequentially assembled on a base plate, and cut into test paper having a width of 4mm, as shown in fig. 3.

Example 2

PBS is used as a buffer solution, 0mM selenose solution is prepared as a sample solution, 100 μ l of the solution is sequentially dripped on a sample pad of the test paper prepared in example 1, is identified by a lectin probe on the binding pad, and forms a T line and a C line on an NC membrane through capillary action, wherein a boric acid group on the T line is bound with the selenose through a covalent bond to form a sandwich structure with the lectin probe, galactose on the C line can be bound with redundant lectin probes (rhodamine-labeled GSL I, BSL I), and residual liquid is absorbed by a water absorption pad. The fluorescence intensity was measured by a fluorescence microscope, and the results are shown in fig. 4 a and b. Fig. 4b is a fluorescence micrograph of a T-line, in which a boronic acid group is used to capture selenoglycose, and the left part is a T-line part which is clearly brighter than the right part without the boronic acid group immobilized (i.e. the part without spots on the NC film), and there is a clear light-dark boundary between the two parts.

Example 3

Example 3 the only difference from example 2 was that the concentration of the selenoglycose solution was 0.1mM, otherwise the same as example 2, and the results are shown in c, d of FIG. 4.

Example 4

Example 4 the only difference from example 2 was that the concentration of the selenoglycose solution was 1mM, the results are shown in e, f of FIG. 4, which is otherwise the same as example 2.

Example 5

Example 5 the only difference from example 2 was that the concentration of the selenoglycose solution was 10mM, the results are shown in g, h of FIG. 4, which is otherwise the same as example 2.

FIG. 4 is a graph of fluorescence intensity of the immunochromatographic test strip prepared in example 1, measured by a fluorescence microscope at different concentrations of selenoglycose molecules. From FIG. 4, it can be seen that as the selenose concentration increases, the fluorescence intensity on the C line decreases, while the fluorescence intensity on the T line increases, indicating that the selenose concentration is proportional to the fluorescence intensity.

Example 6

Example 6 is the same as example 2 except that the concentrations of the selenoglycose solution were 0.1, 1 and 10. mu.M, and the fluorescence intensities on the C line and the T line were obtained by a fluorescence detector, and the results are shown in FIG. 5.

FIG. 5 shows the fluorescence intensity of the immunochromatographic test strip prepared in example 1 at different concentrations of selenoglycose molecules detected by a fluorescence detector, and the area (y) of the fluorescence peak of the T-line at different concentrations (x) of selenoglycose was obtained by baseline integration. From FIG. 5, it can be seen that the fluorescence intensity on the T line increases with the increase of the selenose concentration, indicating that the selenose concentration is proportional to the fluorescence intensity, and the relationship of the fitted curve is-2.693 × (lgx)²+4.252 × lgx +14.257, standard deviation R²1.000; wherein y has a unit of 10⁶U., x is in μ M.

Claims

1. a microchip for identifying selenosaccharide molecule, is characterized in that, with glass slide as solid phase carrier, adopt 3-aminopropyl triethoxysilane to do silanization treatment, make its surface load amine group, Provides an immobilization environment for phenylboronic acid. Using EDC and NHS as the reaction medium, the amide condensation reaction between p-carboxyphenylboronic acid and aminated solid phase carrier is carried out, so that the phenylboronic acid is modified on the substrate to obtain a microchip for identifying selenosaccharide molecules.

2. The microchip for identifying selenosaccharide molecules as claimed in claim 1, wherein the preparation method comprises the steps:

(1) Take a cleaned glass slide for activation, and use absolute ethanol as the reaction medium, and use 3-aminopropyltriethoxysilane to silanize the reaction area reserved for the activated slide glass, The surface is loaded with amine groups.

(2) Using 2-(N-morpholino)ethanesulfonic acid as a buffer solution, sequentially adding N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide to the carboxyphenylboronic acid solution Amine hydrochloride and N-hydroxysuccinimide are mixed evenly to activate the carboxyl group on carboxyphenylboronic acid.

(3) Using the amine-based substrate as a solid-phase carrier, dropwise addition of the activated p-carboxyphenylboronic acid solution in step (2) to generate an amide condensation reaction, and washing after the reaction for ≥ 24 hours to remove unreacted reactants to obtain Phenylboronic acid coated substrate.

3. The microchip for identifying selenosugar molecules as claimed in claim 1, wherein the selenosugar molecules are a kind of sugar molecules containing acetylgalactosamine ligand, and the structural formula is as follows:

4. a qualitative analysis method of selenium sugar, is characterized in that, comprises:

The free selenosaccharide in solution is captured by the covalent bond of the boronic acid group to the cis-adjacent diol in the sugar structure.

Using the affinity recognition of lectin and acetylgalactosamine ligand, to achieve firm and specific entrapment of selenosaccharide in a specific situation, further label the lectin with fluorescent dye, and indicate whether it contains selenosaccharide through microscopic imaging.

5 . The qualitative analysis method of selenosaccharides according to claim 4 , wherein the microchip according to claim 1 is used as a boronic acid-based substrate for identification of selenosaccharides. 6 .

6. A fluorescent immunochromatographic test paper for detecting selenosaccharides, characterized in that it comprises a sample pad, a binding pad, an NC membrane, a T line, a C line, and a water absorption pad. Among them, the sample pad, the binding pad, the NC membrane and the absorbent pad are connected in sequence; the NC membrane is provided with a T line and a C line, the T line is close to the binding pad, and the C line is close to the absorbent pad; bovine serum albumin is used to fix the boronic acid group on the T line. On the line, D-galactose was immobilized on the C line with bovine serum albumin, and rhodamine or fluorescently labeled lectin was stacked on the binding pad; wherein, the sample pad was a cellulose membrane.

7. the fluorescence immunochromatographic test paper for detecting selenosaccharide as claimed in claim 6, is characterized in that, using method comprises the steps:

(1) Drop the solution to be tested on the sample pad, the solution to be tested evenly permeates the sample pad and is filtered, and flows laterally onto the binding pad through capillary action, dissolving the lectin fixed on the binding pad, so that the lectin can react to N-acetyl Galactosamine for specific recognition.

(2) The solution on the binding pad also flows laterally into the NC membrane through capillary action; the boronic acid group on the T line is covalently bound to the vicinal diol on the monosaccharide in the solution to be tested through the capillary action of the NC membrane to achieve the goal. Molecular capture.

(3) The excess lectin flowing through the T line is adsorbed on the C line by affinity recognition of D-galactose, and the excess lectin flowing through the C line is absorbed by the absorbent pad.

(4) Detecting the fluorescence intensity of T line under different seleno sugar molecule concentrations by a fluorescence detector, and realizing the quantitative analysis of seleno sugar according to the relationship between the calibrated seleno sugar concentration x and the fluorescence peak area y of the T line. Among them, the relationship between the calibration x and y is specifically: use a fluorescence detector to detect the fluorescence intensity of the T line under different selenium sugar molecule concentrations, use the baseline integration to obtain the fluorescence peak area y of the T line under different selenium sugar concentrations x, and fit the Get the x-y relation.

8. the fluorescence immunochromatographic test paper for detecting selenosaccharide as claimed in claim 6, is characterized in that, the preparation method comprises the steps:

(1) Using 2-(N-morpholino)ethanesulfonic acid as a buffer solution, N-ethyl-N'-(3-dimethylaminopropyl) was added to the carboxyphenylboronic acid (PBA) solution in turn Carbodiimide hydrochloride and N-hydroxysuccinimide are mixed evenly to activate the carboxyl group on carboxyphenylboronic acid, adjust the pH to neutrality, and then add bovine serum albumin (BSA) to react overnight to prepare BSA-PBA solution.

(2) Using PBS as a buffer solution, add N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxybutyl to the lactobionic acid (Gal) solution in turn The diimide was mixed evenly to activate the carboxyl group on lactobionic acid, and bovine serum albumin (BSA) was added for overnight reaction to prepare a BSA-Gal solution.

(3) The NC film is placed in a constant temperature box for pretreatment under a humidity of 45%-65%, and the BSA-PBA solution prepared in the above step (1) is used for spotting to form a T line on the NC film; Spotting the BSA-Gal solution prepared in the above step (2), forming a C line on the NC film, and then placing it in a 37° C. incubator for drying.

(4) The binding pad is cut to a suitable width, soaked with rhodamine-labeled GSLI and BSLI lectin probes, taken out and placed in a petri dish and dried in a 37°C incubator.

(5) The sample pad, the binding pad, the NC film and the water-absorbing pad, which are cut into a suitable size and processed, are assembled in sequence.

9. The fluorescence immunochromatographic test paper for detecting selenosaccharides as claimed in claim 8, wherein the relational formula between the selenium sugar concentration x and the fluorescence peak area y of the T line is y=-2.693×(lgx) ² + 4.252×lgx+14.257.

10 . The fluorescent immunochromatographic test paper for detecting selenosaccharides according to claim 6 , further comprising a bottom plate; the sample pad, the binding pad, the NC film and the water absorption pad are all arranged on the bottom plate. 11 . The width of the contact surface between the sample pad and the binding pad is about 1-2 mm; the width of the contact surface between the binding pad and the NC film is about 1-2 mm; the width of the contact surface between the NC film and the absorbent pad is about 1-2 mm.