Method for realizing synchronous detection of mercury ions and lead ions based on non-labeled DNA biosensor and DNA probe thereof

Abstract

The application discloses a method for realizing synchronous detection of mercury ions and lead ions based on a non-labeled DNA biosensor and a DNA probe thereof. The method has the advantages of sensitivity, simple operation and low cost. In the detection range, the concentrations of the two ions and the fluorescence signals are in good linear relationship. The application has good specificity for the two ions, and various metal ions such as potassium, copper, nickel, magnesium and calcium have no interference effect on the detection. The detection limits of the mercury ions and the lead ions are 4.094 nM and 3.22 nM respectively, and the whole detection process can be completed at room temperature. The result can be directly obtained through a fluorescence spectrophotometer. The test of the actual tap water sample has been completed, the detection process is convenient and fast, and the method can be used for detecting the mercury ions and the lead ions in actual water bodies.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of environmental analysis, and particularly relates to a method for realizing synchronous detection of mercury ions and lead ions based on a non-labeled DNA biosensor and a DNA probe thereof. BACKGROUND

[0002] With the rapid development of science and technology and industry in recent years, heavy metals are widely used in national defense and military industry, chemical manufacturing and many other industries, and widely exist in human daily production and life. Heavy metal compounds can cause serious pollution of water bodies, among which, mercury ion and lead ion pollution has the most serious influence, has significant characteristics such as great harm and difficult governance, can produce stronger toxicity through enrichment in water bodies, and finally enter the human body through the food chain to cause various diseases such as cardiovascular diseases and neurological diseases, thereby seriously threatening public health and safety. Although traditional methods such as cold atomic absorption spectrometry, inductively coupled plasma (ICP-OES / MS) and the like can meet the demand for synchronous detection of the two ions, have high sensitivity and low detection limit, the detection cost is extremely high, the pretreatment process is complicated, the requirement for the operator is high, and importantly, real-time monitoring cannot be achieved, so the methods are not suitable for application and popularization in actual life.

[0003] Therefore, it is particularly important to develop a DNA probe which is simple to operate, low in cost, high in sensitivity, rapid in detection and capable of meeting the synchronous detection of Hg 2+ and Pb 2+ . SUMMARY

[0004] A first object of the present application is to provide a DNA probe capable of synchronously detecting mercury ions and lead ions in view of the deficiencies of the prior art.

[0005] The technical scheme adopted by the present application is as follows:

[0006] A DNA probe ODN-1 capable of synchronously detecting mercury ions and lead ions, the sequence of which is as follows:

[0007] 5'-TTTTTTCCGGTTGGTGTGGTTGGTTTTTT-3'.

[0008] After the DNA probe ODN-1 is mixed with a sample to be detected, when mercury ions exist in the sample, TT bases of the DNA chain form T-Hg 2+ -T pairing; when lead ions exist in the sample, the lead ions form G-quadruplex interaction with G bases, so that the conformation of T-Hg 2+ -T is changed.

[0009] A second object of the present application is to provide the application of the above DNA probe in the synchronous detection of mercury ions and lead ions in water samples.

[0010] A third object of the present application is to provide a method for synchronously detecting mercury ions and lead ions based on a non-labeled DNA biosensor, which utilizes a non-labeled oligonucleotide chain with a specific sequence, two buffers and an embedded fluorescent agent DAPI to realize the detection of mercury ions and lead ions based on fluorescence method, and has the advantages of low cost and rapid detection.

[0011] A method for synchronously detecting mercury ions and lead ions based on a non-labeled DNA biosensor, which adopts the following technical scheme:

[0012] Step (1), dissolving the above DNA probe in a buffer, then adding a sample to be detected and mixing to obtain a mixed solution, and standing at room temperature for 4-6 min;

[0013] Step (2), adding an embedded fluorescent agent DAPI (4', 6-diamidino-2-phenylindole) to the mixed solution obtained in step (1) and mixing, and standing at room temperature for a period of time;

[0014] Step (3), measuring the fluorescence signal intensity of the solution after standing in step (2).

[0015] As a preferred, the concentration of the DNA probe in step (1) is 20-25 nM.

[0016] As a preferred, when detecting mercury ions, the buffer in step (2) is Tris-HAc buffer;

[0017] The concentration of the Tris-HAc buffer is 25 mM, and the pH is 7.5, which is prepared from tris-hydroxymethyl aminomethane Tris, acetic acid HAc and 100 mM NaAc.

[0018] As a preferred, when detecting lead ions, the buffer in step (2) is Tris-HCl buffer with NaCl added;

[0019] The concentration of the Tris-HCl buffer with NaCl added is 25 mM, and the pH is 8.5, which is prepared from tris-hydroxymethyl aminomethane Tris, hydrochloric acid HCl and 100 mM NaCl.

[0020] As a preferred, when detecting lead ions, 1000-1200 nM of mercury ions is further added after standing in step (2), and mixed and stood for 5-7 min.

[0021] As a preferred, the concentration of DAPI in step (2) is 100-125 nM, and the standing time is 20-22 min.

[0022] The embedded fluorescent agent DAPI used in step (2) shows extremely weak fluorescence signal itself, which can be almost ignored when there is no mercury ion and lead ion in the sample to be detected; when the mercury ion exists, DAPI is embedded into T-Hg 2+ -T complex, showing a strong fluorescence signal; when the lead ion exists, the secondary structure of T-Hg 2+ -T complex changes, and the fluorescence signal is quenched, thereby realizing the function of fluorescence switch.

[0023] The fluorescence detection can be realized by a fluorescence spectrophotometer, and the steps are simple, the cost is low, the result is real-time, the detection range is wide, and the purpose of qualitative and quantitative detection and real-time detection of mercury ion and lead ion can be achieved by judging the opening and closing and intensity of the fluorescence signal.

[0024] The beneficial effects of the present application are:

[0025] (1) The DNA probe has high sensitivity, and the detection limits of mercury ion and lead ion are 4.094nM and 3.22nM respectively, the detection result can be judged by the fluorescence value, the two ions can be quantitatively detected, the operation is simple, and the purpose of real-time detection can be realized.

[0026] (2) The DNA probe has high specificity to mercury ion and lead ion, and common other metal ions do not interfere with the detection.

[0027] (3) The DNA probe can simultaneously realize the detection of mercury ion and lead ion, and when the mercury ion and lead ion exist in the solution at the same time, the single detection of one of them can be realized.

[0028] (4) The DNA probe has completed the test of tap water sample, has high recovery rate, and can be applied and popularized in actual life.

[0029] (5) The DNA probe is simple and convenient to operate, and only needs to use simple instruments for detection, without professional technical personnel and complicated preparation process. DETAILED DESCRIPTION

[0030] Figure 1 The principle diagram for detecting mercury ion and lead ion in the present application; wherein (a) is that when the mercury ion exists, DAPI is embedded into T-Hg 2+ -T complex, the fluorescence signal is turned on; (b) is that when the lead ion exists, the secondary structure of T-Hg 2+ -T complex changes, and the fluorescence signal is turned off;

[0031] Figure 2The fluorescence curve diagram of different concentrations of mercury ions and lead ions and the linear relationship diagram of mercury ion and lead ion concentration and fluorescence intensity, wherein (a) is the relative fluorescence (F-F0) intensity curve diagram of different concentrations of mercury ions; (b) is the linear relationship between the relative fluorescence (F-F0) intensity and the concentration of mercury ions; (c) is the relative fluorescence (F-F0) intensity curve diagram of different concentrations of lead ions; (d) is the linear relationship between the relative fluorescence (F-F0) intensity and the concentration of lead ions;

[0032] Figure 3 The result diagram of specific detection of mercury ions and lead ions under the interference of different metal ions; wherein (a) is the relative fluorescence intensity when mercury ions and other metal ions are detected alone in Tris-HAc buffer solution; (b) is the relative fluorescence intensity when mercury ions and other metal ions are mixed and detected in Tris-HAc; (c) is the relative fluorescence intensity when lead ions and mercury ions and lead ions, mercury ions and other metal ions are mixed and detected in Tris-HCl buffer solution; (d) is the relative fluorescence intensity when mercury ions and other metal ions are mixed and detected in Tris-HCl buffer solution. DETAILED DESCRIPTION

[0033] The application will be further described in conjunction with examples, but is not limited to this.

[0034] The application discloses a DNA probe ODN-1 capable of synchronously detecting mercury ions and lead ions, wherein the DNA probe sequence contains 6 pairs of T bases, and two C bases are beside the T base at the 5' end, and the specific sequence is as follows:

[0035] 5'-TTTTTTCCGGTTGGTGTGGTTGGTTTTTT-3'

[0036] The application adopts the following implementation scheme for the detection of mercury ions:

[0037] (1) 20-25 nM of the probe ODN-1 is dissolved in a Tris-HAc (25 mM, containing 100 mM of NaAc, pH 7.5) buffer solution.

[0038] (2) The sample to be detected is added, and after mixing, the mixture is placed at room temperature for 4-6 minutes.

[0039] (3) 100-125 nM of the intercalating fluorescent agent DAPI is added, and after mixing, the mixture is placed at room temperature for 20-22 minutes.

[0040] (4) The fluorescence intensity is detected by using a fluorescence spectrophotometer.

[0041] The principle diagram of the above implementation scheme is shown in route (a) in Figure 1 ​

[0042] The present application adopts the following implementation for the detection of lead ions:

[0043] (1) Dissolve 20-25 nM of the probe ODN-1 in Tris-HCl buffer (25 mM, containing 100 mM NaCl, pH 8.5) with NaCl added.

[0044] (2) Add 1000-1200 nM of mercury ions, mix, and then place at room temperature for 5-7 minutes.

[0045] (3) Add the sample to be detected at the same time, mix, and then place at room temperature for 4-6 minutes.

[0046] (4) Add 100-125 nM of the intercalating fluorescent agent DAPI, mix, and then place at room temperature for 20-22 minutes.

[0047] (5) Detect the fluorescence intensity using a fluorescence spectrophotometer.

[0048] The schematic diagram of the above implementation is shown in route (b) of Figure 1 .

[0049] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without any creative work are within the scope of protection of the present application.

[0050] Embodiment 1

[0051] The DNA probe ODN-1 is used to detect mercury ions in a sample, and the sequence of the DNA probe ODN-1 is as follows: TTTTTTCCGGTTGGTGTGGTTGGTTTTTT.

[0052] (1) Prepare 25 mM Tris-HAc buffer (containing 100 mM NaAc, pH 7.5), and dissolve the probe ODN-1 in the buffer so that the concentration of the probe ODN-1 is 20 nM.

[0053] (2) Add the sample to be detected, mix, and then place at room temperature for 5 minutes. In this step, if the sample to be detected contains mercury ions, the T base part of the probe sequence ODN-1 will form a T-Hg 2+ -T complex with the mercury ions.

[0054] (3) Add 125 nM of the intercalating fluorescent agent DAPI, mix, and then place at room temperature for 20 minutes. In this step, if the sample to be detected contains mercury ions, the fluorescent agent is intercalated into the T-Hg 2+In the T structure, the fluorescence signal is enhanced to achieve the purpose of detecting mercury ions.

[0055] (4) If the process to be tested sample contains lead ions, lead ions and acetate ions form lead acetate, at this time lead ions have no interference on mercury ion detection.

[0056] Example 2

[0057] The DNA probe ODN-1 is used to detect lead ions in the sample, and the DNA probe sequence ODN-1 is as follows: TTTTTTCCGGTTGGTGTGGTTGGTTTTTT.

[0058] (1) First, add NaCl to the Tris-HCl buffer (25mM, containing 100mM NaCl, pH 8.5) to dissolve the probe ODN-1, so that the concentration of the probe ODN-1 is 20nM.

[0059] (2) Add 1000nM mercury ions, mix well, and place at room temperature for 5 minutes. In this step, the T base part of the probe sequence ODN-1 will form a T-Hg 2+ -T complex.

[0060] (3) Add the sample to be tested, mix well, and place at room temperature for 20 minutes. In this step, if the sample to be tested contains lead ions, lead ions will induce G-quadruplex, thereby changing the secondary structure of the T-Hg 2+ -T complex.

[0061] (4) Add 125nM intercalating fluorescent agent DAPI, mix well, and place at room temperature for 20 minutes. In this step, the fluorescent agent DAPI is released from the structure, and the fluorescence signal is reduced to achieve the purpose of detecting lead ions.

[0062] (5) If the process to be tested sample contains mercury ions, it is verified by experiment that when the concentration of mercury ions is 1000nM, the fluorescence signal has reached saturation, so even if the sample to be tested contains mercury ions, it will not interfere with the detection of lead ions.

[0063] Example 3: Detection of different concentrations of mercury ions and lead ions:

[0064] (1) Prepare mercury ion standard solution with concentrations of 0, 1, 3, 5, 10, 20, 50, 100, 200, 300, 500, 600, 800 and 1000nM.

[0065] (2) Add the above different concentrations of mercury ion solution to the reaction system described in Example 1, and detect the fluorescence signal using a fluorescence spectrophotometer after sufficient reaction. Each reaction is repeated at least 3 times, and the results are as follows: Figure 2(a) and (b) show that the fluorescence signal gradually increases with the increase of mercury ion concentration; the fluorescence signal and mercury ion concentration are linearly related in the range of 0-1000 nM.

[0066] (3) Prepare lead ion standard solutions with concentrations of 0, 0.01, 0.05, 0.1, 0.3, 1, 5, 10, 15, 25, 30, 40 and 50 μM, respectively.

[0067] (4) Add the above lead ion solutions with different concentrations to the reaction system described in Example 1, respectively, and after sufficient reaction, detect the fluorescence signal using a fluorescence spectrophotometer. After repeating each reaction at least 3 times, the results are as follows: Figure 2 (c) and (d) show that the fluorescence signal gradually decreases with the increase of lead ion concentration; the fluorescence signal and lead ion concentration are linearly related in the range of 0-40 μM.

[0068] Example 4: Detection of interference of different metal ions:

[0069] (1) Interference of common metal ions in mercury ion detection:

[0070] Prepare standard solutions of different metal ions, which are K + , Mg 2+ , Cu 2+ , Co 2+ , Ca 2+ , Ni 2+ , Fe 2+ , Mn 2+ , Cd 2 + , Pb 2+ and Ag + , with a metal ion concentration of 6 μM.

[0071] Add the above metal ion standard solutions and 1000 nm mercury ion standard solution to the reaction system described in Example 1, respectively, and after sufficient reaction, detect the fluorescence signal of the solution using a fluorescence spectrophotometer at the maximum emission wavelength of 450 nm. From Figure 3 As can be seen from the detection results of (a) and (b), 6 μM of K + , Mg 2+ , Cu 2+ , Co 2+ , Ca 2+ , Ni 2+ , Fe 2+ , Mn 2 + , Cd 2+ , Pb 2+ and Ag +It does not interfere with any detection method. The fluorescence signal only increases when mercury ions are added, proving that this invention has excellent specificity for the detection of mercury ions.

[0072] (2) Interference from common metal ions in lead ion detection:

[0073] Prepare 50 μM standard solutions of different metal ions, namely Mn 2+ Ni 2+ Ca 2+ Cu 2+ Fe 2+ Mg 2+ Co 2+ and K + .

[0074] The aforementioned metal ion standard solution and 50 μM lead ion standard solution were added to the reaction system described in Example 2, respectively. After complete reaction, the fluorescence signal of the solution was detected using a fluorescence spectrophotometer at the maximum emission wavelength of 450 nm. Figure 3 The detection results in (c) and (d) show that 50 μM Mn 2+ Ni 2+ Ca 2+ Cu 2+ Fe 2+ Mg 2+ Co 2+ and K + It does not interfere with any detection method. The fluorescence signal is quenched only when lead ions are added, proving that this invention has excellent specificity for the detection of lead ions.

[0075] Example 5: Detection of mercury and lead ions in actual samples

[0076] Mercury ion samples with concentrations of 20 nM, 200 nM, and 600 nM and lead ion samples with concentrations of 0.5 μM, 5 μM, and 10 μM were prepared in tap water.

[0077] Four experimental groups were set up, and the configuration and operation were as follows:

[0078] Group 1: The method of Example 1 was used to react tap water samples containing 20 nM, 200 nM, and 600 nM mercury ions, and the fluorescence signal was detected using a fluorescence spectrophotometer.

[0079] The second group: 20 nM of mercury ion tap water sample was mixed with 0.5 μM of lead ion tap water sample, 200 nM of mercury ion tap water sample was mixed with 5 μM of lead ion tap water sample, and 600 nM of mercury ion tap water sample was mixed with 10 μM of lead ion tap water sample, then the three mixed samples were reacted respectively by the method of Example 1, and the fluorescence signals were detected by a fluorescence spectrophotometer;

[0080] The third group: 0.5 μM, 5 μM and 10 μM of lead ion tap water samples were reacted respectively by the method of Example 2, and the fluorescence signals were detected by a fluorescence spectrophotometer;

[0081] The fourth group: 20 nM of mercury ion tap water sample was mixed with 0.5 μM of lead ion tap water sample, 200 nM of mercury ion tap water sample was mixed with 5 μM of lead ion tap water sample, and 600 nM of mercury ion tap water sample was mixed with 10 μM of lead ion tap water sample, then the three mixed samples were reacted respectively by the method of Example 2, and the fluorescence signals were detected by a fluorescence spectrophotometer;

[0082] The results are shown in Tables 1-4.

[0083] Table 1 Hg in tap water sample 2+ Average recovery rate of detection (n=3)

[0084]

[0085] Table 2 Hg in tap water sample (containing lead ion) 2+ Average recovery rate of detection (n=3)

[0086]

[0087] Table 3 Pb in tap water sample 2+ Average recovery rate of detection (n=3)

[0088]

[0089] Table 4 Pb in tap water sample (containing mercury ion) 2+ Average recovery rate of detection (n=3)

[0090]

[0091] From the detection results shown in Tables 1-4, it can be seen that whether the mercury ion or the lead ion exists alone in the sample or the mercury ion and the lead ion exist simultaneously, there is a good recovery rate, which proves that the method has good practical applicability for the detection of mercury ion and lead ion.

[0092] The above embodiments are preferred cases for introducing the present application, and the present application is not limited to the above embodiments. Any obvious changes and improvements made by those skilled in the art without departing from the spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A method for detecting lead ions based on a non-labeled DNA biosensor, characterized in that, The method comprises the following steps: Step (1), dissolving DNA probe ODN-1 in buffer solution, the sequence of the DNA probe ODN-1 is as follows: 5'-TTTTTTCCGGTTGGTGTGGTTGGTTTTTT-3'; Step (2), adding 1000 nM of mercury ions, mixing and then placing at room temperature for 5 minutes; Step (3), adding the sample to be tested, mixing and then placing at room temperature for 20 minutes; Step (4), adding intercalating fluorescent agent DAPI, mixing and then placing at room temperature for 20 minutes; Step (5), measuring the fluorescence signal intensity of the solution after standing in step (4).

2. The method for lead ion detection based on non-labeled DNA biosensor according to claim 1, characterized in that, The concentration of the DNA probe ODN-1 in step (1) is 20-25 nM.

3. The method for lead ion detection based on non-labeled DNA biosensor according to claim 1, characterized in that, The buffer solution in step (1) is Tris-HCl buffer solution with NaCl added.

4. The method for lead ion detection based on non-labeled DNA biosensor according to claim 1, characterized in that, The concentration of DAPI in step (4) is 100-125 nM.

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