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SPDP modified lectin chip for detecting liver cancer serum carbohydrate chains as well as preparation and application thereof

A lectin and serum technology, applied in the field of biomedical technology detection, can solve problems such as low sensitivity, limitations, poor chip geometry, etc., and achieve the effects of improving detection efficiency, reducing detection cost, and combining stability.

Active Publication Date: 2020-06-12
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although lectin chips have been used to study the presence of serum sugar chains in patients with liver cancer, and some glycoproteins are found to be significantly higher than normal, but on the one hand, these studies are almost limited to common glycoproteins containing fucose and sialic acid; On the other hand, most of the above-mentioned research samples are cells cultured in vitro or gross specimens of patients undergoing surgical resection, so they are not suitable for high-throughput screening of the population, and lack effective means for disease progression, curative effect evaluation, and prognosis judgment of liver cancer patients.
In addition, in the past, commercial lectin chips were mostly used in sugar chain research, and glass or gel was used as the coating carrier. The lectin probes were easy to elute, the chip geometry was not good, and the sensitivity was low.

Method used

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  • SPDP modified lectin chip for detecting liver cancer serum carbohydrate chains as well as preparation and application thereof
  • SPDP modified lectin chip for detecting liver cancer serum carbohydrate chains as well as preparation and application thereof
  • SPDP modified lectin chip for detecting liver cancer serum carbohydrate chains as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1, Molecular Self-Assembled Monolayer Formation and Probe Solidification

[0068] Clean the gold foil chips: NH 3 、H 2 o 2 and H 2 O is mixed at a volume ratio of 1:1:5 to form a TL1 cleaning solution, which is placed in a stainless steel cleaning box. Immerse the gold foil chip in a stainless steel cleaning box filled with TL1 cleaning solution, bathe in 82°C water for 6 minutes, take it out and rinse it with ultrapure water 4 times, then soak and clean it with absolute ethanol (3min / time x 2 times), blow dry with nitrogen , put it in a clean and airtight chip box after drying, and store it for later use.

[0069] After cleaning the gold foil chip, dry it with nitrogen gas; spot 1 μL of modification solution per well on the dried gold foil chip, then place it in a dry incubation box and incubate at room temperature for 6 hours, take it out, wash it with DMSO solution, and blow it dry with nitrogen gas. After the modification is completed, the solid phase c...

Embodiment 2

[0071] Embodiment 2, quality control experiment

[0072] SPDP was incubated in a dry incubation box, and all other steps were incubated in a wet box at room temperature. After each step, unbound substances were washed twice with PBST solution for three minutes each time, and the chip was dried with nitrogen gas for next use. one-step incubation.

[0073] 1. SPDP modification concentration quality control

[0074] The following experiments were repeated three times.

[0075] In order to optimize the SPDP coating concentration, the concentrations diluted with DMSO were 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, and 0.001mg / mL gradient SPDP modification solutions were respectively modified according to the method of Example 1. After taking them out, they were washed with DMSO solution and dried with nitrogen to obtain a series of solids. The carrier is ready for use.

[0076] Prepare human IgG (containing 0....

Embodiment 3

[0096] Example 3. Combined lectin chip joint detection of sugar chains in serum of patients with hepatocellular carcinoma

[0097] On the solid phase carrier constructed in Example 1, the lectins AAL, LTL, UEA-1, LCA, JAC, RCA-I, VVL, Con A, NPL, DSA, WGA, PNA, MAL-I, SNA and The concentration of PHA-L probes is 1 mg / mL. Samples are applied according to the distribution diagram, placed in a humid box and incubated at room temperature for 2 hours to construct a combined lectin chip, washed with PBST solution, and dried with nitrogen gas before use.

[0098] Figure 9 (a) is the scanning entity map of 5 cases of hepatocellular carcinoma serum and 1 case of normal human serum, and (b) is the histogram of the detection results of 10 cases of hepatocellular carcinoma serum and 10 cases of normal control serum.

[0099] Table 2 shows that the sera of 10 cases of hepatocellular carcinoma patients and 10 cases of normal people were pretreated and spotted on the combined lectin chip, ...

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Abstract

The invention discloses an SPDP modified lectin chip for detecting liver cancer serum carbohydrate chains as well as preparation and application of the SPDP modified lectin chip. A gold foil chip chemically modified by SPDP is used as a solid-phase carrier, fifteen specific lectin probes are fixed on the surface of the gold foil chip in a dot matrix mode, and joint detection of at least fifteen carbohydrate chains in human serum is achieved by coating fifteen specific lectin. The lectin chip provided by the invention can accurately detect at least fifteen carbohydrate chains in serum of a liver cancer patient, has the advantages of high flux, high selectivity, high specificity and the like, and is more suitable for research of human tumor glycosyl spectrum characteristics.

Description

technical field [0001] The invention belongs to the field of biomedical technology detection, and specifically relates to a lectin chip for detecting fifteen kinds of sugar chains in serum of patients with liver cancer modified by 3-(2-pyridyldimercapto) N-hydroxysuccinimide propionate (SPDP) and its preparation and application. Background technique [0002] Previous tumor biomarker research mainly focused on protein and nucleic acid research, ignoring important biological macromolecules such as sugar chains or glycans. The process in which sugar chains are covalently linked to specific amino acids in the protein polypeptide backbone is called protein glycosylation. Glycosylation is the most common and complex form of protein post-translational modification, which plays an important role in regulating protein structure and function. In recent years, many studies have pointed out that the sugar chain structure of proteins secreted by malignant or diseased tissues and cells ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/574G01N33/544G01N33/531
CPCG01N33/68G01N33/57438G01N33/544G01N33/531G01N2333/4724G01N2800/52
Inventor 杜卫东刘胜胜
Owner ANHUI MEDICAL UNIV
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