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Preparation method of microfluid system for capturing and enriching circulating tumor cells and microfluid device

A microfluidic system and tumor cell technology, applied in the preparation of microfluidic systems and the field of microfluidic devices, can solve the problem of low sensitivity, meet the requirements of reducing interference and quantity, and realize all-round detection and high efficiency. the effect of capturing

Inactive Publication Date: 2020-11-17
侯双
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The main purpose of the present invention is to propose a method for preparing a microfluidic system and a microfluidic device for capturing and enriching circulating tumor cells, aiming to solve the technical problem of low sensitivity of traditional CTC counting

Method used

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  • Preparation method of microfluid system for capturing and enriching circulating tumor cells and microfluid device
  • Preparation method of microfluid system for capturing and enriching circulating tumor cells and microfluid device
  • Preparation method of microfluid system for capturing and enriching circulating tumor cells and microfluid device

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Embodiment 1

[0061] The present invention proposes a preparation method of a microfluidic system for capturing and enriching circulating tumor cells, which is prepared by the following preparation method: (this embodiment uses a 0.5 mm thick medical pathological quartz glass slide as the basis)

[0062] 1) Etching, surface modification and biofunctionalization of silicon nanowire chips

[0063] see Figure 1 to Figure 5 , is the etching process of the silicon nanowire chip, and the specific steps are as follows:

[0064] S1, spin coating PMMA-A8 photoresist glue on the pathological glass slide, the thickness of the PMMA-A8 photoresist glue is 500nm, dry for later use;

[0065] S2, carry out O to the PMMA-A8 photoresist glue of coating 2 Reactive ion etching to form point-like nanostructures, wherein the etching time is 2 minutes;

[0066] S3. Carry out CF4 etching to the dot-shaped nanostructure, etch for 10 minutes, and wash with concentrated sulfuric acid / hydrogen peroxide mixed solut...

Embodiment 2

[0087] 1) Preparation of simulated samples

[0088] Prepare a cell suspension with a density of 10,000 / mL (accurate count) from the culture dish of the non-small cell lung cancer cell line H2228 with a culture abundance of about 95%, and dilute and sort 200 / 20uL of the ready-to-use solution in a gradient, and add 180uL Contains 2*10 5 Jurkat cell suspension (the cells are all diluted and configured with PBS), as a 200uL mock sample. Add 1.5mL solution containing 5umol biotin-labeled EpCAM antibody (anti-EpCAM) to the mock sample, and incubate at room temperature for 45 minutes. Then centrifuge (400g, 5 minutes), after removing the supernatant, wash again with PBS, and constant volume in 200uL PBS solution, as test simulation sample.

[0089] 2) Test and selection of optimal flow rate

[0090] ①Experimental steps:

[0091] Select the simulated sample in experiment 1) as the test object, and test the capture efficiency of H2228 cells at flow rates of 0.1mL / h, 0.2mL / h, 0.5mL / h,...

Embodiment 3

[0111] 1) Testing and application of blood samples from clinical cancer patients:

[0112] ①The blood collection of clinical samples requires BD Vacutainer Glass ACD Solution A tube8.5mL to avoid EDTA anticoagulant damage to the antigen on the surface of the cells in the blood, which will affect the binding of the capture antibody and reduce the capture efficiency; take the first tube of blood draw only Take 2mL and not use it for the test (to detect false positive cells due to epithelial cells exfoliated when the needle is inserted into the blood).

[0113] ② Taking 4mL whole blood as an example, this method adopts gradient density centrifugation to purify the blood of cancer patients initially. First add 4mL PBS solution to dilute the blood sample to an equal volume, mix well, and then slowly add the diluted blood sample into the 15mL centrifuge tube that has been added with 4mL gradient density centrifugation solution (1077); choose 300g, centrifuge for 40 minutes to remove...

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Abstract

The invention discloses a preparation method of a microfluid system for capturing and enriching circulating tumor cells and a microfluid device. The preparation method comprises the following steps: 1) etching, surface modification and biological functionalization of a silicon nanowire chip; 2) preparation of a microfluidic chip; and 3) assembling of the silicon nanowire chip and the microfluid chip. According to the technical scheme, the silicon nanowire chip and the microfluid chip are designed and assembled, so that a technical problem that traditional CTC counting is not high in sensitivity is solved.

Description

technical field [0001] The invention relates to the technical fields of biological materials and clinical detection, in particular to a preparation method of a microfluidic system for capturing and enriching circulating tumor cells and a microfluidic device. Background technique [0002] With the increase of the degree of pollution, the incidence rate of tumor has been higher than one-third of the proportion. The diagnostic methods for tumors mainly include: pathology (biopsy section); imaging (ultrasound, X-ray, CT or PET, etc.) and serology (serum tumor-associated proteins, such as CA-125, CA-199 or CEA, etc.). However, these methods have their own defects and cannot provide help for changing tumors and diagnosis and early warning. The current clinically recognized human peripheral blood circulating tumor cell (Circulating Tumor Cell, CTC) detection has been recognized as one of the best detection methods. In 2004, the US Drug and Food Administration approved the first t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01L3/00
CPCB01L3/502707B01L3/502761B01L2200/0668B01L2300/0861B01L2300/12B01L2300/161B01L2400/0481
Inventor 侯双罗群皓吴冬霞
Owner 侯双
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