CN108929896B

CN108929896B - Copper ion mismatch type universal partition ultrafast amplification colorimetric sensor

Info

Publication number: CN108929896B
Application number: CN201810637640.1A
Authority: CN
Inventors: 许文涛; 罗云波; 黄昆仑; 杜再慧; 田晶晶
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-06-20
Filing date: 2018-06-20
Publication date: 2020-07-28
Anticipated expiration: 2038-06-20
Also published as: CN108929896A

Abstract

The invention belongs to the field of heavy metal detection, and particularly discloses a copper ion mismatched universal partition ultrafast amplification colorimetric sensor. The invention skillfully designs a primer and a template (shown in SEQ ID NO. 1-3), so that the template can be subjected to ultrafast amplification in the presence of copper ions, and an amplification product forms a G quadruplex in a proper environment. The G quadruplex peroxidase-like activity is further utilized for color development, the problem that the traditional PCR product is difficult to detect visually is solved, and the rapid and visual detection of copper ions is realized. Moreover, the sensor and the method provided by the invention have the characteristics of high specificity and high sensitivity on copper ions, and the detection result is more objective and accurate.

Description

Copper ion mismatch type universal partition ultrafast amplification colorimetric sensor

Technical Field

The invention belongs to the field of heavy metal detection, and particularly relates to a copper ion mismatched universal partition ultrafast amplification colorimetric sensor.

Background

Copper is a transition element, Cu, with the chemical notation Cu, atomic number 29, and atomic weight 63.546, belonging to group IB. Pure copper is soft metal, has red orange color band metallic luster when the surface is just cut, and has a purplish red simple substance. The copper ion has good ductility, high thermal conductivity and electrical conductivity, the amount of copper in normal human body is 100-200 mg, the copper ion in human body mainly takes the form of catalytic accessory factors or structures of a plurality of enzymes and proteins, and is widely involved in a plurality of important metabolic processes in vivo, and influences the generation of human blood, the formation of connective tissues, the central nervous system, the metabolism of cholesterol and glucose, the cardiac function, the immune system and the like, and the human body can maintain normal life activities only by trace amount of copper. However, a lack of copper or an excess of copper can have adverse health effects. Copper deficiency is generally accompanied by a deficiency in other nutrients or an excessive uptake of its biological antagonists, affecting the normal function of many enzymes in the cell and, in turn, the metabolic processes of the cell. Copper overdose is often caused by genetic diseases or by environmental heavy metal pollution, by mistaking large amounts of copper-containing food or by inhaling gases with high copper content. Pollution caused by copper (Cu) and its compounds in the environment is mainly generated by mining and smelting of copper-zinc ores, metal processing, machinery manufacturing, steel production and the like, wherein smoke dust discharged by smelting is a main source of atmospheric copper pollution.

At present, a plurality of methods for detecting copper ions are available, and mainly comprise Atomic Absorption Spectrometry (AAS), Atomic Fluorescence Spectrometry (AFS), inductively coupled plasma mass spectrometry, an electrochemical analysis method, a visible spectrophotometry method, a flow injection chemiluminescence method, a differential potential dissolution method and the like. The methods have the advantages of high sensitivity, wide detection range, suitability for analysis of various samples and the like, but the methods also have the defects of complex pretreatment, need of large instruments and professional personnel for operation, high maintenance cost, long detection time, unsuitability for rapid field detection and the like. Therefore, a new method for visually detecting copper ions, which is simple to operate, low in price, sensitive, rapid and accurate, is urgently needed.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a copper ion mismatched universal partition ultrafast amplification colorimetric sensor for realizing rapid and visual detection of copper ions.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides a copper ion mismatched universal blocking ultrafast amplification colorimetric sensor comprising: (1) the kit comprises (1) an sPCR amplification system, (2) a detection system containing ABTS color development liquid, wherein the detection system is used for performing color development detection on a product obtained after a sample to be detected is amplified by the sPCR amplification system;

wherein the sPCR amplification system comprises: template, DNA polymerase, forward primer, reverse primer, dNTP and buffer solution;

the template is as follows:

CCTCAGTATCAGTGCTATACGTC

TTGTTGTTGCGATAGCGCCAGC；

the forward primer is as follows:

GTGGGTAGGGCGGGTTGG-cut-off-CCAACCCGCCCTACCCAC

The reverse primer is as follows:

GTGGGTAGGGCGGGTTGG-cut-off-CCAACCCGCCCTACCCAC

The partition between the forward primer and the reverse primer is poly-hexaethylene glycol.

The partition is connected with bases at two ends in a phosphodiester bond mode.

In the invention, the formula of the ABTS chromogenic solution comprises 1m L DNAzyme substrate buffer solution, 0.933g of citric acid, 100m L of distilled water, 5 mu L of ABTS substrate solution and 1 mu L30% of H₂O₂。

DNAzyme substrate buffer: namely citrate buffer solution with pH 3.6, and the formula is as follows: na (Na)₂HPO₄.12H₂O1.843g, citric acid 0.933g and distilled water 100m L.

ABTS substrate solution 20mg of 2,2' -diaza-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt powder (purchased from Sigma) was dissolved in 1m L DMSO.

The DNA polymerase was Ex Taq DNA polymerase, the Buffer was 10 × Ex Taq Buffer, and both were purchased from Saimer fly Technologies (Thermo Scientific L files) along with the dNTPs.

In the presence of copper ions, the bases in italic font in the forward and reverse primers will successfully pair with the bases in italic font in the template sequence based on the mismatch of copper ions with cytosine, thereby initiating sPCR amplification of the primer with the template. However, continued extension by the DNA polymerase will be prevented by the presence of the cleavage such that the sPCR product carries a single strand at its 5 'and 3' ends with a G-rich sequence.

Further, at K⁺In the presence of (b), the sPCR product will bind chlorine highIron heme forms a G-quadruplex structure with peroxidase-like activity, catalyzing H₂O₂And ABTS color development, and the detection of copper ions is completed through colorimetric detection.

Therefore, based on the above detection principle, the detection system of the present invention comprises: enzyme activity buffer solution, hemin solution.

Wherein the enzyme activity buffer solution is: 100mM Tris, 120mM NaCl, 10mM MgCl₂、100mM KCl，pH8.4。

The hemin solution is hemin diluted solution obtained by mixing 20mM hemin stock solution and the enzyme activity buffer solution according to the proportion of 2 mu L: 1m L.

In a second aspect, the invention provides the use of the aforementioned sensor for detecting copper ions, which detection may be characterized as qualitative or quantitative.

In a third aspect, the present invention provides a method for qualitatively detecting copper ions by using the aforementioned sensor, comprising the following steps:

s1, carrying out ultrafast polymerase chain reaction on the sample to be detected and the negative control sample by using the sPCR amplification system to obtain an sPCR product;

s2, detecting the sPCR product by using the detection system;

carrying out qualitative judgment on copper ions according to the color difference between the sample to be detected and the negative control sample;

the negative control sample was deionized water containing no copper ions.

The sPCR amplification system includes a reducing agent for reducing copper ions to cuprous ions, such as sodium ascorbate, but is not limited thereto.

When the experimental group and the negative group have obvious color change in contrast, judging that the sample to be detected contains copper ions; and when no obvious color change exists, judging that the content of the copper ions in the sample to be detected is lower than the qualitative detection limit.

Further, in the sPCR amplification system, the amount of the forward primer is equal to the sum of the amounts of the reverse primers.

Preferably, the S1 includes:

s11, preparing an sPCR reaction system on ice:

s12, rapidly placing the reactor in an sPCR reaction device for temperature control:

2s at 90-95 ℃, 3s at 55-60 ℃ and 30-40 cycles; preferably 95 ℃ for 2s, 58 ℃ for 3s, 36 cycles.

S13, finishing the sPCR reaction process, and verifying the amplification effect of the sPCR reaction system by using polyacrylamide gel electrophoresis, wherein the reaction conditions are as follows: 120V 2h, photographing system: molecular Imager Gel Doc XR (Bio-Rad).

Further, the detection system comprises an enzyme activity buffer solution and a hemin diluted solution, the enzyme activity buffer solution, the hemin diluted solution and the sPCR product are uniformly mixed according to the volume ratio of 8:1:1 to obtain a mixture, the mixture reacts for 30min at 37 ℃, ABTS color development liquid with the same volume as the mixture is added, the mixture is uniformly mixed, the mixture is incubated for 10min at 37 ℃ in a dark place, and monitoring is carried out by naked eyes.

For example, enzyme activity buffer 80 mu L, hemin diluted solution 10 mu L and sPCR product 10 mu L are taken, the substances are mixed uniformly and react for 30min at 37 ℃, the sPCR product is combined with hemin to form a G-quadruplex structure with peroxidase-like activity, ABTS color developing solution with the volume equal to that of the mixture (100 mu L) is added, the mixture is mixed uniformly and incubated for 10min in dark at 37 ℃, and the monitoring is carried out by naked eyes.

In a fourth aspect, the present invention provides a method for quantitative detection of copper ions by using the aforementioned sensor, comprising the following steps:

SI, standard curve preparation:

constructing an sPCR system with different copper ion concentrations by using a copper ion solution with a known concentration, wherein the amplification and detection steps are the same as those of the qualitative detection method;

then, taking the concentration of copper ions as an abscissa and the OD415 value as an ordinate to draw a standard curve;

wherein the concentration interval of different copper ion concentrations is 0.2-10 μ M; the lowest detection limit is 56nM (the absorbance value corresponding to the lowest detection limit is the average blank absorbance value +3 σ (blank value standard deviation), also called 3 σ rule principle, then the lowest detection concentration is deduced according to the standard curve), in one embodiment of the invention, the standard curve is made by adopting the copper ion concentrations of 0.2 μ M, 0.5 μ M, 1 μ M, 2 μ M, 5 μ M and 10 μ M;

and SII, detecting the sample to be detected according to the qualitative detection method, substituting the measured OD415 value into the standard curve, and calculating to obtain the content of copper ions in the sample to be detected so as to realize quantitative detection of the copper ions.

The invention has the beneficial effects that:

the invention provides a sensor and a method for rapidly detecting copper ions based on a DNA and copper ion mismatch principle, which can carry out ultrafast amplification on a template in the presence of copper ions by skillfully designing a primer and the template, reduce the time consumption of a traditional PCR process of about 3 hours to 10 minutes, and remarkably reduce the time consumption of PCR reaction. The color development is further carried out by combining with the peroxidase-like activity of the G quadruplex, the difficult problem that the traditional PCR product is difficult to detect visually is solved, and the rapid and visual detection of copper ions is realized.

Moreover, the sensor and the method provided by the invention have the characteristics of high specificity and high sensitivity on copper ions, and the detection result is more objective and accurate.

Drawings

FIG. 1 is a polyacrylamide gel electrophoresis of example 1 to verify the amplification effect of the sPCR reaction system; wherein, lane 1: DNA ladder; lane 2: cu²⁺Adding to the sPCR product obtained in the reaction system.

FIG. 2 is a qualitative test in example 1.

FIG. 3 is a standard curve according to example 2 of the present invention.

FIG. 4 shows a specificity test performed in example 3 of the present invention.

FIG. 5 shows the reverse primer mismatch base optimization experiment performed in comparative example 1 of the present invention.

Detailed Description

The present invention is further illustrated by the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the inventive concepts of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The experimental materials used in the present invention are as follows:

SYBR Gold nucleic acid dye, nucleic acid molecular weight standard ultra-low range DNA ladder, dNTP, ExTaq DNA polymerase, 10 × Taq buffer, hemin, cupric chloride, sodium ascorbate, 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diamine salt (ABTS), H₂O₂All purchased from Thermo Scientific L ife technologies.

In addition, materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 qualitative test

This example uses ultrapure water with different concentrations of copper ions added artificially as the sample to be measured to illustrate the use of the sensor and the method of the present invention.

1. Construction of sPCR device

The temperature change of the sPCR device is realized by a high-temperature water bath kettle at 95 ℃ and a medium-temperature water bath kettle at 58 ℃, L light Cycler type capillaries (20u L, 04929292001, Roche) are used as sPCR sample chambers, samples are respectively gathered to one end of each capillary in a rapid centrifugation mode, and the capillaries with the samples are fixed on a special plastic bracket after the centrifugation is finished.

2. sPCR reaction

The sPCR reaction system is shown in the following table:

TABLE 1

Reaction components	Final concentration
		Template	0.01μM
Ex Taq DNA polymerase	1.5U/mL
		Reverse primer	2μM
Forward primer	2μM
		Sample to be tested	2μL
Ascorbic acid sodium salt	50mM
		dNTP	250μM

	10×Ex Taq Buffer	1×
ddH₂O			Make up to 10 μ L

Note: 50mM sodium ascorbate is added into a sample to be tested, and the solution is prepared as before.

sPCR reaction process:

according to the above table, a 10. mu.l reaction system was prepared on ice and rapidly placed in an sPCR reaction apparatus for temperature control: 95 ℃ for 2s, 58 ℃ for 3s, 36 cycles.

The sPCR reaction process was completed, and the amplification effect of the sPCR reaction system was verified by using 20% polyacrylamide gel electrophoresis (see FIG. 1), and the reaction conditions were as follows: 120V 2h, photographing system: molecular Imager Gel Doc XR (Bio-Rad).

Experimental results show that the universal blocking primer can be combined with the template in the presence of target metal ions, and amplification can be completed in a short time.

3. Chromogenic detection of sPCR products

Preparing a detection system:

80 μ L enzyme activity buffer (100mM Tris, 120mM NaCl, 10mM MgCl)₂100mM KCl, pH8.4), 10. mu. L diluted hemin solution (2. mu. L stock hemin (20mM) mixed with 1m L enzyme activity buffer) and 10. mu. L sPCR product.

Mixing, reacting at 37 deg.C for 30min to make sPCR product combine with hemin to form G-quadruplex structure with peroxidase-like activity, adding 100 μ L ABTS color development solution, mixing, incubating at 37 deg.C in dark for 10min, and monitoring with naked eye.

Further, deionized water containing no copper ions was used as a control group in this example to verify the accuracy of the sensor and method provided by the present invention in qualitative detection.

The results are shown in FIG. 2, which shows that the color of the experimental group and the control group is changed significantly.

Example 2 quantitative assay

In this embodiment, on the basis of the qualitative detection described in embodiment 1, the quantitative detection of copper ions in a sample to be detected is realized by making a standard curve using copper ion solutions with different concentrations.

Relative to example 1, this example adds the steps of preparing a standard curve, as follows:

an sPCR reaction system with final copper ion concentrations of 0.2. mu.M, 0.5. mu.M, 1. mu.M, 2. mu.M, 5. mu.M and 10. mu.M was prepared using a copper ion solution of known concentration (the reaction system was the same as in example 1 except that the final copper ion concentration was different from that in example 1), and the sPCR product was allowed to form a G quadruplex under appropriate conditions to catalyze ABTS color development, with the standard curve shown in FIG. 3.

The regression equation is: 0.0658X +0.0203, R²＝0.9997。

The method for amplifying and detecting the sample to be detected is the same as that in embodiment 1, in this embodiment, the OD415 value obtained by detection can be substituted into the regression equation to calculate, so as to realize quantitative detection of the sample to be detected.

Example 3 specificity test

This example serves to verify the specificity of the sensors and methods of the present invention.

This example was carried out by mixing 2. mu.M of Cu²⁺Adding proper amount of sodium ascorbate, and 100 mu M Pb²⁺、Cr³⁺、Zn²⁺、Cd²⁺The specific test is carried out by the method described in example 1 after the specific test is respectively added into the reaction system, and the test result is shown in figure 4, which shows that the sensor and the method of the invention have higher specificity to copper ions.

Example 4 sensitivity test

This example serves to verify the sensitivity of the sensor and method of the present invention.

This example was carried out by mixing 0.2, 1, 5. mu.M of Cu²⁺The labeled samples were added to the reaction system separately for sensitivity experiments, and the results are shown in Table 2.

TABLE 2

It can be seen that the recovery rate of the normalized product obtained by the sensor and method of the present invention is 95-104%.

Comparative example 1

This comparative example is used to illustrate the effect of the number of mismatched bases on the detection accuracy of the reverse primer designed in the present invention.

The invention selects the optimal reverse primer sequence by designing the reverse primers with different mismatched base numbers, which comprises the following steps:

the number of mismatched bases of the reverse primer is respectively designed into two, four and eight groups, and the experiments are carried out in three groups.

The sequence is as follows:

four mismatches:

GTGGGTAGGGCGGGTTGG-cut-off-CCAACCCGCCCTACCCAC

Six mismatches:

GTGGGTAGGGCGGGTTGG-cut-off-CCAACCCGCCCTACCCAC

Eight mismatches:

GTGGGTAGGGCGGGTTGG-cut-off-CCAACCCGCCCTACCCAC

The three sets of reverse primers were added to the reaction system, and the experiment was performed according to the reaction system and method described in example 1, and the results are shown in fig. 5.

It should be understood that the technical solutions of the above embodiments, in which the amounts of reagents or raw materials used are proportionally increased or decreased, are substantially the same as those of the above embodiments.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<120> copper ion mismatch type universal partition ultrafast amplification colorimetric sensor

<141>2018-05-28

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tgataagtca cgattgttgt tgcgatagcg ccagc 95

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<213> Artificial primer (Artificial Sequence)

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gtgggtaggg cgggttggcc aacccgccct acccactcat cgcaccgtca aaggaacc 58

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gtgggtaggg cgggttggcc aacccgccct acccactcgt gacttatcat ggggcccc 58

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Claims

1. A copper ion mismatched universal cut-off amplification colorimetric sensor, comprising: (1) the kit comprises (1) an sPCR amplification system, (2) a detection system containing ABTS color development liquid, wherein the detection system is used for performing color development detection on a product obtained after a sample to be detected is amplified by the sPCR amplification system;

wherein the sPCR amplification system comprises: the kit comprises a template, a forward primer, a reverse primer and a reducing agent for reducing copper ions into cuprous ions;

the template is as follows:

TCATCGCACCGTCAAAGGAACCTCAGTATCAGTGCTATACGTC GATCAGTACCCCCCCCATGATAAGTCACGATTGTTGTTGCGATAG CGCCAGC；

the forward primer is as follows:

GTGGGTAGGGCGGGTTGG-cut-off-CCAACCCGCCCTACCCACTCATCGCACCGTCAAAGGAACC；

The reverse primer is as follows:

GTGGGTAGGGCGGGTTGG-cut-off-CCAACCCGCCCTACCCACTCGTGACTTATCATCCCCCCCC；

The detection system contains K⁺And hemin.

2. The sensor of claim 1, wherein the partition in the forward primer and the reverse primer is poly-hexaethylene glycol.

3. The sensor of claim 1 or 2, wherein the detection system comprises: enzyme activity buffer solution and hemin solution.

4. Use of a sensor according to any one of claims 1 to 3 for detecting copper ions.

5. The use according to claim 4, wherein the detection is a qualitative or quantitative detection.

6. A method for the qualitative detection of copper ions using a sensor according to any one of claims 1 to 3, comprising the steps of:

s1, carrying out polymerase chain reaction on the sample to be detected and the negative control sample by using the sPCR amplification system to obtain an sPCR product;

s2, detecting the sPCR product by using the detection system;

the negative control sample was deionized water containing no copper ions.

7. The method according to claim 6, wherein the detection system comprises enzyme activity buffer solution and hemin diluted solution, the enzyme activity buffer solution, the hemin diluted solution and the sPCR product are uniformly mixed according to the volume ratio of 8:1:1 to obtain a mixture, the mixture is reacted for 20-40min at 35-40 ℃, ABTS color developing solution with the same volume as the mixture is added, the mixture is uniformly mixed, and the mixture is incubated at 35-40 ℃ in a dark place and monitored by naked eyes.

8. A method for quantitative detection of copper ions by using the sensor according to any one of claims 1 to 3, comprising the steps of:

SI, standard curve preparation:

constructing an sPCR system with different copper ion concentrations by using copper ion solutions with known concentrations, wherein the amplification and detection steps are the same as S1 and S2 in claim 6;

drawing a standard curve by taking the concentration of copper ions as an abscissa and taking the OD415 value as an ordinate;

and SII, detecting the sample to be detected according to the method of claim 6, substituting the measured OD415 value into the standard curve, and calculating to obtain the content of copper ions in the sample to be detected so as to realize the quantitative detection of the copper ions.

9. The method according to claim 8, wherein the concentration interval of the different copper ion concentrations is 0.2 to 10 uM.