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

Abstract

The invention discloses a method for constructing a chiral sensor assembled by DNA enzyme for measuring lead ions, which belongs to the field of nanobiological technology detection. The invention includes: silver nanoparticles of 10nm and 20nm are respectively modified with a pair of DNA probes, the modified silver nanoparticles are assembled into an asymmetric chiral dimer under the action of DNA enzyme, and the silver nanoparticle assembly is used as a detection sensor For the detection of Pb ²⁺ , circular dichroism (CD) is used for detection. The invention uses a silver nanoparticle probe, and under the action of Pb2 ⁺ -dependent DNase, large and small silver nanoparticles are assembled into an asymmetric chiral dimer, and the Pb2 ⁺ is detected through a CD signal. The method of the invention can realize ultrasensitive detection of Pb ²⁺ , has high detection sensitivity and good specificity, and can detect trace target objects in aqueous solution at the same time.

Description

A method for constructing a chiral sensor assembled by DNase for measuring lead ions

technical field

The invention relates to a method for constructing a chiral sensor assembled by DNase for measuring Pb ²⁺ , belonging to the field of nanobiological technology detection.

Background technique

Pb ²⁺ is one of the most toxic heavy metal ions. Lead pollution comes from production wastewater and wastes such as mining, smelting, rubber production, dyes, printing, ceramics, lead glass, solder, cables and lead pipes. In addition, tetraethyl lead in automobile exhaust is a highly toxic substance. With the rapid development of industry and agriculture in our country, the pollution of heavy metals in the environment is becoming more and more serious. The poison caused by heavy metal pollution persists and will enter the food chain with the recirculation of soil and water, posing a threat to food safety and endangering human life and health. So far, lead has no known physiological role in living organisms. Any lead in human body is regarded as pollution. Lead poisoning can cause gastrointestinal disorders, liver, kidney and nerve damage. The United States Environmental Protection Agency stipulates that the maximum content of Pb ²⁺ in drinking water shall not exceed 72nM. According to the survey of the Ministry of Environmental Protection, the problem of heavy metal pollution in my country's water is very prominent, and the pollution rate of the bottom of rivers, lakes and reservoirs is as high as 80.1%. According to the characteristics of heavy metal pollution, its discovery and detection is very important.

Traditional Pb ²⁺ detection methods include Inductively Coupled Plasma (ICP) and Atomic Absorption Spectrometry. The personnel have certain professional knowledge, and the analysis cost is high. Chemical and biological sensors have been favored by people because of their high sensitivity, good selectivity, small size, and low cost. In recent years, with the development of nanotechnology, a lot of research and application have been carried out in the detection of food hazard factors based on oligonucleotide functionalized nanoparticles. Since nanoparticles have special properties, such as: optical properties, electrochemical properties, fluorescence properties, paramagnetism, etc., the signals unique to these properties can be used to amplify to achieve the purpose of detecting the target. The chiral nanostructure assembled by plasmonic nanoparticles has a CD signal. This discovery has become a new development in the application of nanomaterials in the detection field. The CD signal of the chiral nanomaterial assembly can be used as a detection signal to detect harmful substances. .

DNase is a functionalized DNA molecule obtained by in vitro screening. This enzyme is different from protease and can be obtained by chemical synthesis. It has good thermal stability and reactivity. At the same time, DNA molecules can modify various functional groups. And it can be immobilized on a solid support. At present, a large number of target objects have DNA enzymes that can be recognized. In the presence of heavy metal ions, the substrate chain of the heavy metal-dependent DNase is cut at the RNA base cleavage site, and the Pb ²⁺ -dependent DNase is called "17 E" DNase, which consists of an enzyme chain Composed with a substrate chain, the Pb ²⁺ detection sensor established by using DNase has aroused widespread research interest.

The present invention assembles silver nanoparticles of large and small sizes modified by DNA probes into asymmetric silver nanoparticle dimers by virtue of the action of DNA enzymes, and the dimers are dissociated in different degrees under the conditions of the presence of different concentrations of Pb ²⁺ Polymerization, as the concentration of Pb ²⁺ increases, the degree of disaggregation of the dimer increases, and the CD signal decreases accordingly. According to the corresponding relationship between the intensity of the CD signal and the concentration of Pb ²⁺ , the Pb ^{2 +} content is tested.

Contents of the invention

The object of the present invention is to provide a kind of chiral sensor method that Pb ²⁺ is detected and quantified, by means of the effect of DNA enzyme, the size silver nanoparticles modified by DNA probes are assembled into asymmetric silver nanoparticle dimers, In the presence of different concentrations of Pb ²⁺ , the dimer depolymerized to different degrees, and finally the silver nanoparticle assembly was measured by CD spectroscopy, so as to indirectly detect the content of the target Pb ²⁺ .

Technical scheme of the present invention: a method for constructing a chiral sensor assembled by DNase for measuring Pb ²⁺ , comprising: silver nanoparticles of 10nm and 20nm are modified with a pair of DNA probes respectively, Under the action, it is assembled into an asymmetric chiral dimer, and the silver nanoparticle assembly is used as a detection sensor for the detection of Pb ²⁺ , and CD spectroscopy is used for detection; the specific steps are:

(1) Silver nanoparticles of 10nm and 20nm were modified with a pair of DNA probes respectively

Concentrate the newly synthesized 10nm and 20nm silver nanoparticles by centrifugation at 10000r/min and 8000r/min respectively, and then resuspend the silver nanoparticles into 10mM Tris-HCl buffer containing 50mM NaCl In the method, the final concentrations of 10nm and 20nm silver nanoparticles were 50 nM and 20 nM respectively; then DNA probes and silver nanoparticles were coupled at a molar ratio of 10:1, specifically as follows: 1 μL of 20 μM S1 nucleic acid The fragments were added to 100 μL of 20 nM 20 nm silver nanoparticles, and 1 μL of 50 μM S2 nucleic acid fragments were added to 100 μL of 50 nM 10 nm silver nanoparticles. After incubation for 12 hours, the successfully coupled silver nanoparticles were centrifuged three times. , the uncoupled DNA was removed, and finally the nanoparticles were resuspended in 50 μL of 25 mM Tris-acetate buffer (pH 8.2, containing 100 mM NaCl), and finally the coupled silver nanoparticles were obtained;

S1: 5'-TCACAGATGA GT-SH-3';

S2: 5'-SH-CACGAGTTGA CA-3';

(2) Silver nanoparticles are assembled into asymmetric chiral dimers under the action of DNase

The whole assembly system includes: 70 μL S1 modified 20 nm silver nanoparticles, 30 μL S2 modified 10 nm silver nanoparticles, 1 μL 100 μM substrate chain (Sub), 2 μL 100 μM enzyme chain (17E); Firstly, the entire reaction system was heated to 70°C, and then naturally cooled to room temperature to promote the assembly of silver nanoparticles; after hybridization for 8 hours, the assembled product was centrifuged three times to obtain a purified asymmetric silver nanoparticle dimer;

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

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

(3) The silver nanoparticle assembly is used as a detection sensor for the detection of Pb ²⁺ , and the CD spectrum is used for detection

Adding a series of different concentrations of Pb ²⁺ to the asymmetric silver nanoparticle dimer, under the action of Pb ²⁺ , the substrate chain is broken, which causes the dimer to be split into individual particles, and the intensity of the CD signal decreases , the reaction system under different target concentrations was detected by CD spectrum, and the CD spectrum of 200nm-800nm was obtained. With the increase of Pb ²⁺ concentration, the number of asymmetric silver nanoparticle dimers gradually decreased, and the CD signal gradually decreased. According to the corresponding relationship between Pb ²⁺ concentration and CD signal intensity, draw the standard curve of Pb ²⁺ concentration and CD signal intensity, so as to use CD signal to quantify the concentration of Pb ²⁺ .

The silver nanoparticles of 10nm and 20nm were synthesized by reducing silver nitrate with sodium borohydride. Synthesis steps: 0.6 mL of 0.1 M sodium borohydride was dissolved in 20 mL of ice water, and then 5 mL of 1% polyvinylpyrrolidone ( PVP) was used as a protective agent, and 600 μL of 1% trisodium citrate was added to obtain 20nm silver nanoparticles, and the above mixture was kept stirring in an ice bath. Then, 5 mL of 1% polyvinylpyrrolidone (PVP) and 5 mL of 10 mM silver nitrate were added simultaneously at a rate of 30 mL/h using two constant-flow pumps. Finally, the reaction solution was incubated at 80°C to remove excess sodium borohydride. After 3 h, the synthesized 10nm silver nanoparticles were obtained, and placed at 4°C for use.

Beneficial effects of the present invention: the present invention provides a chiral sensor method for detecting and quantifying Pb ²⁺ , by means of the action of DNase, silver nanoparticles of large and small sizes modified by DNA probes are assembled into asymmetric silver nanoparticles. In the presence of different concentrations of Pb ²⁺ , the dimer depolymerized to different degrees, and finally the silver nanoparticle assembly was measured by CD spectroscopy, so as to indirectly detect the content of the target Pb ²⁺ .

Description of drawings

Figure 1 CD spectrum of Pb ²⁺ detection;

Figure 2 Standard curve for Pb ²⁺ detection.

Detailed ways

Example 1

Construction of a DNase-assembled chiral sensor for the measurement of Pb ²⁺ . Including: 10nm and 20nm silver nanoparticles are respectively modified with a pair of DNA probes, and the silver nanoparticles are assembled into an asymmetric chiral dimer under the action of DNase, and the silver nanoparticle assembly is used as a detection sensor for Pb For the detection of ²⁺ , CD spectrum is used for detection; the specific steps are:

(1) Silver nanoparticles of 10nm and 20nm were modified with a pair of DNA probes respectively

Concentrate the newly synthesized 10nm and 20nm silver nanoparticles by centrifugation at 10000r/min and 8000r/min respectively, and then resuspend the silver nanoparticles into 10mM Tris-HCl buffer containing 50mM NaCl In the method, the final concentrations of 10nm and 20nm silver nanoparticles were 50 nM and 20 nM respectively; then DNA probes and silver nanoparticles were coupled at a molar ratio of 10:1, specifically as follows: 1 μL of 20 μM S1 nucleic acid The fragments were added to 100 μL of 20 nM 20 nm silver nanoparticles, and 1 μL of 50 μM S2 nucleic acid fragments were added to 100 μL of 50 nM 10 nm silver nanoparticles. After incubation for 12 hours, the successfully coupled silver nanoparticles were centrifuged three times. , the uncoupled DNA was removed, and finally the nanoparticles were resuspended in 50 μL of 25 mM Tris-acetate buffer (pH 8.2, containing 100 mM NaCl), and finally the coupled silver nanoparticles were obtained;

S1: 5'-TCACAGATGA GT-SH-3';

S2: 5'-SH-CACGAGTTGA CA-3';

(2) Silver nanoparticles are assembled into asymmetric chiral dimers under the action of DNase

The whole assembly system includes: 70 μL S1 modified 20 nm silver nanoparticles, 30 μL S2 modified 10 nm silver nanoparticles, 1 μL 100 μM substrate chain (Sub), 2 μL 100 μM enzyme chain (17E); Firstly, the whole reaction system was heated to 70°C, and then naturally cooled to room temperature to promote the assembly of silver nanoparticles; after hybridization for 8 hours, the assembled product was centrifuged three times to obtain a purified asymmetric silver nanoparticle dimer;

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

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

(3) The silver nanoparticle assembly is used as a detection sensor for the detection of Pb ²⁺ , and the CD spectrum is used for detection

Adding a series of different concentrations of Pb ²⁺ to the asymmetric silver nanoparticle dimer, under the action of Pb ²⁺ , the substrate chain is broken, which causes the dimer to be split into individual particles, and the intensity of the CD signal decreases . The reaction system under different target concentrations was detected by CD spectrum, and the CD spectrum of 200nm-800nm was obtained. With the increase of Pb ²⁺ concentration, the number of asymmetric silver nanoparticle dimers gradually decreased, and the CD signal gradually decreased. , according to the corresponding relationship between Pb ²⁺ concentration and CD signal intensity, draw the standard curve of Pb ²⁺ concentration and CD signal intensity, so as to use CD signal to quantify the concentration of Pb ²⁺ .

(4) Research on detection sensitivity

According to the CD signal intensity corresponding to each target Pb ²⁺ concentration, a standard curve was drawn with the DNA concentration as the abscissa and the CD signal intensity as the ordinate, and the detection limit of Pb ²⁺ was calculated as 0.02 ng mL ^{- 1} .

(5) Specificity research

Mn ²⁺ , Zn ²⁺ , Mg ²⁺ , Fe ²⁺ , Ca ²⁺ , Hg ²⁺ , and Cu ²⁺ were used as detection objects for specificity analysis. The concentration was 5 ng mL ^-1 , and the operation method was the same as The operation method of Pb ²⁺ detection is the same. The CD signal of the reaction system is compared with the CD signal of the negative blank sample without adding any heavy metal ions. There is no significant difference between the obtained CD signal and the CD signal of the blank sample. Ions do not recognize Pb ²⁺ -dependent DNase, and the specificity of this method is good.

(6) Add recovery experiment

Add different concentrations of Pb ²⁺ into the negative tap water, and use the Pb ²⁺ detection sensor established by the above method to carry out the addition and recovery determination of the water sample. The final recovery rate ranges from 94% to 100%, which can be used for the actual sample. detection.

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';

the

Claims (1)

1. A method for constructing a chiral sensor that measures Pb ²⁺ DNase assembly, is characterized in that comprising: the silver nanoparticles of 10nm and 20nm are modified with a pair of DNA probes respectively, and the silver nanoparticles are used in the effect of DNase Assembled into an asymmetric chiral dimer, the silver nanoparticle assembly is used as a detection sensor for the detection of Pb ²⁺ , and the CD spectrum is used for detection; the specific steps are: (1) Silver nanoparticles of 10nm and 20nm were modified with a pair of DNA probes respectively Concentrate the newly synthesized 10nm and 20nm silver nanoparticles by centrifugation at 10000r/min and 8000r/min respectively, and then resuspend the silver nanoparticles into 10mM Tris-HCl buffer containing 50mM NaCl In the method, the final concentrations of 10nm and 20nm silver nanoparticles were 50 nM and 20 nM respectively; then DNA probes and silver nanoparticles were coupled at a molar ratio of 10:1, specifically as follows: 1 μL of 20 μM S1 nucleic acid The fragments were added to 100 μL of 20 nM 20 nm silver nanoparticles, and 1 μL of 50 μM S2 nucleic acid fragments were added to 100 μL of 50 nM 10 nm silver nanoparticles. After incubation for 12 hours, the successfully coupled silver nanoparticles were centrifuged three times. , the uncoupled DNA was removed, and finally the nanoparticles were resuspended in 50 μL pH 8.2, containing 100 mM NaCl in 25 mM Tris-acetate buffer, and finally the coupled silver nanoparticles were obtained; S1: 5'-TCACAGATGA GT-SH-3'; S2: 5'-SH-CACGAGTTGA CA-3'; (2) Silver nanoparticles are assembled into asymmetric chiral dimers under the action of DNase The whole assembly system includes: 70 μL S1 modified 20 nm silver nanoparticles, 30 μL S2 modified 10 nm silver nanoparticles, 1 μL 100 μM substrate chain Sub, 2 μL 100 μM enzyme chain 17E; The system was heated to 70°C, and then cooled naturally to room temperature to promote the assembly of silver nanoparticles; after hybridization for 8 hours, the assembled product was centrifuged three times to obtain a purified asymmetric silver nanoparticle dimer; Sub: 5'-ACTCATCTGT GAACTCACTA T(rA)GGAAGAGAT GTGTCAACTC GTG-3'; 17E: 5'-CATCTCTTCT CCGAGCCGGT CGAAATAGTG AGT-3'; (3) The silver nanoparticle assembly is used as a detection sensor for the detection of Pb ²⁺ , and the CD spectrum is used for detection Adding a series of different concentrations of Pb ²⁺ to the asymmetric silver nanoparticle dimer, under the action of Pb ²⁺ , the substrate chain is broken, which causes the dimer to be split into individual particles, and the intensity of the CD signal decreases , the reaction system under different target concentrations was detected by CD spectrum, and the CD spectrum of 200nm-800nm was obtained. With the increase of Pb ²⁺ concentration, the number of asymmetric silver nanoparticle dimers gradually decreased, and the CD signal gradually decreased. According to the corresponding relationship between Pb ²⁺ concentration and CD signal intensity, draw the standard curve of Pb ²⁺ concentration and CD signal intensity, so as to use CD signal to quantify the concentration of Pb ²⁺ .

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