CN103698320A - Construction method of chiral sensor for detecting DNA enzymatic assembly of lead ion - Google Patents

Abstract

The invention provides a construction method of a chiral sensor for detecting DNA enzymatic assembly of a lead ion, belonging to the field of nano-biotechnology detection. The method comprises the steps of modifying a silver nano particle of 10nm and a silver nano particle of 20nm with a pair of DNA probes respectively, and assembling the modified silver nano particles to form an asymmetrical chiral dimer under the action of DNA enzyme, wherein the silver nano particle assembly is taken as a detecting sensor for detecting Pb<2+> by circular dichroism (CD). The construction method is used for detecting the Pb<2+> through a CD signal by the means that the big silver nano particle and the small silver nano particle are assembled to form the asymmetrical chiral dimmer under the action of the Pb<2+>-dependent DNA enzyme and by virtue of the silver nano particle probe. The method can be used for performing ultra-sensitive detection on the Pb<2+>, is high in detecting sensitivity and good in specificity, and is capable of detecting a trace target object in a water solution.

Description

The construction method of the chiral sensor that a kind of DNA enzyme of measuring lead ion is assembled

Technical field

A kind of mensuration Pb ²⁺the construction method of chiral sensor of DNA enzyme assembling, belong to nanometer biotechnology detection field.

Background technology

Pb ²⁺be one of heavy metal ion of tool toxicity, plumbous pollution is from factory effluent and discarded objects such as mining, smelting, rubber production, dyestuff, printing, pottery, lead glass, scolding tin, cable and lead pipes.In addition, the lead tetraethyl in vehicle exhaust is extremely toxic substance.Along with developing rapidly of China's industrial or agricultural, in environment, the pollution of heavy metal is more and more serious.The murder by poisoning that heavy metal pollution causes exists lastingly, can again circulate and enters food chain with soil, water body, food security is constituted a threat to, and harm humans life and health.So far, plumbous do not have known physiological action, the lead in any human body to be all regarded as polluting in vivo, and it is disorderly that saturnism meeting causes stomach, liver, kidney and neurotrosis.Pb in Environmental Protection Agency's regulation potable water ²⁺high-load must not surpass 72nM.The investigation demonstration of national environmental protection portion, China's heavy metal pollution of water body problem is very outstanding, and the pollution rate of river,lake and reservior substrate is up to 80.1%.For the feature of heavy metal pollution, to its discovery and detection, be vital.

Traditional Pb ²⁺detection method comprises Inductively coupled plasma method (ICP) and atomic absorption spectrography (AAS), the advantages such as it is highly sensitive, quantitatively accurate that these methods have, but required instrument is expensive, sample pretreatment is complicated, consuming time, and requires testing staff to possess certain professional knowledge, and analysis cost is high.The features such as chemistry, biology sensor is highly sensitive owing to having, selectivity good, volume is little, cost is low, have been subject to people's favor.In recent years, along with the development of nanometer technology, the nano particle based on oligonucleotides functionalization has carried out a lot of research and application in food Hazard factor context of detection.Because nano particle has special character, as: optical characteristics, electrochemical properties, fluorescent characteristic, paramagnetism etc., can utilize the peculiar signal of these character to amplify to reach the object that detects object.The chirality nanostructured that plasma nano particle is assembled into has CD signal, this discovery becomes a new progress to nano material in the application of detection field, and the CD signal that can apply chirality nanomaterial assembly body detects nuisance as detection signal.

DNA enzyme is the DNA molecular of a kind of functionalization of obtaining through in-vitro screening, this kind of enzyme is different from proteinase, can obtain by chemosynthesis, there is good thermal stability and reactivity, DNA molecular can be modified several functions group simultaneously, and can be fixed on solid support, at present, a large amount of objects has the DNA enzyme that can identify.Under the condition existing in heavy metal ion, the substrate chain of the DNA enzyme that heavy metal relies on is cut open at RNA base cleavage site place, Pb ²⁺the DNA enzyme relying on is known as " 17 E " DNA enzyme, consists of the Pb that application DNA enzyme is set up an enzyme chain and a substrate chain ²⁺detecting sensor has caused that people study interest widely.

The present invention is that the big or small Nano silver grain of DNA probe being modified by means of the effect of DNA enzyme is assembled into asymmetric Nano silver grain dimer, at variable concentrations Pb ²⁺under the condition existing, there is depolymerization in various degree in dimer, along with Pb ²⁺the increase of concentration, dimeric depolymerization degree is larger, thus CD signal decreases, according to the intensity of CD signal and Pb ²⁺the corresponding relation of the foundation between concentration, thereby to Pb ²⁺content detects.

Summary of the invention

The object of the present invention is to provide a kind of to Pb ²⁺detect and quantitative chiral sensor method, the big or small Nano silver grain of DNA probe being modified by means of the effect of DNA enzyme is assembled into asymmetric Nano silver grain dimer, at variable concentrations Pb ²⁺under the condition existing, there is depolymerization in various degree in dimer, finally by CD spectrum, Nano silver grain assembly is measured, thus indirect detection target P b ²⁺content.

Technical scheme of the present invention: a kind of mensuration Pb ²⁺the construction method of chiral sensor of DNA enzyme assembling, comprise: the Nano silver grain of 10nm and 20nm is modified with a pair of DNA probe respectively, Nano silver grain is assembled into asymmetric chirality dimer under the effect of DNA enzyme, and this Nano silver grain assembly is used for Pb as detecting sensor ²⁺detection, application CD spectrum detects; Concrete steps are:

(1) Nano silver grain of 10nm and 20nm is modified with a pair of DNA probe respectively

The Nano silver grain of newly synthetic 10nm, 20nm is passed through respectively to ten times of centrifugal concentratings under the speed conditions of 10000r/min, 8000r/min, then Nano silver grain is resuspended to respectively in the 10mM Tris-HCl damping fluid that includes 50mM NaCl, makes the final concentration of the Nano silver grain of 10nm, 20nm be respectively 50 nM, 20 nM, then DNA probe and Nano silver grain are carried out to coupling according to the molar ratio of 10:1, specific as follows: the 20 μ M S1 nucleic acid fragments of 1 μ L join in the 20 nm Nano silver grains of 100 μ L 20 nM, the 50 μ M S2 nucleic acid fragments of 1 μ L join in the 10 nm Nano silver grains of 100 μ L 50 nM, hatch after 12h, centrifugal three times of the Nano silver grain of success coupling, the DNA of not coupling is removed, last nano particle is all resuspended to that in 50 μ L 25 mM Tris-acetate buffer solutions, (pH 8.2, comprise 100 mM NaCl), finally obtain the Nano silver grain that coupling is good,

S1：5'-TCACAGATGA?GT-SH-3'；

S2：5'-SH-CACGAGTTGA?CA-3'；

(2) Nano silver grain is assembled into asymmetric chirality dimer under the effect of DNA enzyme

Whole assembly system comprises: the Nano silver grain of 20 nm that 70 μ L S1 modify, the Nano silver grain of 10 nm that 30 μ L S2 modify, 1 μ L 100 μ M substrate chains (Sub), 2 μ L 100 μ M enzyme chains (17E); First whole reaction system is heated to 70 ℃, is then naturally cooled to room temperature to promote the assembling of Nano silver grain; After hybridization 8h, will assemble product centrifugal three times, obtain the good asymmetric Nano silver grain dimer of purifying;

Sub：5'-ACTCATCTGT?GAACTCACTA?T(rA)GGAAGAGAT?GTGTCAACTC?GTG-3'；

17E：5'-CATCTCTTCT?CCGAGCCGGT?CGAAATAGTG?AGT-3'；

(3) Nano silver grain assembly is used for Pb as detecting sensor ²⁺detection, application CD spectrum detects

The Pb that adds a series of variable concentrations in asymmetric Nano silver grain dimer ²⁺, at Pb ²⁺effect under, substrate chain fracture, thus cause dimer to be split into single particle, the strength decreased of CD signal, detects the reaction system under different target substrate concentration with CD spectrum, obtain the CD spectrogram of 200nm-800nm, along with Pb ²⁺the increase of concentration, the dimeric quantity of asymmetric Nano silver grain reduces gradually, and CD signal reduces gradually, according to Pb ²⁺corresponding relation between concentration and CD signal intensity, draws Pb ²⁺the typical curve of concentration and CD signal intensity, thus application CD signal is to Pb ²⁺concentration carry out quantitatively.

Described 10nm, the Nano silver grain of 20nm synthesize by the method for sodium borohydride reduction silver nitrate; synthesis step: 0.6 mL 0.1 M sodium borohydride is dissolved in 20 mL frozen water; add again the polyvinylpyrrolidone (PVP) of 5 mL 1% as protective agent; separately add the trisodium citrate of 600 μ L 1% to obtain 20nm Nano silver grain, above potpourri in ice bath in continuing stirring.Then use two constant flow pumps with the speed of 30 mL/h, add respectively the polyvinylpyrrolidone (PVP) of 5 mL 1% and the silver nitrate of 5 mL 10 mM simultaneously.Finally reaction solution is hatched to remove under 80 ℃ of conditions excessive sodium borohydride, after 3 h, obtains the Nano silver grain of synthetic 10nm, and put into 4 ℃ stand-by.

Beneficial effect of the present invention: the invention provides a kind of to Pb ²⁺detect and quantitative chiral sensor method, the big or small Nano silver grain of DNA probe being modified by means of the effect of DNA enzyme is assembled into asymmetric Nano silver grain dimer, at variable concentrations Pb ²⁺under the condition existing, there is depolymerization in various degree in dimer, finally by CD spectrum, Nano silver grain assembly is measured, thus indirect detection target P b ²⁺content.

Accompanying drawing explanation

Fig. 1 Pb ²⁺the CD spectrum detecting;

Fig. 2 Pb ²⁺the typical curve detecting.

Embodiment

Embodiment 1

Measure Pb ²⁺the construction method of chiral sensor of DNA enzyme assembling.Comprise: the Nano silver grain of 10nm, 20nm is modified with a pair of DNA probe respectively, Nano silver grain is assembled into asymmetric chirality dimer under the effect of DNA enzyme, and this Nano silver grain assembly is used for Pb as detecting sensor ²⁺detection, application CD spectrum detects; Concrete steps are:

(1) Nano silver grain of 10nm, 20nm is modified with a pair of DNA probe respectively

The Nano silver grain of newly synthetic 10nm, 20nm is passed through respectively to ten times of centrifugal concentratings under the speed conditions of 10000r/min, 8000r/min, then Nano silver grain is resuspended to respectively in the 10mM Tris-HCl damping fluid that includes 50mM NaCl, makes the final concentration of the Nano silver grain of 10nm, 20nm be respectively 50 nM, 20 nM, then DNA probe and Nano silver grain are carried out to coupling according to the molar ratio of 10:1, specific as follows: the 20 μ M S1 nucleic acid fragments of 1 μ L join in the 20 nm Nano silver grains of 100 μ L 20 nM, the 50 μ M S2 nucleic acid fragments of 1 μ L join in the 10 nm Nano silver grains of 100 μ L 50 nM, hatch after 12h, centrifugal three times of the Nano silver grain of success coupling, the DNA of not coupling is removed, last nano particle is all resuspended to that in 50 μ L 25 mM Tris-acetate buffer solutions, (pH 8.2, comprise 100 mM NaCl), finally obtain the Nano silver grain that coupling is good,

S1：5'-TCACAGATGA?GT-SH-3'；

S2：5'-SH-CACGAGTTGA?CA-3'；

(2) Nano silver grain is assembled into asymmetric chirality dimer under the effect of DNA enzyme

Whole assembly system comprises: the Nano silver grain of 20 nm that 70 μ L S1 modify, the Nano silver grain of 10 nm that 30 μ L S2 modify, 1 μ L 100 μ M substrate chains (Sub), 2 μ L 100 μ M enzyme chains (17E); First whole reaction system is heated to 70 ℃, is then naturally cooled to room temperature to promote the assembling of Nano silver grain; After hybridization 8h, will assemble product centrifugal three times, obtain the good asymmetric Nano silver grain dimer of purifying;

Sub：5'-ACTCATCTGT?GAACTCACTA?T(rA)GGAAGAGAT?GTGTCAACTC?GTG-3'；

17E：5'-CATCTCTTCT?CCGAGCCGGT?CGAAATAGTG?AGT-3'；

(3) Nano silver grain assembly is used for Pb as detecting sensor ²⁺detection, application CD spectrum detects

The Pb that adds a series of variable concentrations in asymmetric Nano silver grain dimer ²⁺, at Pb ²⁺effect under, substrate chain fracture, thus cause dimer to be split into single particle, the strength decreased of CD signal.Reaction system under different target substrate concentration is detected with CD spectrum, obtain the CD spectrogram of 200nm-800nm, along with Pb ²⁺the increase of concentration, the dimeric quantity of asymmetric Nano silver grain reduces gradually, and CD signal reduces gradually, according to Pb ²⁺corresponding relation between concentration and CD signal intensity, draws Pb ²⁺the typical curve of concentration and CD signal intensity, thus application CD signal is to Pb ²⁺concentration carry out quantitatively.

(4) detection sensitivity research

According to each target P b ²⁺under concentration, the CD signal intensity of correspondence, take DNA concentration as horizontal ordinate, and CD signal intensity is that ordinate is made a typical curve, according to typical curve, calculates Pb ²⁺detection be limited to 0.02 ng mL ^-1.

(5) specificity research

With Mn ²⁺, Zn ²⁺, Mg ²⁺, Fe ²⁺, Ca ²⁺, Hg ²⁺, Cu ²⁺for detected object, carry out specificity analyses, add concentration to be 5 ng mL ^-1, method of operating and Pb ²⁺the method of operating detecting is consistent, the CD signal of reaction system and negative blank sample do not add the CD signal of any heavy metal ion to contrast, the CD signal obtaining and the CD signal of blank sample do not have significant difference, show that thus other heavy metal ion can not identify Pb ²⁺the DNA enzyme relying on, the specificity of the method is good.

(6) add recovery experiment

By the Pb of variable concentrations ²⁺join in negative tap water the Pb setting up in order to upper method ²⁺mensuration is reclaimed in the interpolation that detecting sensor is carried out in water sample, and the recovery scope finally obtaining, at 94%-100%, can be carried out the detection of actual sample.

S1：5'-TCACAGATGA?GT-SH-3'；

S2：5'-SH-CACGAGTTGA?CA-3'；

Sub：5'-ACTCATCTGT?GAACTCACTA?T(rA)GGAAGAGAT?GTGTCAACTC?GTG-3'；

17E：5'-CATCTCTTCT?CCGAGCCGGT?CGAAATAGTG?AGT-3'；

 

Claims (1)

1. measure Pb for one kind ²⁺the construction method of chiral sensor of DNA enzyme assembling, it is characterized in that comprising: the Nano silver grain of 10nm and 20nm is modified with a pair of DNA probe respectively, Nano silver grain is assembled into asymmetric chirality dimer under the effect of DNA enzyme, and this Nano silver grain assembly is used for Pb as detecting sensor ²⁺detection, application CD spectrum detects; Concrete steps are:

(1) Nano silver grain of 10nm, 20nm is modified with a pair of DNA probe respectively

The Nano silver grain of newly synthetic 10nm, 20nm is passed through respectively to ten times of centrifugal concentratings under the speed conditions of 10000r/min, 8000r/min, then Nano silver grain is resuspended to respectively in the 10mM Tris-HCl damping fluid that includes 50mM NaCl, makes the final concentration of the Nano silver grain of 10nm, 20nm be respectively 50 nM, 20 nM, then DNA probe and Nano silver grain are carried out to coupling according to the molar ratio of 10:1, specific as follows: the 20 μ M S1 nucleic acid fragments of 1 μ L join in the 20 nm Nano silver grains of 100 μ L 20 nM, the 50 μ M S2 nucleic acid fragments of 1 μ L join in the 10 nm Nano silver grains of 100 μ L 50 nM, hatch after 12h, centrifugal three times of the Nano silver grain of success coupling, the DNA of not coupling is removed, last nano particle is all resuspended to 50 μ L pH 8.2, in the 25 mM Tris-acetate buffer solutions that comprise 100 mM NaCl, finally obtain the Nano silver grain that coupling is good,

S1：5'-TCACAGATGA?GT-SH-3'；

S2：5'-SH-CACGAGTTGA?CA-3'；

(2) Nano silver grain is assembled into asymmetric chirality dimer under the effect of DNA enzyme

Whole assembly system comprises: the Nano silver grain of 20 nm that 70 μ L S1 modify, the Nano silver grain of 10 nm that 30 μ L S2 modify, 1 μ L 100 μ M substrate chain Sub, 2 μ L 100 μ M enzyme chain 17E; First whole reaction system is heated to 70 ℃, is then naturally cooled to room temperature to promote the assembling of Nano silver grain; After hybridization 8h, will assemble product centrifugal three times, obtain the good asymmetric Nano silver grain dimer of purifying;

Sub：5'-ACTCATCTGT?GAACTCACTA?T(rA)GGAAGAGAT?GTGTCAACTC?GTG-3'；

17E：5'-CATCTCTTCT?CCGAGCCGGT?CGAAATAGTG?AGT-3'；

(3) Nano silver grain assembly is used for Pb as detecting sensor ²⁺detection, application CD spectrum detects

The Pb that adds a series of variable concentrations in asymmetric Nano silver grain dimer ²⁺, at Pb ²⁺effect under, substrate chain fracture, thus cause dimer to be split into single particle, the strength decreased of CD signal, detects the reaction system under different target substrate concentration with CD spectrum, obtain the CD spectrogram of 200nm-800nm, along with Pb ²⁺the increase of concentration, the dimeric quantity of asymmetric Nano silver grain reduces gradually, and CD signal reduces gradually, according to Pb ²⁺corresponding relation between concentration and CD signal intensity, draws Pb ²⁺the typical curve of concentration and CD signal intensity, thus application CD signal is to Pb ²⁺concentration carry out quantitatively.

Images