Lead ion fluorescent DNA (Deoxyribose Nucleic Acid) probe and fluorescent determination method for lead ion concentration

A DNA probe, lead ion technology, used in recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of multiple labeling of DNA probes, high requirements for experimental conditions, and many difficulties in operation, and achieves good specificity and avoidance. High fluorescence background, good selectivity

Inactive Publication Date: 2012-07-18
ELECTROMECHANICAL PRODS DETECTING TECH CENT OF SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the existing method of detecting Pb2+ by using 17E deoxyribozyme and substrate chain multi-labeled probe has many difficulties in operation and is not easy to popularize.
[0005] The present invention solves the high requirement of experimental conditions and the need for DNA Due to the shortcomings of multiple labeling and complicated operation of the probe, a single-labeled, highly sensitive new lead-ion fluorescent DNA probe consisting of 17E deoxyribozyme and FAM fluorescein-labeled substrate chain is proposed, using nano-gold colloid as the FAM Fluorescein quencher for fluorescent detection of lead ion concentration

Method used

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  • Lead ion fluorescent DNA (Deoxyribose Nucleic Acid) probe and fluorescent determination method for lead ion concentration
  • Lead ion fluorescent DNA (Deoxyribose Nucleic Acid) probe and fluorescent determination method for lead ion concentration
  • Lead ion fluorescent DNA (Deoxyribose Nucleic Acid) probe and fluorescent determination method for lead ion concentration

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Effect test

Embodiment 1

[0047] Adopt conventional gel electrophoresis experiment method, the feasibility of lead ion fluorescent DNA probe test lead ion method of the present invention is evaluated, experimental result is as follows figure 2 The gel chromatogram electrophoresis is shown. The first and second lanes from the left are the bands of lead ion fluorescent DNA probes, which become lighter and disappear after the addition of lead ions, and the electrophoretic bands that appear in new positions in the third to sixth lanes represent the bands after the addition of lead ions enzyme cleavage products. The addition of lead ions is caused by figure 2 The only reason for the change of the bands shown is that the lead ion fluorescent DNA probe and method of the present invention are suitable for the detection of lead ions.

[0048] In order to determine the optimal addition amount of nano-gold colloid, a sufficient concentration of lead ion solution was added to multiple DNA probe solutions, and ...

Embodiment 2

[0060] Embodiment 2: the mensuration of soluble lead ion in toy sample

[0061] Reference standard: EN71-3:1994+A1:2001

[0062] This embodiment shows the situation when the sample to be tested is a toy, and all accessible parts on the toy sample are tested.

[0063] 1. Sample preparation

[0064] For toys such as textile fabrics, plastic or rubber, use scissors or pliers to cut the material into small pieces of 5mm×5mm as the samples to be tested.

[0065] For toys with paint, ink or similar coatings, use a clean razor blade to scrape off the part as the sample to be tested.

[0066]For toys that are liquid paint or ink, take a part of the paint and apply it on a clean glass plate, let it dry naturally at room temperature, and then scrape it off as the sample to be tested. If the sample to be tested cannot be dried at room temperature, it should be dried in an oven according to the temperature used, and scraped off to obtain the sample to be tested.

[0067] All powdery...

Embodiment 3

[0075] Embodiment 3: Determination of total lead content in the plastic sample

[0076] 1. Pretreatment of samples before testing

[0077] Crush the plastic sample, weigh 0.5g, accurate to 0.1mg, put it into the digestion tank, add 7mLHNO 3 and 1.5mLH 2 o 2 . Close the digestion tank and place it in a microwave digestion system for digestion. The heating program is shown in Table 1 below (reference instrument, Anton Paar Microwave 3000).

[0078]

[0079] The resulting digestion solution was slightly heated on the electric heating plate to drive the acid to about 1mL, then add ultrapure water, and dilute to about 50mL. To measure the acidity of the solution, if the pH value is greater than 7, use 1 mol / L NaOH to neutralize to pH 7.0, and perform fluorescence measurement in Tris-HCl buffer.

[0080] 2. Determination

[0081] Add 20 μL of the sample to be tested in 50 mmol / L Tris-HCl (pH 7.0, 50 mM NaCl) buffer solution containing lead ion fluorescent DNA probe, mix ...

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Abstract

The invention relates to the field of detection and analysis of lead ion concentration and discloses a lead ion fluorescent DNA (Deoxyribose Nucleic Acid) probe. The lead ion fluorescent DNA probe comprises double strands formed by hybridizing 17E deoxyribozyme with a substrate strand, wherein the tail end of the substrate strand is modified by FAM (Carboxyfluorescein). The invention also discloses a fluorescent determination method for the lead ion concentration by using the probe. The fluorescent detection of the lead ion concentration of a sample to be detected is carried out by taking the lead ion fluorescent DNA probe as an identification element of a lead ion, the FAM for modifying the tail end of the substrate strand as a fluorescent signal molecule and a colloidal gold nanoparticle as a fluorescence quencher according to the fluorescent signal difference generated when the FAM and the lead ion fluorescent DNA probe are subjected to single-double-strand combination. The fluorescent determination method disclosed by the invention has the advantages of high sensitivity, favorable selectivity for the lead ion, low cost, convenience in operation, quickness in detection and the like.

Description

Technical field [0001] The invention involves the field of detection and analysis of lead ion concentration, which specifically involves a lead ionic fluorescent DNA probe and the fluorescence measurement method of lead ion concentration with the probe. Background technique [0002] Lead is a widely distributed and accumulated environmental pollutant.Lead is a heavy metal element with stable properties, which is extremely harmful to the human body and especially harms the health of children.With the rapid development of industry and other industries, the large amount of leadership of lead has made lead pollution expand from professional environment to water, atmosphere, food, medicine and other people's daily living environment. [0003] In the process of environmental testing, rapid screening, consumer goods safety, wastewater treatment, and industrial process, the sensitive, fast screening and accurate quantitative quantitative lead in lead are very important.At present, the ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 卫碧文吴继魁林莉高欢张俊玲
Owner ELECTROMECHANICAL PRODS DETECTING TECH CENT OF SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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