Unmarked colorimetric determination metallic lead ion method based on aurum nanometer probe and nuclease

A colorimetric assay and nuclease technology, applied in the field of analytical chemistry, can solve problems such as time-consuming and labor-intensive, and achieve the effect of broadening the application range, simple and sensitive colorimetric detection, and convenient lead ion detection.

Inactive Publication Date: 2009-02-11
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These modification operations are time-consuming and labor-intensive

Method used

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  • Unmarked colorimetric determination metallic lead ion method based on aurum nanometer probe and nuclease
  • Unmarked colorimetric determination metallic lead ion method based on aurum nanometer probe and nuclease
  • Unmarked colorimetric determination metallic lead ion method based on aurum nanometer probe and nuclease

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The steps for unlabeled colorimetric determination of lead ions using gold nanoprobes and nucleases are as follows:

[0035] (1) Reference method synthesizes 13nm gold nanoprobe (reference; Grabar, K.C.; Freeman, R.G.; Hommer, M.B.; Natan, M.J., Preparation and Characterization of Au Colloid Monolayers.Anal.Chem.1995,67, (4 ), 735-743);

[0036] (2) In the Tris-HCl buffer solution containing 140mM NaCl, 5mM KCl and 20mM pH=7.5, prepare the solution of 17E type nuclease and its substrate. The sequence of 17E type nuclease is 5'CAT CTC TTC TCC GAG CCGGTC GAA ATA GTG AGT 3'; 17E type nuclease substrate sequence is 5'ACTCAC TAT rA GGA AGA GAT G 3';

[0037] (3) Mix 1 μM 17E-type nuclease and 1 μM 17E-type nuclease substrate evenly, react in a 90°C water bath for 5 minutes, cool down to room temperature, and obtain 1 μM double-stranded product; store at 4°C for later use; the 1 μM 17E type Nuclease: The volume ratio of 1 μM 17E-type nuclease substrate is 1:1;

[0038] (4)...

Embodiment 2

[0042] In order to investigate the specificity of the method for the unlabeled colorimetric determination of lead ions using gold nanoprobes and nucleases, a comparative experiment was carried out as follows:

[0043] (1) Reference method synthesizes 13nm gold nanoprobes (references: Grabar, K.C.; Freeman, R.G.; Hommer, M.B.; Natan, M.J., Preparation and Characterization of Au Colloid Monolayers.Anal.Chem.1995, 67, (4 ), 735-743);

[0044] (2) In the Tris-HCl buffer solution containing 140mM NaCl, 5mM KCl and 20mM pH=7.5, prepare the solution of 17E type nuclease and its substrate; the sequence of 17E type nuclease is 5' CAT CTC TTC TCC GAG CCGGTC GAA ATA GTG AGT 3'; the sequence of the 17E-type nuclease substrate is 5'ACTCAC TAT rA GGA AGA GAT G 3';

[0045] (3) Mix 1 μM 17E-type nuclease and 1 μM 17E-type nuclease substrate evenly, react in a 90°C water bath for 5 minutes, cool down to room temperature, and obtain 1 μM double-stranded product; store at 4°C for later use; th...

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Abstract

The invention relates to a method for measuring metallic lead ion based on a nano-gold probe and markless color comparison of nuclease. 17E type nuclease forms a double chain structure with the substrate through hybridization. If the specimen to be tested contains lead ion, the lead ion can greatly increase the speed of catalyzing and transecting the substrate by the 17E type nuclease, after the transaction of the substrate, the double chain structure is dissociated to generate free single-chain DNA which can be adsorbed on the surface of the nano-gold probe, in order to protect the nano-gold probe from being inducted by salt and aggregated, therefore, the nano-gold probe solution is in claret red color; if the specimen to be tested does not contain lead ion, the double chain structure of the 17E type nuclease and the substrate thereof can not be damaged, reaction can not happen between the double chain structure and the nano-gold probe, therefore, the nano-gold probe can be inducted by salt and aggregated and in bluish violet color. Through the color change of the nano-gold probe, the detection of the lead ion can be realized. The detection limit to the lead ion is 500 nanomole per liter. By using the method to detect the lead ion, the selectivity is high, the sensitivity is high, the operation is simple, and complex instruments are not needed.

Description

technical field [0001] The invention relates to a method for unlabeled colorimetric determination of metal lead ions based on gold nanoprobes and nucleases, and belongs to the technical field of analytical chemistry. Background technique [0002] Nucleases are oligonucleotides with catalytic function (References: Robertson, D.L.; Joyce, G.F., Selection in vitro of an RNA enzyme that specificallycleaves single-stranded DNA. Nature 1990, 344, (6265), 467-468 ; Joyce, G.F., Directed evolution of nucleic acid enzymes.Annu.Rev.Biochem.2004, 73, 791-836; Breaker, R.R., DNA enzymes.Nat.Biotechnol.1997, 15, (5), 427-431), It can catalyze a wide range of reactions, including: cleavage reactions to nucleic acid base substrates, DNA ligation reactions, phosphorylation reactions, porphyrin metallation reactions, etc. Just as the catalytic activity of some protein-type enzymes is related to metal ion cofactors, the catalytic activity of some nucleases is also closely related to specific...

Claims

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Application Information

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IPC IPC(8): G01N21/25C12Q1/68
Inventor 魏辉李冰凌李敬董绍俊汪尔康
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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