CN114354712A

CN114354712A - Bright green indicator type electrochemical biosensor for detecting cadmium

Info

Publication number: CN114354712A
Application number: CN202111486125.6A
Authority: CN
Inventors: 邹辉; 刘茵茵; 陈世豪; 邱茹涵; 王赪胤; 刘宗平; 袁燕; 卞建春
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-04-15

Abstract

The invention discloses a bright green indicator type electrochemical biosensor for detecting cadmium, wherein bright green BG is used as a DNA hybridization indicator, a carbon paste electrode is used as a working electrode, and Cd is modified on the surface of the carbon paste electrode²⁺Specific sensitive dsDNA; when Cd²⁺Cd interaction with dsDNA due to higher affinity of BG to ssDNA than with dsDNA²⁺The reduction peak current of BG can be changed while the stability of dsDNA is damaged. By detecting the change of BG electrochemical signals, the Cd is detected²⁺Specific detection of (3).

Description

Bright green indicator type electrochemical biosensor for detecting cadmium

Technical Field

The invention belongs to the technical field of animal product heavy metal detection, and particularly relates to a bright green indicator type electrochemical biosensor for detecting cadmium.

Background

With the rapid development of urbanization and industrialization in China, heavy metal environmental pollution becomes a very prominent problem, and the quality safety of livestock and poultry products is indirectly influenced. The heavy metal cadmium and the compound thereof have obvious biological toxicity to animals and human beings due to environmental pollution. Once entering into the body, heavy metals can cause some chronic diseases, deformity, cancers, etc. With the rapid development of the large-scale livestock and poultry breeding industry, on one hand, the excrement and heavy metals in some breeding wastes cannot be digested in situ due to the adoption of a high-density and large-scale breeding mode in a farm; on the other hand, in order to improve the utilization rate of the feed, prevent diseases, promote growth and the like, a great amount of trace elements such as copper, zinc, arsenic and the like are added into the feed, and heavy metal cadmium is also doped due to the special properties of the trace elements, wherein most heavy metals in the breeding waste are discharged along with livestock and poultry manure, so that the risk of environmental pollution caused by the heavy metals in the breeding waste due to insufficient investment of treatment technologies and equipment facilities of the waste in the breeding farm for a long time is caused. Once heavy metals cause serious pollution to the surrounding environment, the environment is irreversible and long-term, particularly, the soil is difficult to recover in a short period due to heavy metal pollution, and the heavy metals can enter human bodies and animal bodies through related food chains to cause toxic damage to the bodies.

The World Health Organization (WHO) ranks cadmium as a potential carcinogen, with a secondary carcinogenic risk to humans. The international agency for research on cancer (IARC) also classifies cadmium as a carcinogen and causes serious health damage to the human body; cadmium is listed as a seventh substance harmful to human health by the U.S. poison and disease registry (ATSDR) and the U.S. Environmental Protection Agency (EPA); china also ranks cadmium as one of the key monitoring indicators for total emission control. In 2019, 7 and 23, cadmium and cadmium compounds are listed in the national poisonous and harmful water pollution record (the first batch), and the comprehensive discharge of sewage is controlled (below 0.1 mg/L). According to the source of cadmium intake, the U.S. Food and Drug Administration (FDA) stipulates that the concentration of cadmium ions in drinking water does not exceed 0.005mg/L, while the limit value of WHO is 0.003mg/L, and GB5749-2006 sanitary Standard for Drinking Water in China also stipulates that the concentration of cadmium does not exceed 0.005 mg/L.

The heavy metal cadmium has poor migration and strong residue in the environment, is accumulative and difficult to degrade, and can be enriched in human bodies and animal bodies through food chains, thereby causing chronic poisoning. Therefore, quantitative analysis of heavy metals is becoming especially important in the field of food safety detection. At present, the detection aiming at heavy metal cadmium pollution is respectively an optical analysis method, an electrochemical analysis method and a biological analysis method. The optical analysis method is used as an official detection means, and mainly comprises an atomic absorption method (AAS), an ion chromatography method, an inductively coupled plasma emission spectrometry (ICP-MS), an ultraviolet spectrophotometry (UV), a High Performance Liquid Chromatography (HPLC), an X-ray fluorescence spectrometry (XRF) and the like. A standard method for detecting trace cadmium in food is issued based on AAS in China (GB 5009.15-2014). These methods are capable of accurately quantifying metal content, but such assays have certain limitations, such as complex pretreatment, cumbersome and expensive instrumentation, long assay times, and the need for professional laboratory operations. In addition, theoretically, there are electrochemical analysis and biological analysis. The electrochemical analysis method comprises an ion selective electrode method, a voltammetry method and an anodic potential stripping method, and the method inherits the advantages of wide detection range and high accuracy of an optical analysis method, has good selectivity, can detect various heavy metals simultaneously, realizes continuous measurement, but has troublesome pretreatment and can cause secondary pollution. Biological analysis comprises an enzyme analysis method, an immunoassay method and a biosensor, and mainly detects by means of biological materials, and the methods have the advantages of high detection speed, simple operation, specificity and lower sensitivity and accuracy.

Disclosure of Invention

The purpose is as follows: in order to solve the defects of the prior art, the invention provides the bright green indicator type electrochemical biosensor for detecting cadmium, which is economic, convenient and effective.

The adopted technical solution is as follows:

in the first aspect, the DNA electrochemical sensor is provided, wherein bright green BG is used as a DNA hybridization indicator, a carbon paste electrode is used as a working electrode, and Cd is modified on the surface of the carbon paste electrode²⁺Specific sensitive dsDNA; wherein the sequences of the two oligonucleotide strands of the dsDNA are:

ssDNA1：poly-G:5’-GGGGGGGGGG-3’；

ssDNA2：poly-C:5’-CCCCCCCCCCC-3’。

in a second aspect, there is provided a dsDNA modified carbon paste electrode prepared by a method comprising: immersing the activated carbon paste electrode into a dsDNA solution by an electrochemical enrichment principle, applying voltage for a period of time to fix the dsDNA on the surface of the carbon paste electrode, and washing with ultrapure water to obtain a dsDNA modified carbon paste electrode;

wherein the sequences of the two oligonucleotide strands of the dsDNA are:

ssDNA1：poly-G:5’-GGGGGGGGGG-3’；

ssDNA2：poly-C:5’-CCCCCCCCCCC-3’。

in some embodiments, the carbon paste electrode surface is activated under a cyclic voltammetry scan of 1.5V.

In some embodiments, the solvent of the dsDNA solution is 0.1mol/L Tris-HCl, pH 8.0;

in some embodiments, the activated carbon paste electrode is immersed in the dsDNA solution and a voltage of 0.5V is applied for 5min to immobilize the dsDNA.

A DNA electrochemical sensor of the first aspect comprising said dsDNA modified carbon paste electrode.

In a third aspect, a Cd based on a DNA electrochemical sensor is provided²⁺The detection method of (3), comprising:

step (1) immersing the dsDNA-modified carbon paste electrode of claim 2 in a sample solution to be tested to allow Cd to pass through²⁺Interacting with dsDNA, and washing the carbon paste electrode with ultrapure water after the action is finished;

accumulating brilliant green BG on the surface of the dsDNA modified carbon paste electrode treated in the step (1);

step (3) modifying the dsDNA treated in the step (2)The carbon paste electrode is used as a working electrode, is placed in an electrochemical measuring pool of Tris-HCl buffer solution with the pH value of 7.4, takes a saturated calomel electrode as a reference electrode, takes a platinum electrode as a counter electrode to form a three-electrode measuring system, is connected with an electrochemical workstation, adopts differential pulse voltammetry for detection, and reacts Cd by measuring the change of BG reduction peak current²⁺The concentration of (c).

In some embodiments, in step (1), the solvent of the sample solution to be tested is Tirs-HCl buffer solution, and the pH is 7.4.

In some embodiments, step (2) comprises: immersing the dsDNA modified carbon paste electrode treated in the step (1) into 1.0 multiplied by 10^-3And (3) stirring the hybridization indicator BG solution for 5min, washing the surface of the electrode by ultrapure water after the adhesion is finished, and wiping.

In some embodiments, in step (3), the electrode is scanned at a cathodic potential of 0.7-0.2V, with an amplitude of 0.04995, a modulation time of 0.05s, at intervals of 0.5s, and a step potential of 0.01005V.

In addition, the invention also provides the DNA electrochemical sensor or the dsDNA modified carbon paste electrode on cadmium Cd²⁺To the use of (1) in the detection of (3). Cd in particular in animal tissue²⁺Application in the detection of cadmium content.

Has the advantages that: according to the bright Green indicator type electrochemical biosensor for detecting cadmium, disclosed by the invention, according to the fact that heavy metal residues in animal tissues are detected in China, Bright Green (BG) is used as a DNA hybridization indicator, and a simple, rapid and high-sensitivity Cd detection method is constructed on the surface of a Carbon Paste Electrode (CPE)²⁺An electrochemical biosensor. The method can be used for quickly measuring the cadmium content of drinking water, feed and animal products of livestock and poultry, and provides technical support for effectively controlling the harm and pollution of heavy metals to the environment, human beings, animals and animal products in the production link of livestock and poultry. A novel hybridization indicator is used, single-strand and double-strand DNA has obvious affinity difference and can be specifically combined with a base site acted by Cd and the DNA. The experimental operation is simple, the reaction time is short, and the detection efficiency is improved.

Drawings

FIG. 1 is a schematic diagram of an electrochemical biosensor for detecting heavy metal cadmium DNA according to an embodiment of the present invention;

FIG. 2 shows a biosensor of 1X 10 according to an embodiment of the present invention^-5mol/L Hg²⁺,Pb²⁺,Zn²⁺,Ca²⁺,Ag⁺,Cu²⁺And 1X 10^-9mol/L Cd²⁺DPV peak of the solution.

Detailed Description

The present invention will be further described with reference to the following examples. The following examples are only for illustrating the performance of the present invention more clearly and are not limited to the following examples.

1. Principle of electrochemical biosensor for detecting heavy metal cadmium DNA

Construct a method for detecting Cd²⁺The hybridization indicator formulation DNA electrochemical sensor of (a). The sensor takes Brilliant Green (BG) as a DNA hybridization indicator, and a high-sensitivity Cd detection is constructed on the surface of a Carbon Paste Electrode (CPE)²⁺An electrochemical biosensor. When Cd²⁺Cd interaction with dsDNA due to higher affinity of BG to ssDNA than with dsDNA²⁺The reduction peak current of BG can be changed while the stability of dsDNA is damaged. By detecting the change of BG electrochemical signals, the Cd is detected²⁺The principle of the specific detection is shown in figure 1 of the accompanying drawings.

Firstly, two designed oligonucleotide short chains are synthesized into dsDNA, and the dsDNA is assembled on the surface of CPE by a voltage enrichment method. When the detected solution contains Cd²⁺Purine and Cd in the dsDNA²⁺The action causes the dsDNA to destabilize and spin to form two ssDNAs, the hybridization indicator BG specifically inserts into the purine position of the ssDNA, the electrochemical signal of BG changes, and the reduction peak value of BG can be detected by Differential Pulse Voltammetry (DPV). Cd in solution²⁺The concentration of (b) is different, the degree of ssDNA generated by unwinding dsDNA is different, so that the cadmium ion can be quantitatively detected.

The method uses a novel hybridization indicator, the single-strand and double-strand DNA has obvious affinity difference and can be specifically combined with the base site acted by Cd and DNA. The experimental operation is simple, the reaction time is short, and the detection efficiency is improved.

Preparation of DNA sensor

2.1 reagents and materials

2.1.1 DNA sequences

A 10 base oligonucleotide strand used to prepare dsDNA. The sequences of the oligonucleotides used are as follows:

ssDNA1：poly-G:5’-GGGGGGGGGG-3’

ssDNA2：poly-C:5’-CCCCCCCCCCC-3’

2.1.2 Primary reagents

AgNO₃、CuCl₂、CaCl₂、Zn(NO₃)₂、NaCl、HgCl₂、CdCl₂、NaAc、HAcHNO₃(GR)、HCl Tris、Brilliant Green。

2.1.3 Main Instrument

2.2 methods and procedures

2.2.1 preparation and activation of carbon paste electrodes

Graphite powder and liquid paraffin are mixed according to a certain proportion, and then the mixture is filled into a PVC tube to prepare a Carbon Paste Electrode (CPE). As a working electrode, the carbon paste electrode surface was activated under cyclic voltammetry sweep of 1.5V.

2.2.2 activation of saturated calomel electrode

The saturated calomel electrode was immersed in 0.5mol/L acetate buffer solution (pH 4.8) containing 0.02mol/L NaCl for 5min without stirring.

2.2.3 immobilization of DNA

The activated carbon paste electrode was immersed in dsDNA solution (0.1mol/L Tris-HCl, pH 8.0) by electrochemical enrichment principle, and a voltage of 0.5V was applied for 5min to immobilize dsDNA. Finally, the carbon paste electrode was rinsed with ultrapure water.

2.2.4 Effect with cadmium ions

Immersing the dsDNA-modified carbon paste electrode into Cd²⁺Adding into solution (Tirs-HCl buffer solution, pH 7.4), stirring gently for 5min, and waiting for Cd²⁺And dsDNA, this process does not require the application of an electrical potential to the electrodes. After the treatment, the carbon paste electrode was rinsed with ultrapure water.

2.2.5 attachment of BG

To combine dsDNA with Cd²⁺After interaction, BG accumulated on the surface of the carbon paste electrode, which was immersed in a 1.0X 10 bath^- ³And stirring the hybridization indicator BG solution for 5min in mol/L. In this step, no potential needs to be applied. After completion of the attachment, the electrode surface was rinsed with ultrapure water and wiped clean with filter paper.

2.2.6 electrochemical method-differential pulse voltammetry DPV

To be dsDNA and Cd²⁺After interaction, Cd was reacted by measuring the change in BG reduction peak current by differential pulse voltammetry in Tris-HCl buffer (pH 7.4)²⁺The concentration of (c). The electrode was scanned at a cathodic potential of 0.7-0.2V (vs. sce), amplitude 0.04995, modulation time 0.05s, interval 0.5s, and step potential 0.01005V (vs. sce).

The selectivity experiment of metal ions of the biosensor is also to detect the current peak value change of BG marker signals after the interaction of interfering metal ions and dsDNA under the same condition treatment.

Biosensor pair Cd²⁺Selectivity experiment compares Cd²⁺And Hg²⁺、Pb²⁺、Zn²⁺、Ca²⁺、Ag⁺、Cu²⁺And (3) solution. As shown in fig. 2, even at a relatively high concentration (1 × 10)^-5mol/L) did not change significantly in the DPV current peaks of BG of dsDNA-modified CPEs. Low Cd²⁺Concentration (1X 10)^-9mol/L) can significantly change the current peak of BG. The result shows that the sensor pair Cd²⁺Has good selectivity.

2.2.7 recovery test (beneficial effect)

The experimental blank chicken samples were from poultry institute of agricultural sciences (Yangzhou), China. Digesting the blank group of chicken without cadmium treatment by microwave, and adjusting the pH of the digestion solution to prepare the liquid to be detected. Adding 0.5X 10 respectively^-9mol/L，1.0×10^- ⁹mol/L and 1.5X 10^-9And (3) detecting the cadmium content of the mol/L cadmium solution by respectively adopting an inductively coupled plasma mass spectrometer (IPC-MS) and a DNA electrochemical sensor, and calculating the recovery rate.

In the recovery experiments of the biosensor (table 1), the DNA biosensor has good recovery rates, emphasizing that the detection of cadmium by the biosensor is consistent in both pure solution and complex liquid environments.

TABLE 1 Cd in Chicken tissue²⁺Recovery experiment

Application example:

rats were fed 0mg/L, 25mg/L and 75mg/L cadmium ion solutions for 3 months, respectively. After neck removal and sacrifice, 200mg of rat tail was taken and added with 2ml of GR-grade nitric acid, volume was adjusted to 30ml by microwave digestion, and pH was adjusted to 7.4. The DNA electrochemical biosensor is used for detecting the cadmium content in the solution. Meanwhile, the sample is used for measuring the cadmium content by an inductively coupled plasma mass spectrometer (ICP-MS). Detection of cadmium in practical samples as shown in table 2, the DNA biosensor was more sensitive than inductively coupled plasma mass spectrometry in detecting ultra-low concentration cadmium solutions. ICP-MS detection requires expensive instruments and is complicated to operate. The biosensor is relatively simple to operate, and the required instrument electrochemical workstation is also convenient to carry, so that the DNA biosensor has strong practicability in practical application.

TABLE 2 rat tail tissue sample test results

The present invention has been disclosed in terms of the preferred embodiment, but it is not intended to be limited to the embodiment, and all technical solutions obtained by substituting or converting the equivalent embodiments fall within the scope of the present invention.

Claims

1. The DNA electrochemical sensor is characterized in that bright green BG is used as a DNA hybridization indicator, a carbon paste electrode is used as a working electrode, and Cd is modified on the surface of the carbon paste electrode²⁺Specific sensitive dsDNA; wherein the sequences of the two oligonucleotide strands of the dsDNA are:

ssDNA1：poly-G: 5’-GGGGGGGGGG-3’；

ssDNA2：poly-C: 5’-CCCCCCCCCCC-3’。

2. a dsDNA-modified carbon paste electrode, the method of preparation comprising: immersing the activated carbon paste electrode into a dsDNA solution by an electrochemical enrichment principle, applying voltage for a period of time to fix the dsDNA on the surface of the carbon paste electrode, and washing with ultrapure water to obtain a dsDNA modified carbon paste electrode;

wherein the sequences of the two oligonucleotide strands of the dsDNA are:

ssDNA1：poly-G: 5’-GGGGGGGGGG-3’；

ssDNA2：poly-C: 5’-CCCCCCCCCCC-3’。

3. the dsDNA modified carbonaceous paste electrode of claim 2, wherein the carbonaceous paste electrode surface is activated under a cyclic voltammetry scan of 1.5V.

4. The dsDNA modified carbon paste electrode of claim 2, wherein said dsDNA solution has a solvent of 0.1mol/L Tris-HCl, pH 8.0;

the activated carbon paste electrode was immersed in the dsDNA solution and a voltage of 0.5V was applied for 5min to immobilize the dsDNA.

5. Cd based on DNA electrochemical sensor²⁺Is detected byA method, characterized in that it comprises:

and (3) taking the dsDNA modified carbon paste electrode treated in the step (2) as a working electrode, placing the working electrode in an electrochemical measuring pool of Tris-HCl buffer solution with the pH of 7.4, taking a saturated calomel electrode as a reference electrode and a platinum electrode as a counter electrode to form a three-electrode measuring system, connecting the three-electrode measuring system with an electrochemical workstation, detecting by adopting a differential pulse voltammetry, and reacting Cd by measuring the change of BG reduction peak current²⁺The concentration of (c).

6. The detection method according to claim 5, wherein in the step (1), the solvent of the sample solution to be detected is Tirs-HCl buffer solution, and the pH value is 7.4.

7. The detection method according to claim 5, wherein the step (2) comprises: immersing the dsDNA modified carbon paste electrode treated in the step (1) into 1.0 multiplied by 10^-3And (3) stirring the hybridization indicator BG solution for 5min, washing the surface of the electrode by ultrapure water after the adhesion is finished, and wiping.

8. The detecting method according to claim 5, wherein in the step (3), the electrode is scanned at a cathode potential of 0.7 to 0.2V, the amplitude is 0.04995, the modulation time is 0.05s, the interval is 0.5s, and the step potential is 0.01005V.

9. The DNA electrochemical sensor of claim 1 in cadmium Cd²⁺To the use of (1) in the detection of (3).

10. The dsDNA modified carbon paste electrode of claim 2 in cadmium Cd²⁺To the use of (1) in the detection of (3).