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A click chemistry-based copper ion detection method and detection kit

A copper ion detection and click chemistry technology, applied in the field of environmental analytical chemistry, can solve the problems of expensive instruments and interference detection sensitivity, and achieve the effects of low cost, high sensitivity and good selectivity

Active Publication Date: 2019-10-18
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the instruments used in these methods are expensive and require professional operators, which limits their use in primary laboratories
Cu-based 2+ The identified specific nuclease sequence can also be used for copper ion detection, but it involves specific nucleic acid sequence and secondary structure transformation, which has a high background value and interferes with detection sensitivity

Method used

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  • A click chemistry-based copper ion detection method and detection kit
  • A click chemistry-based copper ion detection method and detection kit
  • A click chemistry-based copper ion detection method and detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0080] A method for detecting copper ions based on click chemistry is carried out as follows:

[0081] (1) Tris-HCl buffer (20 mM, pH 7.5, containing 100 mM NaCl and 15 mM MgCl 2 ) to dissolve nucleic acid sequences A, B, C, D, E, and F, respectively. 300 nM A, 300 nM B, and 500 nM C were mixed thoroughly at room temperature, and reacted for 30 minutes to form an A-B-C complex;

[0082] (2) Different concentrations of copper ions (Cu 2+ ) solution, and a five-fold concentration of sodium ascorbate solution were thoroughly mixed, added to the A-B-C complex, and reacted at room temperature for 60 minutes;

[0083] (3) Heat at 95°C for 5 minutes to separate C and A-B, and immediately ice-bath for 10 minutes to keep C and A-B single-chain. Remove the supernatant after magnetic bead separation, and keep the A-B chain;

[0084] (4) Add 300 nM D and E, mix thoroughly, and react at room temperature for 30 minutes;

[0085] (5) Add 500 nM F and react at room temperature for 90 min...

Embodiment 2

[0087] A copper ion detection kit based on click chemistry, comprising the following components:

[0088] (1) Nucleic acid sequences A, B, C, D, E, F, their sequences and corresponding modification groups are as follows:

[0089] A: 5'-NH 2 -CGAATCCTGAGT-N 3 -3' (SEQ ID NO: 1);

[0090] B: 5'-CH≡CH-CTACAAATACCTA-3' (SEQ ID NO: 2);

[0091] C: 5'-TAGGTATTTGTAGACTCAGGATTCG-3' (SEQ ID NO: 3);

[0092] E: 5'-TAGGTATTTGTAGGTTACACCCATGTTACTCT-3' (SEQ ID NO: 4);

[0093] D: 5'-GATATCAGCGATTAACACTCAGGATTCG-3' (SEQ ID NO: 5);

[0094] F: 5'-FAM-AGAGTATrAGGATATC-BHQ-3' (SEQ ID NO: 6);

[0095] (2) Sodium ascorbate solution;

[0096] (3) Hydroxyl-modified magnetic bead solution;

[0097] (4) 20 mM Tris-HCl buffer, pH 7.5, containing 100 mM NaCl and 15 mM MgCl 2 .

Embodiment 3

[0099] Detection of different concentrations of copper ions:

[0100] Prepare copper ion standard solutions with concentrations of 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 500 nM and 800 nM respectively and store at room temperature. different concentrations of Cu 2+ Solution is added in the reaction system described in embodiment 1 respectively, detects fluorescence intensity after sufficient reaction, as figure 2 As shown, with Cu 2+ As the concentration increases, the corresponding fluorescence intensity gradually increases. When Cu 2+ Saturation is gradually reached when the concentration exceeds 500 nM. Take Cu 2+ The logarithm of the concentration (lgC) is the abscissa, the fluorescence intensity is the ordinate, draw the standard curve, the two have a good linear relationship, the linear range is from 10 pM to 500 nM, the linear equation is: F=302 + 157 lgC (R 2 =0.975)( F is the fluorescence intensity, C is the copper ion concentration), according to the standard o...

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Abstract

The invention discloses a copper ion detection method based on click chemistry and a detection kit. The bivalent copper ion is reduced to the cuprous ion under the effect of a reducing agent, so that the click chemistry reaction is activated; two sections of nucleotide sequences are connected with each other; a catalytic nucleic acid structure with catalytic activity can be further assembled after the magnetic bead separation; a substrate nucleic acid can be cut, so that fluorescence groups and quenching groups at the two ends of the substrate nucleic acid can be separated; and the copper ion concentration can be identified through fluorescence detection. The copper ion detection method has higher sensitivity; a detection limit to copper ions is 2pM; the detection has excellent specificity; the other conventional interfering ions have no influence on the detection; and the detection process is simple in operation, low in cost and suitable for quick detection for the copper ion concentration in practical samples.

Description

technical field [0001] The invention belongs to the field of environmental analytical chemistry, and in particular relates to a click chemistry-based copper ion detection method and a detection kit. Background technique [0002] Copper ions (Cu 2+ ) is one of the essential nutrients for the human body, but excessive intake will cause symptoms of poisoning, Cu 2+ Enrichment in the human body will cause serious damage to the brain, liver, pancreas, and heart muscle. The copper ingested by the human body is mainly obtained from water, food and the environment. Therefore, for Cu 2+ The analysis and determination of content is of great significance. [0003] Common methods for the determination of trace copper mainly include graphene atomic absorption spectrometry and inductively coupled plasma mass spectrometry. However, the instruments used in these methods are expensive and require professional operators, which limits their use in primary laboratories. Cu-based 2+ The id...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/58G01N33/53G01N21/64
Inventor 陈俊华李芳柏
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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