Micro-fluidic chip according to time-resolved fluorescent technique, preparation method of micro-fluidic chip and application of micro-fluidic chip

A time-resolved fluorescence, microfluidic chip technology, applied in the field of immunology, to improve efficiency, avoid interference, save sample cost and time cost

Inactive Publication Date: 2018-05-29
成都微康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no microfluidic immune chip that uses time-resolved fluorescence to complete the detection on the microfluidic chip

Method used

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  • Micro-fluidic chip according to time-resolved fluorescent technique, preparation method of micro-fluidic chip and application of micro-fluidic chip
  • Micro-fluidic chip according to time-resolved fluorescent technique, preparation method of micro-fluidic chip and application of micro-fluidic chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Procalcitonin (PCT) microfluidic chip combined with time-resolved fluorescence technology

[0054] A PCT microfluidic chip combined with time-resolved fluorescence technology, the microfluidic chip includes a sample loading area, a filter area, a time-resolved fluorescent microsphere antibody or antibody complex binding area, a micro-mixer, a reaction area and a waste liquid area. like figure 1 shown.

Embodiment 2

[0055] Example 2 Preparation method of PCT microfluidic chip combined with time-resolved fluorescence technology

[0056] 1) Preparation of the microfluidic chip: the microfluidic chip is composed of a hydrophilic substrate and a cover sheet, and the substrate and the cover sheet are uniformly injection-molded and then surface-modified to obtain a microfluidic chip;

[0057] 2) Preparation of time-resolved fluorescent microsphere antibody or antigen complex:

[0058] a. Take 1 mg of rare earth fluorescent microspheres (purchased from bangslabs, item number: ), centrifuge, remove the supernatant, and wash twice with 400ul of coating buffer. Coating buffer formula: Weigh 9.76g MES, dissolve in 1L distilled water, adjust pH=6.0 with 5M NaOH;

[0059] b. Activation: Add 50ul of EDC (concentration: 0.1mg / ML) to the washed microspheres, shake at room temperature for 1 hour;

[0060] c. Labeled antibody: centrifuge the activated latex to remove the supernatant, wash twice with 10mM...

Embodiment 3

[0066] Embodiment 3 Sensitivity detection

[0067] The 1 mg / ml PCT antigen was serially diluted with 10 mM PBS to obtain (10 ng, ml, 5 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml) gradient calibrator. Draw 30ul gradient calibrator and add it to the microfluidic chip. After reacting for 5 minutes, measure it with a microfluidic fluorescence quantitative analyzer to obtain a set of data corresponding to the fluorescence intensity value and the concentration gradient, and prepare a standard curve: y= -7E-15x 3 +2E-09x 2 +8E-05x+0.0064, see figure 2 .

[0068] Pipette 30ul of 10mM PBS buffer solution, add it to the microfluidic chip, react for 5min, measure the fluorescence intensity value when the concentration is 0, repeat sample addition and measurement 20 times, and obtain the detection result.

[0069] After taking the average value + 2 times SD of 20 groups of detection results into the standard curve for calculation, the obtained concentration value is the analytical sensiti...

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Abstract

The invention belongs to the field of immunology, and particularly relates to a micro-fluidic chip according to a time-resolved fluorescent technique, a preparation method of the micro-fluidic chip and an application of the micro-fluidic chip. The micro-fluidic chip according to the time-resolved fluorescent technique comprises a sample adding area, a filtering area, a time-resolved fluorescent micro-sphere antibody or antibody compound binding area, a micro-mixer, a reaction area and a waste liquid area. According to the micro-fluidic chip, antigens or antibodies marked by time-resolved fluorescent nano-particles, light stability is strong, sensitivity is high, and interference of samples can be effectively avoided. The antigens or antibodies of a plurality of items to be detected can besimultaneously marked on the micro-fluidic chip, a plurality of items of a single sample can be simultaneously detected, efficiency is improved, and sample cost and time cost are saved. The preparation method of the micro-fluidic chip is simple, detection accuracy is high, low difference between batches and high stability are ensured, and full quantitative detection of the samples can be realized.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to a microfluidic chip combined with time-resolved fluorescence technology and its preparation method and application. Background technique [0002] At present, most of the detection kits on the in vitro diagnostic products (IVD) market use the principle of specific combination of antigen and antibody, using different fluoresceins as labeled antibodies or antigens, and the antibodies or antigens on the stationary phase are specific. Antigens or antibodies in the specific and the sample to be tested form a complex, and finally detect the fluorescence intensity of the complex to complete the determination of the concentration of the analyte in the sample. However, this common fluorescein (such as FITC, Alexa Fluor647, etc.) has a small stock displacement, and it is not easy to distinguish the laser light from the emitted light. A special narrow-band filter is needed to distinguis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01L3/00G01N33/533
Inventor 唐勇
Owner 成都微康生物科技有限公司
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