CN111273027A

CN111273027A - SPDP modified lectin chip for detecting sugar chains in liver cancer serum as well as preparation and application of SPDP modified lectin chip

Info

Publication number: CN111273027A
Application number: CN202010110912.XA
Authority: CN
Inventors: 杜卫东; 刘胜胜
Original assignee: Anhui Medical University
Current assignee: Anhui Medical University
Priority date: 2020-02-24
Filing date: 2020-02-24
Publication date: 2020-06-12
Anticipated expiration: 2040-02-24
Also published as: CN111273027B

Abstract

The invention discloses an SPDP modified lectin chip for detecting sugar chains in liver cancer serum and preparation and application thereof. The lectin chip can accurately detect at least fifteen sugar chains in the serum of a liver cancer patient, has the advantages of high flux, high selectivity, high specificity and the like, and is more suitable for the research of the characteristics of the human tumor glycosyl spectrum.

Description

SPDP modified lectin chip for detecting sugar chains in liver cancer serum as well as preparation and application of SPDP modified lectin chip

Technical Field

The invention belongs to the technical field of biomedical detection, and particularly relates to a lectin chip for detecting fifteen sugar chains in serum of a liver cancer patient modified by 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester (SPDP), and preparation and application thereof.

Background

In recent years, many studies indicate that sugar chain structures on proteins secreted by malignant or diseased tissues and cells are often obviously different from sugar chains on proteins secreted by normal tissue cells, in fact, it has been observed that serum of hepatocellular carcinoma patients contains a large number of sugar chain structures which cannot be observed in normal mature hepatocytes, and the activity of glycosyltransferase synthesizing a specific sugar chain structure is increased or decreased, for example, liver cancer marker-AFP has a much higher fucose index than that of benign liver disease, and AFP produced by liver cancer contains much more than α → 6, because of the difference in reactivity with lentil Lectin (LCA), it can be classified into AFP-1, AFP-2 and AFP-36L 3982, wherein AFP-36L is derived from the clinical diagnosis of hepatocellular carcinoma, and AFP-4634 is derived from the clinical diagnosis of hepatocellular carcinoma, and liver cancer derived from liver cancer cells, and the research results of liver cancer diagnosis of liver cancer derived from the theoretical liver cancer receptor of pregnant women are considered as a diagnostic marker of liver cancer.

However, the complexity and diversity of sugar chains pose a great challenge to related structure and function studies, and the development of sugar science is far behind genomics and proteomics. In order to research the glycosyl change of important biomolecule markers in physiological and pathological states, a series of glycosylation research technologies including mass spectrometry, capillary electrophoresis, lectin chips and other mainstream methods exist, but mass spectrometry and capillary electrophoresis detection systems have high technical requirements, are complex to operate and have expensive reagents, so that the popularization and application of the mass spectrometry and capillary electrophoresis detection systems are limited.

Lectin (Lectin) refers to a glycoprotein or a sugar-binding protein purified from various plants, invertebrates and higher animals, which has a high specific binding to a carbohydrate on a glycoprotein because it can agglutinate erythrocytes (including blood group-containing substances), and is often used in laboratories for the isolation and purification of glycoproteins. Thus, lectins can be used as a probe to study specific sugar groups on cell membranes or glycoproteins. Compared with other traditional methods, the lectin chip has the advantages of high flux, high selectivity, high specificity and the like, and is suitable for researching the characteristics of the glycosyl spectrum of the liver cancer. The lectin chip is mainly applied to various diseases such as gastric cancer, endometrial cancer, ovarian cancer, breast cancer, liver cancer, prostatic cancer, pancreatic cancer, lung cancer, reproductive medicine and the like at present. Although the lectin chip is used for researching the existence of the sugar chains in the serum of a liver cancer patient at present and finding that the content of some glycoproteins is obviously higher than that of normal people, on one hand, the research is almost limited to the research on the content of common glycoproteins containing fucose, sialic acid and the like; on the other hand, most of the above research specimens are cells cultured in vitro or general specimens excised by surgery of patients, so that the research specimens cannot be applied to high-throughput screening of people, and an effective means for disease progress, curative effect evaluation and prognosis judgment of liver cancer patients is lacked. In addition, commercial lectin chips are often used in sugar chain research in the past, and glass or gel is used as a coating carrier, so that lectin probes are easy to elute, the geometrical pictures of the chips are poor, and the sensitivity is low. Therefore, the development of a method with stable performance, high throughput, high sensitivity, high specificity and better adaptability to the characteristics of the glycospectrum of liver cancer is urgently needed.

Disclosure of Invention

The invention aims to provide a combined lectin chip for detecting the serum sugar chain level of a common human population and a liver cancer patient, and a preparation method and a use method thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the invention firstly discloses an SPDP modified lectin chip for detecting sugar chains in serum, which is characterized in that: fifteen specific lectin probes are fixed on a surface lattice of a solid phase carrier of the lectin chip, and combined detection of at least fifteen sugar chains in human serum is realized by coating fifteen specific lectins;

the solid phase carrier is a gold foil chip chemically modified by SPDP, and immobilized specific agglutinin is used as a probe;

the fifteen specific lectin probes were AAL, LTL, UEA-1, LCA, JAC, RCA-I, VVL, ConA, NPL, DSA, WGA, PNA, MAL-I, SNA, PHA-L, respectively, and Table 1 is a table showing the control of each lectin and its binding specific sugar chain.

TABLE 1

The preparation method of the lectin chip comprises the following steps:

step 1, carrying out chemical modification on the gold foil chip to obtain the solid phase carrier.

Taking a DMSO solution with the concentration of 0.1mg/mL SPDP as a modification solution;

cleaning the gold foil chip, and drying the gold foil chip by nitrogen; spotting 1 mu L of modification solution on each hole of a dried gold foil chip, then placing the gold foil chip in a dried incubation box for incubation for 6 hours at room temperature, taking out the gold foil chip, cleaning the gold foil chip by using a DMSO solution, and drying the gold foil chip by using nitrogen to finish modification, thereby obtaining a solid phase carrier for later use;

step 2, fixing fifteen specific lectins as probes

Weighing 0.2383g of HEPES powder and 0.0011g of anhydrous calcium chloride, dissolving in 100mL of pure water, and adjusting the pH value to 8.5 to obtain HEPES buffer solution; then dissolving fifteen kinds of lectins in the HEPES buffer solution respectively, and enabling the final concentration of each lectin to be 1mg/mL to obtain corresponding lectin solutions; BSA was added to each lectin solution to make the BSA concentration by mass 0.001% for blocking unbound-NHS groups;

and (2) spotting each lectin solution on the solid phase carrier obtained in the step (1), spotting one lectin solution in each of the 1 st to 15 th wells, spotting a PBST-BSA solution in the 16 th well as a blank control, wherein each well is 1 mu L, then placing the mixture in a wet box, incubating the mixture for 2 hours at room temperature, taking the mixture out, washing the mixture with a PBST buffer solution, and drying the mixture with nitrogen to obtain the lectin chip for the combined detection of at least fifteen sugar chains in human serum.

Specifically, the method for cleaning the gold foil chip in the step 1 comprises the following steps: reacting NH₃、H₂O₂And H₂O is mixed according to the volume ratio of 1: 1: 5 mixing to form TL1 cleaning solution, immersing the gold foil chip in a stainless steel cleaning box containing TL1 cleaning solution, and adding 82 ℃ waterBathing for 6 minutes, taking out, washing with ultrapure water, cleaning with absolute ethyl alcohol, and drying with nitrogen.

The invention also discloses a using method of the lectin chip, which comprises the following steps:

step 1, removing high-abundance protein in serum

Taking 33 mu L of serum to be detected, diluting the serum to be detected to 100 mu L by using PBS (phosphate buffer solution) with the concentration of 0.01M, pH ═ 7.4, and then removing albumin and globulin in the serum to be detected through a chromatographic column to obtain deproteinized serum to be detected; determining the protein concentration C of the deproteinized serum to be detected in a unit of mg/mL by using a BCA protein concentration determination kit;

step 2, fluorescent label detection

Uniformly mixing 20 mu L of the serum to be detected treated in the step 1, a Cy3 solution with the volume of V, 80 mu L of PBS buffer solution with the concentration of 0.01M, pH-7.4 and 100 mu L of 0.1M sodium bicarbonate buffer solution, and then incubating for 1h at room temperature in a dark place to finish the Cy3 fluorescent labeling of the serum to be detected; removing redundant Cy3 in the serum to be detected after the fluorescent labeling by using a G-25 chromatographic column to finish the pretreatment of the serum to be detected;

the volume V of the Cy3 solution satisfied formula (1) in mL:

in the formula, M₁Molecular weight, M, of fluorescent marker Cy3₂Is the protein molecular weight, M₁＝829.03、M₂＝65000；

Spotting the pretreated serum to be detected into holes of the lectin chip, which contain specific lectin probes and PBST-BSA solution used as a blank control, wherein each hole is 1 mu L, then placing the holes into a wet box, incubating the holes for 1h at room temperature, washing the holes by PBST buffer solution, and detecting the holes by using a chip instrument;

the serum of the healthy human is pretreated and detected in the same way as the serum of the liver cancer patient to be detected, and is used as a negative control.

The invention further discloses a kit for combined detection of at least fifteen sugar chains in human serum, which is characterized in that the kit comprises: the lectin chip described above; PBS buffer at a concentration of 0.01M, pH ═ 7.4; PBST buffer; 0.1M sodium bicarbonate buffer; cy3 solution; a chromatographic column for removing high-abundance protein in serum; BCA protein concentration determination kit; g-25 chromatographic column;

specifically, the method comprises the following steps: the PBST buffer solution is prepared by mixing PBS buffer solution with the concentration of 0.01M, pH-7.4 and Tween20, and the volume concentration of Tween20 in the PBST buffer solution is 0.1%; the 0.1M sodium bicarbonate buffer solution is prepared by dissolving 1.06g of sodium carbonate and 0.84g of sodium bicarbonate in 100mL of deionized water, and the pH value is adjusted to 8.5 before use; the Cy3 solution was obtained from 1mg Cy3 NHS dissolved in 50. mu.L DMSO; the PBST-BSA solution is formed by mixing PBS buffer solution with the concentration of 0.01M, pH-7.4, Tween20 and fetal bovine serum BSA, wherein the volume concentration of Tween20 in the PBST-BSA solution is 0.1%, and the mass concentration of the fetal bovine serum BSA is 0.1%.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention prepares SPDP novel chemical modified gold foil chip as a substrate. The combination of the gold foil and the chemical substances is firmer than that of the traditional glass sheet and silicon wafer, the glass or gel is used as a coating carrier, the lectin probe is easy to elute, the geometric picture of the chip is not good, and the sensitivity is lower. The inert gold foil has low biological affinity and is not easy to generate nonspecific adsorption with substances such as genes or proteins. Meanwhile, SPDP is a novel chemical modification method, and has never been reported in the use of a chip combined with lectin, the modification can be directly combined with lectin by utilizing terminal-NHS ester, and the method is simple to operate, stable in combination and high in sensitivity.

2. The present invention simultaneously detects fifteen kinds of lectin-bound sugar chains in serum of a liver cancer patient under substantially the same conditions.

3. The present invention uses a 2 x 96(8 rows and 12 columns) well chip format, allowing simultaneous detection of multiple samples (multiple replicates, or samples taken at different time points to obtain dynamic values, or different samples each). The lectin chip disclosed by the invention adopts a novel lectin chip combination to realize high-flux detection, so that the detection cost is reduced and the detection efficiency is improved on the whole.

Drawings

FIG. 1 is a schematic diagram of gold foil chip spotting arrangement;

FIG. 2 is a planar representation of a gold foil chip before and after chemical modification by atomic force microscope scanning, wherein (a) is the gold foil chip before modification; (b) the modified gold foil chip;

FIG. 3 is a graph showing the effect of the concentration of SPDP surface chemical modification on the fluorescence intensity of human IgG, where: (a) carrying out surface chemical modification on SPDP with different concentrations to obtain IgG fluorescence scanning images; (b) obtaining a fluorescence intensity curve graph;

FIG. 4 is a graph of the effect of SPDP surface chemistry modification on fluorescence intensity at different temperatures and times, where: (a) scanning fluorescence intensity of surface chemical modification temperature and time for different SPDPs; (b) obtaining a fluorescence intensity curve graph;

FIG. 5 is a quality control experiment of SPDP modified chip protein IgG coating detection line, in which: (a) detecting a physical map for the chip; (b) a chip detection curve graph is obtained;

FIG. 6 is a SPDP modified chip assay for the gradient quality control of the concentration of Cy 3-labeled IgG, in which: (a) detecting a physical map for the chip; (b) a chip detection curve graph;

FIG. 7 shows the lectin Con A immunospecific quality control, in which: (a) fluorescence scans obtained for immunospecific detection of different concentrations of Con A and different concentrations of anti-Con A; (b) obtaining a fluorescence intensity curve graph;

fig. 8 is a lectin Con a incubation temperature-time quality control experiment, in which: (a) fluorescence intensity profiles for different Con A incubation temperatures versus time; (b) obtaining a fluorescence intensity curve graph;

FIG. 9 shows the results of sugar chain detection in serum of liver cancer patients with lectin combination chips, wherein: (a) fluorescence intensity scans of 5 liver cancer patients and 1 normal control; (b) the obtained 10 liver cancer patients and 10 normal control fluorescence intensity mean value histograms are obtained.

Detailed Description

The following embodiments of the present invention will be described in detail with reference to the accompanying drawings, which are provided for implementing the technical solution of the present invention, and provide detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following embodiments.

The sources and preparations of the materials and reagents used in the following examples are as follows:

1. gold foil chip

A gold foil chip from Ulm Interactiva, Germany is used as a base plane, the chip is made of a glass plate as a substrate, the surface of the chip is covered with a layer of pure gold (purity 99.9%) with the thickness of 0.1 μm, and a TEFLON membrane array (96 holes multiplied by 2, 8 rows multiplied by 12 columns) with the thickness of 50 μm is covered on the chip, the aperture of the array is 1.25mm, and the maximum solvent per hole can be 1 μ L.

2. Surface chemical modification

SPDP is available from Macrocyclics, Inc. (USA);

taking DMSO solution with concentration of 0.1mg/mL SPDP as modifying solution.

3. Lectin, antibody, and fluorescein

Lectins AAL, LTL, UEA-1, LCA, JAC, RCA-I, VVL, Con A, NPL, DSA, WGA, PNA, MAL-I, SNA, PHA-L are available from Vector corporation (USA);

human IgG was purchased from abcam, england;

the Cy 3-labeled donkey anti-human IgG antibody and the Cy 3-labeled donkey anti-rabbit IgG antibody are purchased from Shanghai Biotech engineering Co., Ltd;

cy 3-labeled rabbit anti-ConA was purchased from Sigma;

cy3 fluorescent dye was purchased from AAT Bioquest, Inc. (USA).

4. Buffer solution

HEPES powder, PBS powder, Tween20, and fetal Bovine Serum (BSA) powder were purchased from Sigma;

hydrochloric acid, sodium bicarbonate, sodium hydroxide (analytical grade) were purchased from Shanghai Zhengqi chemical reagent, Inc. (China).

PBST buffer: commercial PBS powder was dissolved in deionized water to form PBS buffer at a concentration of 0.01M, pH ═ 7.4; and adding Tween-20, and mixing uniformly to obtain a PBST solution, wherein the volume concentration of Tween20 in the PBST solution is 0.1%.

HEPES solution: 0.2383g of HEPES powder and 0.0011g of anhydrous calcium chloride were weighed out and dissolved in 100mL of pure water, and the pH was adjusted to 8.5 to obtain a HEPES buffer solution.

0.1M sodium bicarbonate buffer: 8.4g of sodium bicarbonate and 10.6g of sodium carbonate powder were dissolved in 1000mL of pure water, at which time the pH was about 8.0.

5. Serum sample pretreatment

A chromatographic column, namely a protein Prep affinity Albumin and DG deletion Kit for removing high-abundance protein in serum, which is purchased from Sigma company; the BCA protein concentration determination kit is purchased from Biyun (China); PDMiniTrap G-25 column was purchased from GE.

In the research and development, serum samples of 10 patients with hepatocellular carcinoma before and after operation and 10 normal human serum samples are collected for research and development, and all the serum is subpackaged and stored at-80 ℃ to keep the activity of protein and avoid repeated freeze thawing.

Example 1 molecular self-assembled monolayer formation and Probe curing

Cleaning the gold foil chip: reacting NH₃、H₂O₂And H₂O is mixed according to the volume ratio of 1: 1: 5 mixing to form TL1 cleaning solution, and placing in a stainless steel cleaning box. Immersing the gold foil chip into a stainless steel cleaning box filled with TL1 cleaning solution, carrying out water bath at 82 ℃ for 6 minutes, taking out, washing with ultrapure water for 4 times, soaking and cleaning with absolute ethyl alcohol (3 min/times multiplied by 2 times), blowing by nitrogen, drying, placing into a clean and sealed chip box, and preserving for later use.

Cleaning the gold foil chip, and drying the gold foil chip by nitrogen; spotting 1 mu L of modification solution on each hole of a dried gold foil chip, then placing the gold foil chip in a dried incubation box for incubation for 6 hours at room temperature, taking out the gold foil chip, cleaning the gold foil chip by using a DMSO solution, and drying the gold foil chip by using nitrogen to finish modification, thereby obtaining a solid phase carrier for later use; the SPDP modified chips can be stored for several months.

The characterization of the gold foil chip before and after modification was observed by atomic force microscope and shown in FIG. 2(a) and FIG. 2(b), respectively. The SPDP modified gold surface was found to be rougher than the unmodified surface using Nanoscope analysis software, indicating that the modified chemical groups were covalently attached to the gold surface.

Example 2 quality control experiment

SPDP incubation was performed in a dry incubation chamber, all remaining steps were incubated in a wet chamber at room temperature, and after each step, unbound material was washed twice with PBST solution for three minutes each time and the chip was dried with nitrogen for further incubation.

1. Quality control of SPDP modified concentration

The following experiment was repeated three times.

To optimize the SPDP coating concentration, a series of solid phase carriers were obtained by modifying a SPDP modified solution diluted with DMSO to a concentration of 0.8mg/mL, 0.4mg/mL, 0.2mg/mL, 0.Lmg/mL, 0.05mg/mL, 0.025mg/mL, 0.013mg/mL, 0.007mg/mL, 0.004mg/mL, 0.002mg/mL, or 0.001mg/mL, respectively, according to the method of example 1, washing with a DMSO solution, and blowing with nitrogen gas.

Preparing human IgG (containing 0.1% BSA) with the concentration of 50 mug/mL, spotting 0.84 mug L of the IgG in each hole onto each solid phase carrier, sealing by a sealing film, and incubating and coating in a wet box at room temperature for 2 hours; mu.g/mL of the donkey anti-human IgG antibody labeled with Cy3 at 2.5. mu.g/mL was applied to the wells, protected from light, incubated in a wet box at room temperature for 1h, and then scanned and detected on the chip using a Luxscan 10K-A (Boo Bio, China) chip apparatus, and the results are shown in FIG. 3 (a). FIG. 3(b) is a graph in which the mean value of the fluorescence intensity obtained by modifying the coated IgG at the same concentration with SPDP in each line in FIG. 3(a) is plotted on the ordinate and the concentration with SPDP is plotted on the abscissa. As can be seen from the figure, the fluorescence value of each group decreases with the decrease of the SPDP modification concentration, and the IgG fluorescence approaches saturation when the SPDP concentration is 0.1mg/mL, so the optimal SPDP modification concentration should be 0.1 mg/mL.

2. SPDP modified temperature-time quality control

The following experiments were repeated three times under the same conditions. The step of the surface chemical modification of SPDP in example 1 is performed at ambient temperatures of 25 ℃ and 37 ℃ for 24 hours, 12 hours, 6 hours, 3 hours, 1.5 hours, and 0 hour to complete the chip modification, and solid phase carriers with different modification temperatures and times are obtained.

Respectively spotting 50mg/mL human IgG (containing 0.1% BSA) on two solid phase carriers (each spot is 0.83 μ L), sealing with a sealing film, and incubating at room temperature for 2 h; coating a Cy 3-labeled donkey anti-human IgG antibody on the sample application holes, keeping out of the sun, and incubating for 1h at room temperature in a wet box; finally, PBST solution is used for cleaning and nitrogen is used for blow-drying. The above chips were scanned and examined by a chip scanner Luxscan 10K-A (Boo Co., Ltd., China), and the results are shown in FIG. 4 (a). FIG. 4(b) is a graph in which the average of the fluorescence intensities obtained at the same concentration at different experimental temperatures in FIG. 4(a) is plotted on the ordinate and the number of wells in the horizontal row is plotted on the abscissa. As can be seen from the figure, the difference between the fluorescence values is not large, and 25 ℃ is more beneficial to the experimental operation, so that 25 ℃ is selected as the optimal environmental temperature.

3. Quality control experiment of protein IgG coating detection line of SPDP modified chip

Human IgG solutions, which were diluted in a gradient of 200. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.13. mu.g/mL, 1.56. mu.g/mL, 0.78. mu.g/mL, 0.39. mu.g/mL, 0.19. mu.g/mL, were spotted onto the chips prepared in example 1, incubated in a wet cell at room temperature for 2 hours, washed with PBST buffer, and blown dry with nitrogen. The anti-human IgG antibody solution labeled with Cy3 diluted at 2.5. mu.g/mL was incubated on the antibody-containing chip, and the chip was taken out and washed with PBST for 3 times, 2 minutes each, and dried with nitrogen.

The chip was scanned by a chip scanner, which is a quality control experiment of the SPDP modified chip protein IgG coating detection line, and the results are shown in FIG. 5. As can be seen from the figure: within a certain range, the intensity of the generated fluorescence signal changes along with the change of the human IgG concentration, and the intensity of the fluorescence signal is obviously different from that of the fluorescence signal generated by the blank control. Therefore, the SPDP modified chip can be well combined with protein biomolecules.

4. SPDP modified chip detection antibody Cy3 labeled anti-human IgG antibody concentration gradient quality control experimental graph

Soaking the gold box chip subjected to surface chemical modification in the example 1 as a solid phase carrier in a human IgG solution with the concentration of 50 mu g/mL, incubating for 2 hours at room temperature (25 ℃), taking out, washing for 3 times by PBST, each time for 2 minutes, and drying by nitrogen; furthermore, Cy 3-labeled anti-human IgG antibody solutions diluted at 10. mu.g/mL, 5. mu.g/mL, 2.5. mu.g/mL, 1.25. mu.g/mL, 0.63. mu.g/mL, 0.32. mu.g/mL, 0.16. mu.g/mL, 0.08. mu.g/mL, 0.04. mu.g/mL, 0.02. mu.g/mL, 0.01. mu.g/mL, PBST-BSA were incubated on the antibody-bearing chip, and then washed 3 times with PBST, 2 minutes each, and blown dry with nitrogen.

The chip was scanned by a chip scanner, which was a quality control experiment of the fluorescein-labeled anti-IgG antibody, and the results are shown in fig. 6. As can be seen from the figure: under the condition of unchanged human IgG incubation condition, when the incubation concentration of the Cy3 labeled goat anti-human IgG antibody is more than 2.5 mu g/mL, the intensity of the generated fluorescence signal is obviously different from that of the negative control. It was thus shown that the optimal concentration of incubated Cy 3-labeled goat anti-human IgG antibody should be above 2.5. mu.g/mL.

5. Lectin immune specificity quality control

For the detection of lectins, which have been confirmed to coat the modified solid phase carrier, rabbit anti-Con a labeled with lectin Con a and its antibody Cy3 was selected as the subject.

The lectin was diluted with HEPES buffer, and BSA was added to the diluted lectin solution so that the BSA mass concentration was 0.001%.

A gradient (2mg/mL-0.002mg/mL) solution of Con A (containing 0.001% BSA) was spotted onto the chips prepared in example 1 at the same concentration in each well at 0.84. mu.L per well, incubated in a wet cell at room temperature for 2 hours, washed with PBST buffer and blown dry with nitrogen. Carrying out gradient dilution (1:1000-1:8000) on Cy 3-labeled rabbit anti-Con A (containing 0.001% BSA) according to the same sample application of each horizontal row concentration, wherein each well is 0.83 mu L, incubating for 1h at room temperature, then washing with PBST buffer solution, and drying with nitrogen, wherein each well is provided with a plurality of wells; cy 3-labeled donkey anti-rabbit IgG antibody (1:200, containing 0.1% BSA) was coated onto the wells at 0.84. mu.L per well, protected from light, incubated at room temperature for 1h, washed with PBST for 3 min/2 times, and air-dried with nitrogen.

The above chips were scanned and examined by a chip scanner Luxscan TM 10K-A (Boo Co., Ltd., China), and the results are shown in FIG. 7 (a). FIG. 7(b) is a graph in which the fluorescence value of Cy 3-labeled rabbit anti-Con A bound at the same concentration as Con A is plotted on the ordinate and the concentration of Con A is plotted on the abscissa. The results show that the fluorescence value gradually decreases with decreasing Con a concentration; also as the concentration of Cy 3-labeled rabbit anti-Con A decreased, the fluorescence decreased gradually. The results indicated that lectin Con a had been coated on the chip plane established in example 1 and specifically bound to Cy 3-labeled rabbit anti-Con a.

6. Lectin Con A incubation time-temperature quality control

Spotting a lectin Con A solution with the concentration of 1mg/mL on the solid phase carrier of the gold box chip with the surface being chemically modified by SPDP in example 1, and incubating the solid phase carrier at 37 ℃, room temperature (25 ℃) and 4 ℃ for 8 hours, 4 hours, 2 hours, 1 hour, 0.5 hour and 0 hour respectively to obtain lectin Con A chips coated under different temperature and time conditions; the chip is cleaned by PBST and dried by nitrogen, and the concentration is 1: a1000-diluted solution of rabbit anti-Con A antibody was spotted onto a solid support incubated with a Con A probe, incubated at room temperature (25 ℃) for 1 hour, taken out, washed 3 times with PBST for 2 minutes each, and dried with nitrogen. And dissolving Cy 3-labeled goat anti-rabbit anti-IgG antibody with the dilution concentration of 2.5 mu g/mL in a PBST-BSA solution, spotting the solution on the solid phase carrier incubated with the antibody, incubating the solution for 1 hour in the dark at room temperature, taking out the solution, washing the solution for 3 times with PBST-BSA for 2 minutes each time, and drying the solution with nitrogen.

Scanning the solid phase carrier by a chip scanner, namely a quality control experiment of the antigen incubation temperature-time, and the result is shown in fig. 8. Wherein, FIG. 8(a) is a fluorescence scanning diagram obtained by coating Con A with the same concentration on a chip for different time and at different temperature; FIG. 8(b) is a graph showing the fluorescence intensity of Con A coated on the chip for different times and at different temperatures. As can be seen from the figure:

(1) the fluorescence intensity is increased with the increase of the incubation time, and when the incubation time is more than 2 hours, the change of the fluorescence intensity with the increase of the incubation time is not obvious. Therefore, in practical tests, in order to shorten the incubation time, it is recommended that the incubation time for preparing the probe is more than 2 hours to meet the requirement.

(2) The incubation temperature, the fluorescence intensity is obviously stronger than the fluorescence intensity under the condition of 4 ℃ at 37 ℃ and room temperature (25 ℃), the difference between the fluorescence intensity under the conditions of 37 ℃ and room temperature (25 ℃) is not large, and in order to achieve the principle of simple operation and cost saving, the incubation experiment is recommended to be carried out under the condition of room temperature (25 ℃) in the actual detection.

Example 3 Combined lectin chip for Combined detection of sugar chains in serum of hepatocellular carcinoma patients

Lectin AAL, LTL, UEA-1, LCA, JAC, RCA-I, VVL, Con A, NPL, DSA, WGA, PNA, MAL-I, SNA and PHA-L probes are coated on the solid phase carrier constructed in the example 1, the concentration of each probe is 1mg/mL, the probes are spotted according to a distribution diagram, the probes are placed in a wet box and incubated at room temperature for 2 hours to construct a combined lectin chip, and PBST solution is cleaned and dried by nitrogen for later use.

FIG. 9(a) is a solid-state image of 5 cases of hepatocellular carcinoma sera and 1 case of normal human sera, and (b) is a histogram of the results of detection of 10 cases of hepatocellular carcinoma sera and 10 cases of normal control sera.

Table 2 shows that 10 hepatocellular carcinoma patient sera and 10 normal human sera were spotted on the combined lectin chip after pretreatment, and incubated in a wet chamber for 1h under dark at room temperature. While the last well in each column was blanked with PBST instead of serum. The fluorescence value is found by statistical analysis and research that the level of sugar chains combined with the fifteen kinds of lectins in the serum of the liver cancer patient is obviously higher than that of the normal population (P < 0.05).

TABLE 2

The results show that the lectin chip is suitable for human serum oncology screening, early tumor discovery, treatment evaluation, relapse or progress monitoring and survival early warning of patients.

The present invention is not limited to the above exemplary embodiments, and any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An SPDP modified lectin chip for detecting sugar chains in liver cancer serum, which is characterized in that: fifteen specific lectin probes are fixed on a surface lattice of a solid phase carrier of the lectin chip, and combined detection of at least fifteen sugar chains in human serum is realized by coating fifteen specific lectins;

the solid phase carrier is a gold foil chip chemically modified by 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester SPDP, and is used for fixing specific agglutinin as a probe;

the fifteen specific lectin probes are respectively AAL, LTL, UEA-1, LCA, JAC, RCA-I, VVL, Con A, NPL, DSA, WGA, PNA, MAL-I, SNA and PHA-L.

2. A method for producing the lectin chip of claim 1, comprising the steps of:

step 1, carrying out surface chemical modification on a gold foil chip to obtain a solid phase carrier

cleaning the gold foil chip, and drying the gold foil chip by nitrogen; spotting 1 mu L of the modification solution on each hole of a dried gold foil chip, then placing the gold foil chip in a dried incubation box for incubation for 6 hours at room temperature, taking out the gold foil chip, cleaning the gold foil chip by using a DMSO solution, and drying the gold foil chip by using nitrogen to finish modification;

step 2, fixing fifteen specific lectins as probes

spotting each lectin solution on the solid phase carrier obtained in the step 1, spotting one lectin solution in each of the 1 st to 15 th wells, spotting a PBST-BSA solution in the 16 th well as a blank control, wherein each well is 1 mu L, then placing the mixture in a wet box, incubating the mixture for 2 hours at room temperature, taking out the mixture, washing the mixture with a PBST buffer solution, and drying the mixture with nitrogen to obtain the lectin chip for the combined detection of at least fifteen sugar chains in human serum;

the PBST buffer solution is prepared by mixing PBS buffer solution with the concentration of 0.01M, pH-7.4 and Tween20, and the volume concentration of Tween20 in the PBST buffer solution is 0.1%;

the PBST-BSA solution is formed by mixing PBS buffer solution with the concentration of 0.01M, pH-7.4, Tween20 and fetal bovine serum BSA, wherein the volume concentration of Tween20 in the PBST-BSA solution is 0.1%, and the mass concentration of the fetal bovine serum BSA is 0.1%.

3. The method of claim 2, wherein: the method for cleaning the gold foil chip in the step 1 comprises the following steps: reacting NH₃、H₂O₂And H₂O is mixed according to the volume ratio of 1: 1: 5 to form TL1 cleaning solution, immersing the gold foil chip into a stainless steel cleaning box containing TL1 cleaning solution, carrying out water bath at 82 ℃ for 6 minutes, taking out the gold foil chip, washing the gold foil chip with ultrapure water, cleaning the gold foil chip with absolute ethyl alcohol, and drying the gold foil chip with nitrogen.

4. A method of using the lectin chip of claim 1, comprising the steps of:

step 1, removing high-abundance protein in serum

and 2, detecting the fluorescent label.

the volume V of the Cy3 solution satisfied formula (1) in mL:

The Cy3 solution was obtained from 1mg Cy3 NHS dissolved in 50. mu.L DMSO;

the 0.1M sodium bicarbonate buffer solution is prepared by dissolving 1.06g of sodium carbonate and 0.84g of sodium bicarbonate in 100mL of deionized water, and the pH value is adjusted to 8.5 before use;

the PBST buffer solution is prepared by mixing PBS buffer solution with the concentration of 0.01M, pH-7.4 and Tween20, and the volume concentration of Tween20 in the PBST buffer solution is 0.1%.

5. A kit for the combined detection of at least fifteen sugar chains in human serum, which is characterized in that the kit comprises: the lectin chip according to claim 1; PBS buffer at a concentration of 0.01M, pH ═ 7.4; PBST buffer; 0.1M sodium bicarbonate buffer; cy3 solution; a chromatographic column for removing high-abundance protein in serum; BCA protein concentration determination kit; g-25 chromatographic column;

the Cy3 solution was obtained from 1mg Cy3 NHS dissolved in 50. mu.L DMSO.