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A lectin chip for joint detection of ten kinds of sugar chains in human serum and its preparation and use method

A combined detection and lectin technology, applied in measurement devices, instruments, analytical materials, etc., can solve the problems of screening, low sensitivity, poor chip geometry, etc., to achieve high throughput, stable combination, and increased detection. cost effect

Active Publication Date: 2021-06-29
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the above-mentioned research specimens are selected from the gross materials of patients’ surgery or cultured cells in vitro, so high-throughput screening of large-scale samples from the general population cannot be carried out, and there is a lack of effective means for judging the progress, curative effect and prognosis of cancer patients
[0006] In the past, sugar chain research mostly used commercial lectin chips, using glass or gel as the coating carrier. The lectin probes were easy to elute, the chip geometry was not good, and the sensitivity was low, especially when the changes in serum sugar chains in patients were closely related to tumors. Lack of research on invasion, metastasis and correlation with patient survival

Method used

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  • A lectin chip for joint detection of ten kinds of sugar chains in human serum and its preparation and use method
  • A lectin chip for joint detection of ten kinds of sugar chains in human serum and its preparation and use method
  • A lectin chip for joint detection of ten kinds of sugar chains in human serum and its preparation and use method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1, Molecular Self-Assembled Monolayer Formation and Probe Solidification

[0076] 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.

[0077]After cleaning the gold foil chip, immerse it in modification solution 1, and incubate with shaking at room temperature for 12 hours in the dark, so that 16-amino-1-hexadecanethiol can be assembled into the gold foil chip through the Au-S bond. Clean the solution and dry it with nitrogen to complete the first layer of modification; spot 0.78 μL of...

Embodiment 2

[0080] Embodiment 2, quality control experiment

[0081] DOTA-NHS-ester 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 nitrogen gas Dry the chips for the next incubation.

[0082] 1. DOTA-NHS-ester modified concentration quality control

[0083] The following experiments were repeated three times.

[0084] In order to optimize the coating concentration of DOTA-NHS-ester, the DOTA-NHS-ester solution diluted with DMSO with a concentration of 20mM, 10mM and 5mM gradients were respectively spotted and incubated on the modified first layer obtained according to Example 1. (0.78 μL per well, one horizontal row for each concentration), and then placed in a dry incubation box to incubate at room temperature for 12 hours. After taking it out, wash it with DMSO solution and dry it with nitrogen, so that the solid-phase c...

Embodiment 3

[0089] Embodiment 3, lectin immune specificity experiment

[0090] In order to detect that the lectin has been confirmed to be coated on the modified solid phase carrier, the rabbit anti-ConA labeled with the lectin ConA and its antibody Cy3 is selected as the research object of Example 3.

[0091] The lectin was diluted with HEPES buffer, and BSA was added to the diluted lectin solution, so that the mass concentration of BSA was 0.001%.

[0092] ConA (containing 0.001% BSA) solution of gradient dilution (2mg / mL-0.002mg / mL), 0.84 μL per well, was spotted on the chip prepared in Example 1 according to the same concentration in each column, and placed in a wet box After incubating at room temperature for 2 hours, wash with PBST buffer and blow dry with nitrogen. Apply the Cy3-labeled rabbit anti-ConA (containing 0.001% BSA) after serial dilution (1:1500-1:12000) according to the same concentration in each row, 0.84 μL per well, incubate at room temperature for 1 hour, and then ...

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Abstract

The invention discloses a lectin chip for the joint detection of ten kinds of sugar chains in human serum and its preparation and use method, which is that ten kinds of specific lectin probes are immobilized on the surface of a solid phase carrier in lattice, and coated Ten specific lectins are used to realize the joint detection of ten sugar chains in human serum; the solid phase carrier is 16‑amino‑1‑hexadecanethiol and gold foil chip chemically modified by DOTA‑NHS‑ester, and DOTA‑ The three carboxyl groups at the end of the NHS‑ester are activated with EDC and NHS to immobilize specific lectins as probes. The chip of the present invention has the advantages of high throughput, high selectivity, high specificity, etc., and is more suitable for the research on the characteristics of the glycan spectrum of human tumors.

Description

technical field [0001] The invention relates to a lectin chip used for detecting sugar chain levels in human serum, which belongs to the field of biotechnology. Background technique [0002] According to the "2017 China Cancer Registration Annual Report", 10,000 people are diagnosed with cancer every day in China, and about 7 people are diagnosed with cancer every minute. When the life expectancy is 86 years, the cumulative cancer risk is as high as 36%. The cancers with the highest mortality rates are mainly lung cancer and digestive system cancers. Among them, the morbidity and mortality of gastric cancer are ranked second in China's cancer annual report, and gastric cancer has been ranked first in the incidence of tumors in Anhui Province for many years. Although medical technology and detection methods have been greatly improved, the early detection rate of tumors has not been high due to people's worries and fears about radiation, gastrointestinal endoscopy and other ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/574
CPCG01N33/574G01N2400/00
Inventor 杜卫东高毅
Owner ANHUI MEDICAL UNIV
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