Luciferase complementary quantum dot biosensor as well as construction method and application thereof

A biosensor, luciferase technology, applied in the field of biosensing, to achieve the effects of simple method, high sensitivity and high efficiency

Active Publication Date: 2016-07-27
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] A homogeneous detection method that can fully combine the luciferase complementary technology with the optical advantages of quantum dot biosensors to achieve highly sensitive detection of multiple components has not yet been reported.

Method used

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  • Luciferase complementary quantum dot biosensor as well as construction method and application thereof
  • Luciferase complementary quantum dot biosensor as well as construction method and application thereof
  • Luciferase complementary quantum dot biosensor as well as construction method and application thereof

Examples

Experimental program
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Embodiment 1

[0089] 1. Cloning of the complementary fragment of Rluc8 luciferase, expression and purification of the complementary fragment

[0090] (1) Luciferase can be highly expressed in Escherichia coli through codon optimization of its encoded gene. For polypeptide linkers less than 30 amino acids, directly insert the corresponding gene fragment into the primer in one step; for longer polypeptide linkers of 35 to 50 amino acids, compile the Rluc8 fluorescence by overlapping extension polymerase chain reaction (overlappingPCR) The genes of primease and complementary fragments are introduced into the gene fragments of polypeptide linkers.

[0091] (2) Using the NcoI and Sal1 shear sequences introduced in the PCR process, the gene fragments with cohesive ends were obtained by simultaneous treatment with corresponding shear enzymes.

[0092] (3) Use T4 DNA ligase to connect the sticky end gene to the E. coli expression vector (pBAD) with the same z sticky end, and then transform it into...

Embodiment 2

[0104] 1. Cloning of the complementary fragment of Gluc1 luciferase, expression and purification of the complementary fragment

[0105] (1) Introduce a histidine tag (6×Histag) at the C-terminus of the Gluc1-N fragment and introduce a natural disulfide bond peptide chain SEP-Tag at the N-terminus (gene sequence: GDDD–GDDD–GDDD or GDDD-GDDD ), while introducing a histidine tag at the N-terminus of the Gluc1-C fragment and a SEP-Tag at the C-terminus, and introducing hydrophobic and stretchable tags of different lengths between the introduced histidine tag and the two fragments. The flexible peptide chain GGGS.

[0106] (2) Using the NdeI and NcoI cut sequences introduced during the PCR process, the gene fragments with cohesive ends were obtained by simultaneous treatment with the corresponding cutting enzymes.

[0107] (3) Use T4 DNA ligase to incubate and connect the gene fragment with the cohesive end with the E. coli expression vector PET28a with the same cohesive end at 16...

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Abstract

The invention discloses a luciferase complementary quantum dot biosensor as well as a construction method and application thereof, belonging to the technical field of biosensing. The luciferase complementary quantum dot biosensor comprises a quantum dot, a luciferase amino terminal fragment, a luciferase carboxyl terminal fragment, a probe of specifically recognizable determinand and a substrate which have bioluminescence reaction with luciferase. With the combination of optical advantages of the quantum dot sensor, a catalysis function can be reconstructed through induction complementation of the luciferase amino terminal fragment and the luciferase carboxyl terminal fragment on the surface of the quantum dot, a novel high-sensitivity biosensor can be constructed, and the biosensor is low in background noise, high in sensitivity, stable in structure and easy to use, can be used in high-target detection on multiple biological markers, and is applicable to accurate quantification on target testing articles in homogeneous systems.

Description

technical field [0001] The invention belongs to the technical field of biosensing, and relates to a luciferase complementary quantum dot biosensor, its construction method and its application. Background technique [0002] The rapid development of the national economy has greatly enhanced the public's attention to health. In order to achieve a low cost of health while achieving a high standard of health, there is an urgent need to develop novel diagnostic techniques or drug screening methods. Bioluminescence refers to the phenomenon that various organisms can produce light under the catalysis of enzymes. Compared with the general fluorescence detection method, it does not require an excitation light source, and the luminescence is completely dependent on the oxidation process of fluorescein, so it has a higher signal-to-noise ratio. Based on this, an analysis method of bioluminescence resonance energy transfer (BRET) has been rapidly developed in recent years. In this met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N21/64
Inventor 金宗文罗擎颖袁静
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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