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Lactic acid optical probe as well as preparation method and application thereof

An optical probe and lactic acid technology, applied in the field of optical probes, can solve problems such as in-situ, real-time, dynamic, high-throughput and high-spatial-resolution detection, cell crushing, separation, extraction and purification, and unsuitability for living cell research. , to achieve large dynamic changes in fluorescence, easy to mature, and save the effect of sample processing steps

Active Publication Date: 2021-06-22
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are not suitable for living cell research, and there are many defects: time-consuming sample processing is required, such as cell disruption, separation, extraction and purification, etc.; in situ, real-time, dynamic, high Detection of throughput and high spatiotemporal resolution

Method used

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  • Lactic acid optical probe as well as preparation method and application thereof
  • Lactic acid optical probe as well as preparation method and application thereof
  • Lactic acid optical probe as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0141] Embodiment 1, lactic acid binding protein particles

[0142] The LldR (80-258) gene in the Escherichia coli gene was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with BamHI and EcoRI, and the pCDFDuet1 vector was subjected to corresponding double digestion. After ligation with T4 DNA ligase, the product was used to transform MachI, and the transformed MachI was spread on LB plates (streptomycin sulfate 50ug / mL), and cultured at 37°C overnight. After plasmid extraction of the growing MachI transformants, PCR identification was performed. After the positive plasmid is correctly sequenced, the subsequent plasmid construction is carried out.

Embodiment 2

[0143] Example 2, Expression and Detection of cpYFP Optical Probes at Different Insertion Sites

[0144] In this example, based on pCDFDuet-LldR(80-258), the following site was selected to insert cpYFP according to the crystal structure of lactate-binding protein, and the corresponding pCDFDuet-LldR(80-258)-cpYFP plasmid was obtained: 93 / 94, 93 / 95, 93 / 96, 93 / 97, 94 / 95, 94 / 96, 94 / 97, 95 / 96, 95 / 97, 96 / 97, 119 / 120, 119 / 121, 120 / 121, 137 / 138 , 137 / 139, 137 / 140, 137 / 141, 138 / 139, 138 / 140, 138 / 141, 139 / 140, 139 / 141, 140 / 141, 158 / 159, 158 / 160, 158 / 161, 159 / 160, 159 / 161, 160 / 161, 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190 , 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, 190 / 191, 208 / 209, 208 / 210, 209 / 210, 230 / 231, 230 / 232 or 231 / 232. The amino acid sequences of exemplary optical probes are shown in Table 1.

[0145] Table 1. Sequences of Optical Probes

[0146]

[0147]

[0148] The ...

Embodiment 3

[0151] Example 3, Expression and detection of cpGFP optical probes at different insertion sites

[0152] According to the method in Example 2, cpYFP was replaced with cpGFP to construct a fluorescent probe for lactate green fluorescent protein, expressed and detected according to the method in Example 2. Such as image 3 As shown, the detection results show that the optical probes responding to lactic acid more than 1.5 times are at the 188 / 190 and 189 / 190 sites.

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Abstract

The invention relates to a lactic acid optical probe and a preparation method and an application thereof. In one aspect, the present invention relates to an optical probe comprising a lactic acid-sensitive polypeptide or functional variant thereof and an optically active polypeptide or functional variant thereof wherein the optically active polypeptide or functional variant thereof is located within the sequence of the lactic acid-sensitive polypeptide or functional variant thereof. The invention also relates to a preparation method of the probe and an application of the probe in detection of lactic acid.

Description

technical field [0001] The invention relates to the technical field of optical probes, in particular to a lactic acid optical probe and its preparation method and application. Background technique [0002] Lactic acid is an alpha-hydroxy acid that can ionize a hydrogen ion H in water + , while forming CH 3 CH(OH)COO-lactate ion. Lactic acid is a chiral molecule consisting of two enantiomers, D-(-)-lactic acid and L-(+)-lactic acid. Lactic acid may exist in two different configurations in the human body, of which L-lactic acid accounts for the vast majority. When the L-lactic acid concentration in plasma is greater than 5mM and the blood pH is lower than 7.35, it may be lactic acidosis. The causes of lactic acidosis include lack of pyruvate transport network in mitochondria, defect of pyruvate dehydrogenase complex, citric acid cycle disorder, congenital mitochondrial respiratory chain disorder, tissue hypoxia and other reasons. Lactate homeostasis is related to glucose ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62G01N33/68G01N33/58
CPCC07K14/245G01N33/68G01N33/582C07K2319/60G01N2333/245C12N15/62C07K14/00C07K2319/00G01N33/533C12N15/63C12N2320/10
Inventor 杨弋赵玉政李写陈念张则一张秀泽黄立
Owner EAST CHINA UNIV OF SCI & TECH
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