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Microfluidic chip group used for screening formyl peptide receptor agonist and screening method

A technology of microfluidic chips and formyl peptide receptors, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of cumbersome experimental processes, and achieve the effect of saving analysis time and reducing detection costs

Inactive Publication Date: 2010-07-21
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has high sensitivity, the experimental process is cumbersome, especially the process of laying and taking the film is completely manual and requires a certain amount of experience.

Method used

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  • Microfluidic chip group used for screening formyl peptide receptor agonist and screening method
  • Microfluidic chip group used for screening formyl peptide receptor agonist and screening method
  • Microfluidic chip group used for screening formyl peptide receptor agonist and screening method

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Experimental program
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Effect test

Embodiment 1

[0036] BME was taken out from the -20°C refrigerator and placed on ice. When it melted into a liquid state, pipette 0.1 μL, and figure 1The chip shown is placed under an optical microscope, and the suction head is aligned with the entrance of the basement membrane channel, and liquid BME is slowly injected into the channel. After leaving for 5 minutes, add 50% ethanol (v / v), PBS and cell culture medium to the channel of the chip in sequence, place the chip in a 37° C. incubator for 15 minutes, and the BME is completely solidified. Place the chip under an optical microscope, add 7 μL of medium to each sample pool, extract 1 μL of medium from the cell waste pool in the outer circle, and immediately inject 1 μL of cell suspension into the cell inlet, most of the cells enter the perfusion channel and stay, Only a very small number of cells enter the migration channel and place the chip in the incubator. After the cells adhered to the wall, the chip was taken out to take pictures,...

Embodiment 2

[0038] RBL-FPR cell suspension is injected from the cell inlet figure 2 For the chips shown, after the cells evenly entered each micro-culture chamber and settled, the chips were placed in the incubator overnight, and the cells fully adhered to the wall and stretched. The compound solutions (fMLF-QD and GSH-QD) labeled with quantum dots (quantum dot, QD) were introduced into the chip to stimulate the cells, and the chip was placed at 37°C CO 2 After 45 minutes in the incubator, the washing solution was introduced into the chip, and the cells were rinsed for about 45 seconds. Finally, the chip was imaged under a fluorescent microscope to detect the endocytosis of the formyl peptide receptor. The excitation wavelength of the mercury lamp was set to 330-385nm, and the detection wavelength was set to For more than 420nm, take fluorescence photos and bright field photos in the same field of view. The interaction time, concentration and washing time of compound-quantum dots and RB...

Embodiment 3

[0040] RBL-FPR cell suspension is injected from the cell inlet image 3 For the chips shown, after the cells settled, the chips were placed in the incubator overnight, and the cells fully adhered to the wall and stretched. Drain the culture medium in the sample pool, inject HBSS solution into the chip to wash the cells twice, add the calcium ion fluorescent dye Fluo-4AM diluted to 5 μg / mL with HBSS in advance, put the chip in the incubator and incubate for 45 minutes, and drain the sample pool Inject the dye in the chip, wash the cells twice by injecting HBSS solution into the chip again, and record the instantaneous flow process of calcium ions caused by the stimulation of the cells with a fluorescence microscope. Compound stimulation was applied immediately after the light source, and the CCD shutter was opened, and 50 photos were continuously taken at a speed of 1 s / frame. The effects of fMLF or GSH at the concentrations of 0nM, 4nM, 40nM and 400nM on inducing intracellula...

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Abstract

The invention provides a microfluidic chip group used for screening a formyl peptide receptor agonist and a screening method. The microfluidic chip group consists of three microfluidic chip units, i.e. a cell chemotaxis detection unit, a receptor endocytosis detection unit and a calcium ion transient flow process monitoring unit. The invention designs a group of chips by aiming to the characteristics of different cell function analytical experiments, can respectively carry out cell chemotaxis, receptor endocytosis detection and calcium ion transient flow (release) process monitoring, and judges biologic activity thereof by comprehensively evaluating influence on the formyl peptide receptor signal conduction path by an alternative compound. Compared with the traditional technology, the invention realizes real-time monitoring of the intracellular calcium ion releasing process and saves a series of complex operations, such as film laying, film obtaining, cell doctoring, fixing, dying andthe like, thereby greatly saving the analysis time and lowering the detection cost.

Description

technical field [0001] The invention relates to a cell-level drug screening technology, and in particular provides a microfluidic chip set and a screening method for screening formyl peptide receptor agonists. Background technique [0002] Formyl peptide receptor (FPR) plays an important role in the body's defense against exogenous pathogenic microorganisms. When pathogenic microorganisms invade the body, their secreted N-formyl peptide combined with FPR can recruit phagocytic leukocytes to migrate and gather at the lesion during inflammation and immune emergency response to fight and eliminate pathogenic microorganisms. Studies in recent years have confirmed that FPR is not only expressed in leukocytes with phagocytic function, but also expressed in hepatocytes, dendritic cells, astrocytes and microglial cells, etc. FPR agonists also include Non-N-formyl peptide substances such as endogenous polypeptides that appear in the early stage of inflammation. Therefore, the funct...

Claims

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Application Information

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IPC IPC(8): G01N33/483G01N21/76
Inventor 秦建华叶囡楠林炳承
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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