CN111273027B - SPDP modified lectin chip for detecting sugar chains in liver cancer serum as well as preparation and application of SPDP modified lectin chip - Google Patents

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

Background

The previous research on tumor biomarkers mainly focuses on protein and nucleic acid research, and important biological macromolecules such as sugar chains or glycans are ignored. The process by which sugar chains are attached to specific amino acids of the protein polypeptide backbone by covalent bonding is called protein glycosylation. Glycosylation is the most common and complex form of post-translational modification of proteins, and has important regulatory effects on the structure and function of proteins. Many studies in recent years have indicated that sugar chains on proteins secreted from malignant or diseased tissues and cells often have a structure significantly different from that of sugar chains on proteins secreted from normal tissue cells. In fact, it has been observed that in the serum of hepatocellular carcinoma patients, there are contained a large number of expressions of sugar chain structures which are not observed in normal mature hepatocytes, and that the activity of glycosyltransferase synthesizing a certain sugar chain structure increases or decreases. For example, liver cancer marker-AFP, liver cancer produces AFP with a much higher alpha 1 → 6 fucose index than benign liver disease, and can be classified as AFP-L1, AFP-L2 and AFP-L3 due to their different reactivity with lentil Lectin (LCA) lectin, wherein AFP-L1 is mainly from benign liver disease, AFP-L2 is mainly from pregnant women, and AFP-L3 is a fucosylated form of alpha fetoprotein, mainly from HCC. There are currently patented results studying serum carbohydrate chains as markers for hepatocellular carcinoma: trisialylated (trisialylated) sugar chains that disappear or decrease with the onset of hepatocellular carcinoma are labeled and used as hepatocellular carcinoma markers for detection thereof. Therefore, the research on characteristic sugar chains related to the liver cancer has important theoretical significance and clinical application significance on diagnosis, disease course monitoring, prognosis evaluation and targeted therapy of the liver cancer, and can be used as an important index in liver cancer diagnosis and prognosis application.

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 frequently 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 geometric 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 dot matrix of a solid phase carrier, and the combined detection of at least fifteen sugar chains in human serum is realized by coating fifteen specific lectins;

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

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

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 a solid phase carrier.

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

after the gold foil chip is cleaned, 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 a 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 is added into each lectin solution to ensure that the mass concentration of the BSA is 0.001 percent and the BSA is used for blocking unbound-NHS groups;

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 with the volume of 1 mu L per well, placing the wells in a wet box, incubating for 2 hours at room temperature, taking out the wells, washing with a PBST buffer solution, and drying with nitrogen gas to obtain the lectin chip for 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 NH ₃ 、H ₂ O ₂ And H ₂ O is mixed according to the volume ratio of 1:1:5 to form TL1 cleaning liquid, immersing the gold foil chip into a stainless steel cleaning box filled with the TL1 cleaning liquid, carrying out water bath at 82 ℃ 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 buffer solution with the concentration of 0.01M and the pH =7.4, and then removing albumin and globulin in the serum to be detected through a chromatographic column to obtain the 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 and the pH =7.4 and 100 mu L of 0.1M sodium bicarbonate buffer solution, and incubating for 1h at room temperature in a dark place to complete 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 satisfies formula (1) in mL:

in the formula, M ₁ Molecular weight, M, of fluorescent marker Cy3 ₂ Is the protein molecular weight, M ₁ ＝829.03、M ₂ ＝65000；

Spotting the preprocessed serum to be detected in 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 in 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 by comprising the following components in percentage by weight: 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 a PBS buffer solution with the concentration of 0.01M and the pH =7.4 with Tween20, and the volume concentration of the 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 in 50. Mu.L DMSO; the PBST-BSA solution is formed by mixing PBS buffer solution with the concentration of 0.01M and the pH =7.4, tween20 and fetal bovine serum BSA, wherein the volume concentration of the Tween20 in the PBST-BSA solution is 0.1 percent, and the mass concentration of the fetal bovine serum BSA is 0.1 percent.

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

1. the invention prepares SPDP novel chemically 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 a lectin-combined chip is never reported, and the modification method can be used for directly combining lectin by utilizing terminal-NHS ester, is simple to operate, is stable in combination and has high sensitivity.

2. The present invention simultaneously detects fifteen kinds of lectin-bound sugar chains in the 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, so that high-flux detection is realized, the detection cost is reduced on the whole, and the detection efficiency is improved.

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) is a 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) Performing surface chemical modification on SPDP with different concentrations to obtain IgG fluorescence scanning; (b) the resulting fluorescence intensity profile;

FIG. 4 is a graph showing the effect of SPDP surface chemical 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) the resulting fluorescence intensity profile;

FIG. 5 is a quality control experiment of SPDP modified chip protein IgG coating detection line, in which: (a) is a chip detection entity diagram; (b) is a chip detection curve graph;

FIG. 6 is a SPDP modified chip assay for detecting antibody Cy 3-labeled IgG concentration gradient quality control experiment, in which: (a) is a chip detection entity diagram; (b) a chip detection curve graph;

FIG. 7 shows the specific quality control of lectin Con A immunity, wherein: (a) Fluorescence scans obtained by immunospecific detection for different concentrations of Con A and different concentrations of anti-Con A; (b) the resulting fluorescence intensity profile;

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) the resulting fluorescence intensity profile;

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 resulting histograms for the mean fluorescence intensity of 10 patients with liver cancer and 10 normal controls were obtained.

Detailed Description

The following embodiments are described in detail with reference to the accompanying drawings, and the following embodiments are implemented on the premise of the technical solution of the present invention, and give detailed embodiments and specific operation 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. Agglutinin, antibody, and fluorescein

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

human IgG was purchased from abcam, england;

cy 3-labeled donkey anti-human IgG antibody and Cy 3-labeled donkey anti-rabbit IgG antibody are purchased from Shanghai Biotechnology engineering, inc.;

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

cy3 fluorescent dyes were 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, and sodium hydroxide (analytically pure) were purchased from Shanghai Zhenju chemical reagent, inc. (China).

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

HEPES solution: HEPES buffer was obtained by dissolving 0.2383g of HEPES powder and 0.0011g of anhydrous calcium chloride in 100mL of pure water and adjusting the pH to 8.5.

0.1M sodium bicarbonate buffer: 8.4g of sodium bicarbonate and 10.6g of sodium carbonate powder are dissolved in 1000mL of pure water, the pH value then being about 8.0.

5. Serum sample pretreatment

A chromatographic column, protein Prep affinity Albumin and IgG deletion Kit, for removing high-abundance proteins in serum, purchased from Sigma; the BCA protein concentration determination kit is purchased from Biyun (China); PD MiniTrap 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

And (3) cleaning the gold foil chip: reacting NH ₃ 、H ₂ O ₂ And H ₂ O is mixed according to a volume ratio of 1:1:5 mixing to form TL1 cleaning solution, and placing the cleaning solution 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.

In order to optimize the SPDP coating concentration, the SPDP modified solutions diluted with DMSO and having a gradient 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, 0.001mg/mL were modified according to the method of example 1, and after being taken out, the solutions were washed with DMSO solution and dried with nitrogen to obtain a series of solid phase carriers for use.

Preparing human IgG (containing 0.1% of BSA) with a concentration of 50 μ g/mL, spotting 0.84 μ L per well onto each solid phase carrier, sealing with a sealing membrane, and incubating and coating at room temperature for 2h; the donkey anti-human IgG antibody labeled with Cy3 of 2.5. Mu.g/mL is added to the well, protected from light, incubated in A wet box for 1h at room temperature, and then scanned and detected by A Luxscan (TM) 10K-A (Boo biological, chinA) chip instrument, and the result is 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 0.1mg/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.

50mg/mL human IgG (containing 0.1% BSA) was spotted onto two solid phase carriers (0.83. Mu.L per spot), and after sealing with a sealing film, wet-box incubation was carried out at room temperature for 2 hours; coating a Cy 3-labeled donkey anti-human IgG antibody on the sample application holes, keeping out of the sun, and incubating at room temperature for 1h 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 (TM) 10K-A (Boo 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 SPDP modified chip protein IgG coating detection line

Human IgG solutions 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 Cy 3-labeled anti-human IgG antibody solution diluted to 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; the antibody-containing chip was incubated with Cy 3-labeled anti-human IgG antibody solutions diluted to 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, and then washed 3 times with PBST, each for 2 minutes, 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 that the human IgG incubation condition is not changed, when the incubation concentration of the Cy 3-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 fluorescence signal generated by 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%.

0.84. Mu.L of a Con A (containing 0.001% BSA) solution diluted in a gradient (2 mg/mL-0.002 mg/mL) was spotted on the chip prepared in example 1 in each column at the same concentration in each well, incubated in a wet cell at room temperature for 2 hours, washed with PBST buffer, and blown dry with nitrogen. After gradient dilution (1; cy 3-labeled donkey anti-rabbit IgG antibody (1.

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 as the Con a concentration decreases, the fluorescence value decreases gradually; also as the concentration of Cy 3-labeled rabbit anti-Con A decreased, the fluorescence decreased gradually. The results indicate that lectin Con a has 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 a 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 in the dark at room temperature for 1 hour, taking out the solution, washing the solution with PBST-BSA for 3 times, each time for 2 minutes, and drying the solution with nitrogen.

The solid phase carrier is scanned by a chip scanner, which is 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 that under the condition of 4 ℃ at 37 ℃ and room temperature (25 ℃), the difference between the fluorescence intensity under the condition of 37 ℃ and room temperature (25 ℃) is not large, and in order to have 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 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 for 2 hours at the inner room temperature to construct a combined lectin chip, and the PBST solution is cleaned and dried by nitrogen for later use.

FIG. 9 (a) is a solid chart of the scanning of 5 cases of hepatocellular carcinoma sera and 1 case of normal human sera, and (b) is a bar chart of the detection results 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 PBST was substituted for serum in the last well of each column as a blank. 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 life cycle early warning of patients.

The present invention is not intended to be limited to the exemplary embodiments but rather to cover all modifications, equivalents, and improvements falling within the spirit and scope of the present invention.

Claims (5)

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 preparing 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

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 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

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 a 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;

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 with 1 mu L per well, placing the mixture in a wet box, incubating the mixture for 2 hours at room temperature, taking out the mixture, washing the mixture by using a PBST buffer solution, and drying the mixture by using nitrogen to obtain the lectin chip for combined detection of at least fifteen sugar chains in human serum;

the PBST buffer solution is prepared by mixing a PBS buffer solution with the concentration of 0.01M and the pH =7.4 with Tween20, and the volume concentration of the 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 and the pH =7.4, tween20 and fetal bovine serum BSA, wherein the volume concentration of the 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 according to claim 2, wherein the reaction mixture is heated to a temperature in the reaction mixture: the method for cleaning the gold foil chip in the step 1 comprises the following steps: reacting NH ₃ 、H ₂ O ₂ And H ₂ O is mixed according to the volume ratio of 1:1:5 to form TL1 cleaning liquid, immersing the gold foil chip into a stainless steel cleaning box filled with the TL1 cleaning liquid, carrying out water bath at 82 ℃ for 6 minutes, taking out, washing with ultrapure water, cleaning with absolute ethyl alcohol, and drying 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

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

and 2, detecting the fluorescent label.

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 and the 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, thus finishing 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 satisfies formula (1) in mL:

in the formula, M ₁ Molecular weight, M, of fluorescent marker Cy3 ₂ Is the protein molecular weight, M ₁ ＝829.03、M ₂ ＝65000；

The Cy3 solution was obtained from 1mg Cy3 NHS 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;

spotting the preprocessed serum to be detected in 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 in 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 PBST buffer is prepared by mixing a PBS buffer with a concentration of 0.01M and pH =7.4 with Tween20, and the volume concentration of Tween20 in the PBST buffer 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; a 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 PBST buffer solution is prepared by mixing a PBS buffer solution with the concentration of 0.01M and the pH =7.4 with Tween20, and the volume concentration of the 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 in 50. Mu.L DMSO.

Images