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Kit for detecting thrombin based on microfluidic chip and G-four-strobila-protoheme DNA (Deoxyribonucleic Acid) enzyme as well as preparation method and application thereof

A microfluidic chip and quadruplex technology, applied in the field of micro-total analysis systems, can solve the problems of high throughput and high sensitivity, and achieve the effect of not relying on complex equipment and low cost

Active Publication Date: 2018-05-01
HUNAN INSTITUTE OF ENGINEERING
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the development of traditional dynamic microarrays still cannot overcome the problems of high-throughput and high-sensitivity characteristics under the condition of micro-sample

Method used

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  • Kit for detecting thrombin based on microfluidic chip and G-four-strobila-protoheme DNA (Deoxyribonucleic Acid) enzyme as well as preparation method and application thereof
  • Kit for detecting thrombin based on microfluidic chip and G-four-strobila-protoheme DNA (Deoxyribonucleic Acid) enzyme as well as preparation method and application thereof
  • Kit for detecting thrombin based on microfluidic chip and G-four-strobila-protoheme DNA (Deoxyribonucleic Acid) enzyme as well as preparation method and application thereof

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preparation example Construction

[0059] The invention provides a preparation method of the kit, including a preparation method of a microfluidic detection chip coated with functionalized microspheres and a gold nanoparticle whose surface is modified with a second thrombin nucleic acid aptamer and an initiating probe. Preparation.

[0060] The preparation method of the microfluidic detection chip coated with functionalized microspheres described in the present invention comprises the following steps:

[0061] 1) Mix and incubate the avidin-modified microsphere solution with a mass concentration of 1% to 3% after affinity washing and 0.3 μmol / L biotin-labeled first thrombin nucleic acid aptamer solution, and wash to obtain functionalized Microspheres;

[0062] 2) Put the functionalized microspheres in the step 1) into the microchambers in the chip through the microsphere loading channel, fix them, peel off the microbead loading film base, and attach the reagent delivery film base and the microsphere fixed arra...

Embodiment 1

[0102] Use 15 μm avidin-modified polystyrene microspheres as the solid phase interface for Aptamer-1 immobilization, take 100 μL avidin-modified microspheres with a concentration of 2% in a centrifuge tube, wash with 100 μL affinity eluent (20 mM Tris pH 7.5, 1M NaCl, 1mMEDTA, 0.0005% TritonX-100) washed twice, centrifuged at 3500rpm for 5min, and removed the supernatant; respectively added 44 μL of affinity eluent and 3 μL of 0.3 μM biotin-modified Aptamer-1 (see Table 1), incubate at room temperature for 10-15 min; remove unbound molecules by centrifugal washing method and suspend the functionalized microspheres in 100 μL affinity eluent. Aptamer-1 specifically binds the modified biotin to avidin on the surface of the microsphere and immobilizes on the surface of the microsphere, thereby forming a functionalized polystyrene microsphere with the ability to detect target molecules. Each microsphere surface can be modified 3×10 7 a biotinylated molecule.

[0103] The designed...

Embodiment 2

[0110] Preparation of standard curve

[0111] Serum samples were serially diluted 1000 times with 0.4% BSA solution, and thrombin was added to obtain gradient concentrations of 0.1pg / mL, 0.5pg / mL, 1pg / mL, 10pg / mL, 50pg / mL, 80pg / mL and 100pg / mL. Take 15uL of the above-mentioned serum standards of different concentrations of thrombin into the microchamber, and react at 37°C for 1h. Rinse with 1% BSA washing solution for 5 min, then add 5 uL of 1.5 nM functionalized gold nanoparticle reagent, react at 37° C. for 1 h, and wash with 1% BSA washing solution for 5 minutes. 10 uL of a mixed solution of 0.5uM hairpin 1 and 0.5uM hairpin 2 was respectively poured into the miniature chamber, and incubated at 30°C for 6 hours. Wash with 10 nM HEPES buffer for 5 min to remove unreacted hairpin 1 and hairpin 2. Take 20uL luminescence system (75nMhemin, 0.5mMluminol, 30mMH 2 o 2 ) into the detection area for luminescent detection (425nm), and finally use the fluorescent microscope CCD to...

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Abstract

The invention provides a kit for detecting thrombin based on a microfluidic chip and a G-four-strobila-protoheme DNA (Deoxyribonucleic Acid) enzyme as well as a preparation method and application thereof and belongs to the technical field of biological molecular detection. The kit for detecting the thrombin is prepared from a microfluidic chip coated with functional microspheres, a gold nanoparticle solution with the surface modified by a second thrombin aptamer and an initiation probe, a first hairpin probe reagent, a second hairpin probe reagent and a luminous system, wherein the functionalmicrospheres are obtained by modifying a first thrombin aptamer on the surfaces of microspheres through a biotin-avidin system. The invention provides application of the reagent to detection of the thrombin. The kit provided by the invention can be used for overcoming the difficulties in the technical field of micro-medical analysis or minimally invasive and non-invasive diagnosis fields that highflux and high sensitivity are difficult to realize at the same time under a micro-sample condition relatively well.

Description

technical field [0001] The invention belongs to the technical field of micro-total analysis systems, and in particular relates to a kit for detecting thrombin based on a microfluidic chip and G-quadruplex-heme DNase, and a preparation method and application thereof. Background technique [0002] Biomacromolecules are the basic substances that constitute life, including proteins, nucleic acids, hydrocarbons, etc. At present, the detection technologies for the analysis of biological macromolecules mainly include: (1) microarray chip technology (Microarray); (2) static microfluidic array chip technology; (3) conventional molecular biology technology. However, these types of technologies generally require expensive and sophisticated detection equipment, long detection time and generally unsatisfactory sensitivity, cannot achieve high-throughput detection of trace samples, and have high costs, which restricts the application of these technologies in the field of clinical diagnosi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N35/00
CPCG01N35/00
Inventor 张何赵智粮傅昕
Owner HUNAN INSTITUTE OF ENGINEERING
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