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Integrated micro-fluidic chip used for fluorescence detection of enzyme catalysis product and application thereof

A microfluidic chip and fluorescence detection technology, applied in the direction of fluorescence/phosphorescence, laboratory containers, chemical instruments and methods, etc., can solve the problems of affecting enzyme-catalyzed reactions, changing reactions, and difficulty in obtaining fluorescent substrates, etc., to achieve Improve sensitivity, reduce analysis errors, and avoid mutual interference effects

Inactive Publication Date: 2014-05-21
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Stable fluorescent substrates are usually not easy to obtain; and the conversion of products into fluorescent derivatives in the environment of enzyme-catalyzed reactions often affects the enzyme-catalyzed reaction and even changes the reaction

Method used

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  • Integrated micro-fluidic chip used for fluorescence detection of enzyme catalysis product and application thereof
  • Integrated micro-fluidic chip used for fluorescence detection of enzyme catalysis product and application thereof
  • Integrated micro-fluidic chip used for fluorescence detection of enzyme catalysis product and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032]Connect the needles of the dual-channel micro-injection pump to the chip input ports C1 and C2 respectively, the input ports C3 and C4 are opened, and the liquid flows out of the chip through the input ports C3 and C4 and the waste liquid port C5. Rinse the channel with 0.1M NaOH for 30min, wash with distilled water and then rinse with 0.1M HCl for 30min, and finally rinse the chip channel with 0.01PBS buffer solution.

[0033] After the flushing of the above-mentioned channels is completed, the input ports C3 and C4 are closed, and the liquid flows out of the chip through the waste liquid port C5. Through input ports C1 and C2, BSA (1 mg / mL) was input into the microchannel network on the lower layer of the chip at a constant flow rate of 10 μL / min, and left to stand for 3.5 hours. Then, a mixed solution of protein A (20 μg / mL) and 1% glutaraldehyde, and a solution of rabbit immunoglobulin IgG (5 μg / mL dissolved in 0.01M PBS) were sequentially injected, and incubated for...

Embodiment 2

[0036] According to the operation steps described in Example 1, urease was fixed in the channel of the microfluidic chip, the chip was placed in a constant temperature bath at 37°C, and the needles of the two dual-channel micro-injection pumps were respectively connected to the input ports C1, C2; C3 on the chip. , C4 connection pipeline connected. Two 0.2M phosphate buffer solutions with different concentrations of urea (0mM, 0.5mM) were input into the lower microchannel network at a constant flow rate of 10 μL / min through input ports C1 and C2. Six columns of urea solutions with different concentrations are generated by the concentration gradient generation unit, and contact with the urease immobilized on the channel for enzymatic reaction. After incubation for 3 minutes, the urea is hydrolyzed by the enzyme to generate ammonia, which is brought to the upper microchannel network through 6 interconnected holes. At the same time, the OPA reagent (i.e. the fluorescent derivati...

Embodiment 3

[0038] According to the operation steps described in Example 1, urease was fixed in the channel of the microfluidic chip, the chip was placed in a constant temperature bath at 37°C, and the needles of the two dual-channel micro-injection pumps were respectively connected to the input ports C1, C2; C3 on the chip. , C4 connection pipeline connected. From the input ports C1 and C2, two different concentrations of acetylhydroxamic acid (0mM, 0.1mM) and 0.2M phosphate buffer solution of the same concentration of urea (0.1mM) were injected into the lower microchannel at a constant flow rate of 10μL / min The internet. The concentration gradient generation unit generates 6 rows of solutions of acetyl hydroxamic acid and the same concentration of urea, which contact with the urease immobilized on the channel for enzymatic reaction, and at the same time, acetyl hydroxamic acid inhibits urease. After incubation for 3 minutes, the urea is hydrolyzed by the enzyme to generate ammonia, whi...

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Abstract

The invention provides an integrated micro-fluidic chip used for fluorescence detection of an enzyme catalysis product. The chip is mainly composed of two layers of micro-channel network; as shown in a figure 1, the lower-layer micro-channel network is composed of roundabout broken line channels and used as a concentration gradient generation unit; as shown in a figure 2, the upper-layer micro-channel network in the chip is composed of branch channels symmetrically increasing layer by layer, connected with six columns of broken line channels and six expansion channels and used as an equivalent distribution unit, a fluorescence derivative reaction unit and a fluorescence detection window; and the upper-layer micro-channel network is communicated with the lower-layer micro-channel network. The micro-fluidic chip integrates an automatic concentration gradient generation function, an equivalent distribution function, an enzyme area immobilizing function, an enzymic catalytic reaction product separating function, a product fluorescence derivatization function and a fluorescence detection function into one, can be used on a standard ELIASA for acquisition of analysis data and provides a solution for related analysis in fields like the industry, agriculture, medicine and environmental science.

Description

technical field [0001] The invention relates to enzyme-catalyzed reaction analysis technology, in particular to an integrated microfluidic chip for fluorescence detection of enzyme-catalyzed products and its application. Background technique [0002] Enzyme catalysis technology is widely used in various fields such as industry, agriculture, medicine, and environmental science. The analysis of enzyme catalysis products is a key technology for the study of enzyme catalysis reactions and the screening of enzyme inhibitors. Fluorescence analysis is widely used in the analysis of enzyme-catalyzed reactions, and has the advantages of strong specificity and high sensitivity. However, the application of fluorescence analysis in enzyme-catalyzed reaction analysis must meet certain conditions: a specific substrate with fluorescence can be prepared; or the product can be converted into a fluorescent derivative under the conditions of enzyme-catalyzed reaction. Stable fluorescent subst...

Claims

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Application Information

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IPC IPC(8): B01L3/00G01N21/64
Inventor 成志毅张琴唐修雯
Owner SUN YAT SEN UNIV
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