CN115855928A - Mercury ion detection method and kit based on nucleic acid macroarray and bifunctional molecules - Google Patents
Mercury ion detection method and kit based on nucleic acid macroarray and bifunctional molecules Download PDFInfo
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Abstract
The invention discloses a mercury ion detection method based on a nucleic acid macroarray and bifunctional molecules, which comprises the following steps: providing a nucleic acid macro array and a signal probe, wherein a fixed probe in the nucleic acid macro array can specifically capture mercury ions, the signal probe comprises gold nanoparticles and a bifunctional molecule modified on the surface of the gold nanoparticles, and the bifunctional molecule can specifically bind the mercury ions and has a Raman characteristic spectrum; capturing mercury ions with an immobilized probe; the signal probe is specifically combined with the captured mercury ions to form a mercury ion detection system; and (3) detecting the mercury ions by testing color and/or Raman spectrum. The method for detecting the mercury ions provided by the invention utilizes the characteristics that the bifunctional molecules modified on the surfaces of the gold nanoparticles can combine the mercury ions and generate Raman signals, and the nucleic acid fixed probe and the bifunctional molecules capture and identify the mercury ions so as to carry out color development and/or Raman detection, so that the detection of the mercury ions is more convenient and sensitive.
Description
Technical Field
The invention relates to the technical field of heavy metal detection, in particular to a mercury ion detection method and a kit based on a nucleic acid macroarray and a bifunctional molecule.
Background
Mercury ion (Hg) 2+ ) The heavy metal element is one of heavy metal elements with the strongest toxicity in the environment, and can cause serious harm to the health of human bodies even under the condition of low concentration. Water-soluble mercury is one of the most common and relatively stable forms of presence, causing serious mercury contamination due to its high toxicity and bioaccumulation. Therefore, hg with high sensitivity and high selectivity 2+ Micro-detection method for detecting Hg in aquatic ecosystem in real time 2+ The content has great significance for controlling environmental pollution and protecting human health.
Researchers provide a gold nanorod for detecting Hg based on sulfhydryl DNA modification 2+ The principle of the method is that the gold nanorods modified by the sulfhydryl DNA can react with Hg 2+ Formation of T-Hg 2+ A sandwich structure of-T, thereby constructing a Cy5 nucleotide-gold nanorod fluorescence resonance energy transfer system and finally achieving the paired Hg 2+ The detection of (3). However, the inventors found that this method requires nucleic acid modification of the surface of gold nanoparticles, and that the preparation is difficult, and that this method is effective for Hg 2+ The sensitivity of the detection still remains to be improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a nucleic acid macro array and a method and a kit for detecting mercury ions of bifunctional molecules.
The invention aims to provide a novel rapid detection method, which utilizes a bifunctional molecule which can complex mercury ions and can be used as a Raman signal molecule, combines the specific binding action of a nucleic acid fixed probe and the mercury ions, and achieves the aims of visual qualitative detection of the mercury ions and quantitative detection of surface enhanced Raman sensitization by forming a specific detection system.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the invention provides a mercury ion detection method based on a nucleic acid macro array and bifunctional molecules, which comprises the following steps:
providing a nucleic acid macro array and a signal probe, wherein the nucleic acid macro array comprises a plurality of fixed probes arranged in an array form, the fixed probes are oligonucleotide chains capable of specifically capturing mercury ions, the signal probe comprises gold nanoparticles and bifunctional molecules modified on the surfaces of the gold nanoparticles, and the bifunctional molecules are Raman signal molecules and can be specifically combined with the mercury ions;
contacting the nucleic acid macroarray with a sample to be detected so that the immobilized probe captures mercury ions in the sample to be detected;
contacting the nucleic acid macro array capturing mercury ions with the signal probe so that the mercury ions captured by the nucleic acid macro array are specifically combined with the signal probe to form a mercury ion detection system;
and detecting the mercury ions in the sample to be detected by testing the color and/or Raman spectrum of the mercury ion detection system.
In some preferred embodiments, the present invention further provides improvements: before performing color and/or Raman spectrum test on the mercury ion detection system, performing silver staining sensitization treatment on the mercury ion detection system; wherein, the silver staining sensitization treatment specifically comprises the following steps:
providing a silver staining solution, wherein the silver staining solution contains 1.2-1.8 mol/L silver ions and 0.1-0.3 g/mL reducing agent;
and contacting the silver staining solution with the mercury ion detection system and carrying out silver deposition reaction, thereby depositing a silver nano material on the surface of the mercury ion detection system.
In another aspect, the present invention also provides a kit for facilitating the use of the above detection method, comprising:
a nucleic acid macroarray comprising a plurality of immobilized probes arranged in an array on a surface of a solid support, the immobilized probes being oligonucleotide chains capable of specifically capturing mercury ions;
the signal probe comprises gold nanoparticles and bifunctional molecules modified on the surfaces of the gold nanoparticles, wherein the bifunctional molecules are Raman signal molecules and can be specifically combined with mercury ions.
Based on the technical scheme, compared with the prior art, the invention has the beneficial effects that at least:
the mercury ion detection method provided by the invention utilizes the characteristic that the bifunctional molecules modified on the surfaces of the gold nanoparticles can combine with mercury ions and generate Raman signals, and the nucleic acid fixed probe and the bifunctional molecules are used for carrying out color development and/or Raman detection on the recognition and capture of the mercury ions, thereby avoiding the existing detection method based on T-Hg 2+ The method for detecting mercury ions by using the-T' structure requires modifying nucleic acid on the surface of gold nanoparticlesThe method has the advantages of more convenience, sensitivity and higher accuracy for the detection of the mercury ions.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to enable those skilled in the art to more clearly understand the technical solutions of the present invention and to implement them according to the content of the description, the following description is made with reference to the preferred embodiments of the present invention and the detailed drawings.
Drawings
FIG. 1 is a schematic diagram of the principle of a mercury ion detection method provided in some exemplary embodiments of the present invention;
FIG. 2 is a schematic diagram showing comparison of detection results of two mercury ion detection methods provided by an exemplary embodiment of the present invention;
FIG. 3 is a corresponding Raman spectrum provided by an exemplary embodiment of the present invention for detecting different concentrations of mercury ions using Raman;
fig. 4 is a calibration curve established for a series of samples according to raman signal intensity versus mercury ion concentration provided by an exemplary embodiment of the present invention.
Detailed Description
In view of the defects in the prior art, the inventor of the present invention has made extensive research and practice to propose the technical solution of the present invention. The technical solution, its implementation and principles, etc. will be further explained as follows.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as specifically described herein, and thus the scope of the present invention is not limited by the specific embodiments disclosed below.
Moreover, relational terms such as "first" and "second," and the like, may be used solely to distinguish one element or method step from another element or method step having the same name, without necessarily requiring or implying any actual such relationship or order between such elements or method steps.
Referring to fig. 1, an embodiment of the present invention provides a method for detecting mercury ions based on a nucleic acid macro-array and bifunctional molecules, including:
providing a nucleic acid macro array and a signal probe, wherein the nucleic acid macro array comprises a plurality of fixed probes arranged in an array form, the fixed probes are oligonucleotide chains capable of specifically capturing mercury ions, the signal probe comprises gold nanoparticles and bifunctional molecules modified on the surfaces of the gold nanoparticles, and the bifunctional molecules are Raman signal molecules and can be specifically combined with the mercury ions.
And contacting the nucleic acid macro array with a sample to be detected so that the immobilized probes capture mercury ions in the sample to be detected.
And contacting the nucleic acid macro array capturing the mercury ions with the signal probe so that the mercury ions captured by the nucleic acid macro array are specifically combined with the signal probe to form a mercury ion detection system.
And detecting the mercury ions in the sample to be detected by testing the color and/or Raman spectrum of the mercury ion detection system.
The prior art provides some related technical schemes for detecting by using two oligonucleotide chains to form a sandwich structure by matching mercury ions, wherein the two oligonucleotide chains have larger molecular weights, have steric hindrance effect and are not beneficial to preparation and detection; meanwhile, in addition to steric hindrance caused by the oligonucleotide chain, there are disadvantages as follows: 1. compared with the modified bifunctional molecules provided by the invention, the nucleic acid modified on the surface of the gold nanoparticles has larger molecular weight, so the bifunctional molecules have relatively larger molar ratio modified on the surface of the gold nanoparticles, and the probability of being combined with target mercury ions in the detection process is far greater than the probability of being combined with T bases in the nucleic acid; 2. using T-Hg 2+ The sandwich structure of-T relates to the design of two nucleic acid sequences to avoid the occurrence of false positive and ensure the mercury ion to T-Hg 2+ The effective formation of the T structure, the number and sequencing of T bases in two sections of nucleic acid probes are difficult to control, but the invention only relates to the design of a section of nucleic acid sequence rich in T bases, reduces the design difficulty and is not easy to generate false positive.
In the above technical scheme, the nucleic acid macroarray technology is a method in which oligonucleotide chains with a known sequence are used as immobilized probes, the immobilized probes are fixed on the surface of a solid phase carrier to prepare nucleic acid probes arranged in an array form, the nucleic acid probes are subjected to hybridization reaction with a sample to be detected by using the base complementation principle, and fluorescent molecules, precious metal nanoparticles, quantum dots and the like are used as chromogenic markers, so as to obtain a required analysis signal. The solid phase carrier is not limited, and any carrier may be used as long as it can realize array-type immobilization of probes, such as nitrocellulose membrane, which is commonly used in the following examples, but is not limited thereto. The array refers to a fixed probe array arranged on the surface of a carrier, and the arranged pattern can be a single region or a plurality of regions arranged macroscopically like the following embodiments, so as to carry out a series test or a parallel test.
And some bifunctional molecules, such as 4-mercaptopyridine (4-MPY), are used as Raman signal molecules, and the sulfydryl contained in the bifunctional molecules can be covalently combined with the gold nanoparticles, so that the bifunctional molecules are stably modified to the surfaces of the gold nanoparticles to functionalize the gold nanoparticles; on the other hand, N in the pyridine group can be bonded with Hg through a polyvalent N bond 2+ Coordinated to form Hg- (pyridine) 2 The complex can be applied to the mercury ion detection of the surface enhanced Raman method. Single-stranded DNA rich in thymine (T) base as an immobilized probe at the same time, in Hg 2+ When present, can capture Hg 2+ T-Hg can be formed finally 2+ The structure of 4-MPY, which is shown in the upper right part of FIG. 1, is stable and specific.
The gold nanoparticles can be used as a substrate with a Raman enhancement effect, can develop color due to the optical characteristics of the gold nanoparticles, and can be used as a surface enhanced Raman substrate, so that the Raman signal of the bifunctional molecule is greatly improved.
In Hg 2+ When present, through the formation of stable T-Hg 2+ The structure of the 4-MPY is that the immobilized probe is combined with the bifunctional molecule through mercury ions, the gold nanoparticles modified with the 4-MPY are captured on a solid phase carrier, a signal with the gold nanoparticles as a color marker is generated, and the signal can be qualitatively or quantitatively characterized and determined through naked eye observation or Raman spectroscopy.
Thus, in some embodiments, the bifunctional molecule may have a pyridine group and a plurality of thiol groups; the immobilized probe comprises a plurality of T bases.
In some embodiments, the bifunctional molecule may include any one or a combination of two or more of 4-mercaptopyridine, 2-mercaptopyridine, and 2-mercapto-3-pyridinol, which is not limited thereto, and satisfies the requirement of having a raman characteristic spectrum, and both the functional molecule having a coordinating functional group, such as a pyridine group, and a functional group, such as a mercapto group, for connecting the gold nanoparticle may achieve the technical effects of the present invention.
In some embodiments, the nucleic acid sequence of the immobilized probe may be: 5 '(Bio) -ttcgcctctctttgtgtttttgctttgtt-3' may also be exemplified by the following examples: 5 '(Bio-ttctcctctctttgtgttattgctttgtt-3', the invention does not limit the nucleic acid sequence of the immobilized probe, and various alternative nucleic acid sequences of the immobilized probe are provided in the prior art, which can realize the selective adsorption of mercury ions, and can realize the detection of mercury ions based on the same principle, but are not limited to the specific sequences specifically enumerated in the invention.
In some embodiments, the immobilized probe comprises a plurality of T bases and/or the signal probe is obtained by subjecting a coupling reaction system containing gold nanoparticles and bifunctional molecules to a coupling reaction at 2-8 ℃ for 5.5-6.5 h, the average particle diameter of the gold nanoparticles may be 25-30 nm, and the mass ratio of the gold nanoparticles to the bifunctional molecules may be 0.4 to 0.5.
While, as shown in the right part of FIG. 1, the present inventors found that Ag is catalyzed by silver-staining sensitization under the catalysis of gold nanoparticles and the reduction of hydroquinone + Reducing the Ag atoms into core on the surface of the gold nanoparticles, and enhancing the color development intensity and Raman signals, thereby improving the detection sensitivity. Thus, in some embodiments, the mercury ion detection method may further comprise: before testing color and/or Raman spectrum, carrying out silver staining sensitization treatment on the mercury ion detection system; the silver staining sensitization partAnd depositing the silver nano material on the surface of the mercury ion detection system.
In some embodiments, the silver-staining sensitization treatment may specifically comprise: and (3) contacting a silver staining solution containing silver ions and a reducing agent with the mercury ion detection system to perform silver deposition reaction, and optionally removing the silver staining solution by washing to stop the silver deposition reaction so as to accurately control the reaction time.
In some embodiments, the conditions for the preferred silver-staining sensitization treatment are preferably: the concentration of silver ions in the silver staining solution can be 1.2-1.8 mol/L, and the concentration of a reducing agent can be 0.1-0.3 g/mL; the silver deposition reaction may be carried out for a period of time of 2 to 3 min at a temperature generally normal to the normal temperature, for example 15 to 40 c, and preferably 15 to 37 c.
In some embodiments, the method of formulating the silver staining solution may comprise:
reducing agent solution and reducing buffer solution are added 25-35 min before silver staining sensitization treatment to obtain the final product (2.5-3.5): 1, and then keeping the temperature of 35-40 ℃ for incubation to obtain a mixed solution, wherein the original agent solution contains hydroquinone with the concentration of 0.05-0.06 g/mL, and the reduction buffer solution contains citric acid with the concentration of 0.25-0.30 g/mL and sodium citrate with the concentration of 2.3-2.4 g/mL; when the silver staining sensitization treatment is carried out, the mixed solution and the silver nitrate solution are fully mixed and then are immediately used.
Wherein, the immediate use means that after the mixture is observed to have no non-uniform phenomenon or after the mixture is judged to be uniformly mixed through sufficient oscillation, the prepared silver staining solution is contacted with the mercury ion detection system within several seconds, for example, less than ten seconds, and the contact mode includes but is not limited to soaking, spraying, coating and the like, so that the silver deposition reaction occurs.
According to the technical scheme, on the basis of a mercury ion detection system prepared in the previous period, a silver layer is continuously deposited, and further sensitization can be realized. By continuously depositing a silver layer, on one hand, the color on the mercury ion detection system is deepened, and the detection sensitivity of visual observation is improved; on the other hand, the surface enhanced Raman effect of the silver nano material is stronger than that of the gold nano particles, so that the Raman signal of a bifunctional molecule such as 4-MPY is further enhanced, and the quantitative detection sensitivity of mercury ions in the detection method is correspondingly improved.
In some embodiments, the method for preparing the signaling probe specifically may comprise:
and mixing the colloid Jin Fensan solution and the solution of the bifunctional molecules to form a coupling reaction system.
And (3) carrying out coupling reaction on the coupling reaction system at the temperature of 2-8 ℃ for 5.5-6.5 h to obtain the signal probe.
In some embodiments, the mercury ion detection method may further include:
a step of eluting free mercury ions not captured by the immobilized probes with an eluent before contacting the nucleic acid macroarray capturing mercury ions with the signaling probes.
In some embodiments, the qualitative detection limit of the color-based mercury ion detection method is 0.4 to 0.6 nmol/L; the qualitative detection limit of the mercury ion detection method based on Raman spectrum is 0.04-0.06 nmol/L, and the quantitative detection limit is 0.02-0.03 nmol/L.
As some typical application examples of the above exemplary generalized technical solution, the mercury ion detection method may be implemented through the following specific steps:
(1) Preparation of nucleic acid macroarrays:
the nitrocellulose membrane (NC membrane) was cut into sheets of about 1.5 × 1.5 cm size for use. After 3. Mu.L of 100. Mu.M 5' -end was mixed with 3. Mu.L of 5 mg/mL streptavidin and 14. Mu.L of 10mMPBS (pH 7.4) using a biotin-modified immobilized probe, the mixture was incubated in a shaker at room temperature for 2 h, and then spotted on the prepared NC membrane at the amount of 0.3. Mu.L per spot, and five spots were uniformly and symmetrically placed on the prepared NC membrane, and then the spotted array was placed in a 37 ℃ oven for 3 h to stably immobilize the immobilized probe on the array, wherein the sequence of the immobilized probe was 5' (Bio) -ttcgcctctctttgtgtttttgcttgt-3'.
(2) Coupling colloidal gold with 4-mercaptopyridine:
adding 1 mL prepared colloidal gold solution, 30 mu L of potassium carbonate solution with the concentration of 0.1mol/L and 10 mu L of 4-mercaptopyridine solution with the concentration of 0.1mol/L into each 1.5 mL centrifuge tube respectively, rotating and incubating for 15 min on an incubator, taking down and placing in a refrigerator at 4 ℃ for aging 6 h, and fully coupling the materials; after the coupling is finished, the mixture is centrifuged for 10min at 9000 rpm, the supernatant is discarded, and the volume of the supernatant is recovered to 1/10 of the original volume by using sterile water to form the gold nanoparticle-4-mercaptopyridine compound, and the compound is preserved at 4 ℃ for later use.
(3) And (3) detecting heavy metal mercury ions:
100 mu L of Hg with each concentration is respectively dripped on the surface of the nucleic acid macro array nucleic acid array 2+ And (3) using the solution to capture mercury ions by the immobilized probe, after fully reacting for half an hour, eluting free mercury ions in the solution by using an eluent, adding 100 mu L of gold nanoparticle-4-mercaptopyridine compound, reacting for 10min, and flushing the nucleic acid array with double distilled water for three times to observe the color development effect by naked eyes.
(4) Carrying out silver staining sensitization treatment on the nucleic acid array after reaction:
in a light-proof environment. Silver staining is carried out on the mercury ion detection system obtained after the reaction, and AgNO is used as the solution 3 Hydroquinone, citric acid, respectively:
a.0.5 gAgNO 3 /2 mLH 2 O。
b.1.7 g Hydroquinone/30 mLH 2 O。
c.2.55 g citric acid/2.35 g trisodium citrate/10 mLH 2 O。
And the liquid b and the liquid c are mixed in a ratio of 3:1, and when silver staining is carried out, 120 mu LAgNO is added 3 And (3) quickly mixing the mixture evenly, immediately adding the mixture into each culture dish, namely 2 mL of each culture dish, reacting for a period of time, then adding ultrapure water, stopping the reaction, and repeatedly washing with ultrapure water to finally obtain the silver-dyed nucleic acid macroarray.
Wherein, the sequence of the immobilized probe can be: 5 '(Bio) -ttcgcctctctttgtgtttttgctttgtt-3'; the immobilized probe can capture Hg 2+ (ii) a The colloidal gold can be coupled with 4-mercaptopyridine; the composition of the eluent can be 10mM Tris-HCl +1% PEG +1% Tween-20, and can ensure free mercury ions in the elution solution; silver staining and increasingIn the sensitization treatment, the solution b and the solution c are prepared fresh to ensure no crystallization and no color, and the solution b and the solution c are mixed in a volume of 3000 μ L (1000 μ L), and are incubated at 37 ℃ 30 min before silver staining.
In the above application examples, the probe immobilized on the nitrocellulose membrane was finally passed through "T-Hg 2+ The structure of-4-MPY' captures and fixes the gold nanoparticles modified with 4-MPY on the surface of the nitrocellulose membrane, realizes color development under the conditions of higher mercury ion concentration and more captured metal nanoparticles, and can realize qualitative detection through visual observation. When the concentration of the target object is low, the number of the captured gold nanoparticles is small, the gold nanoparticles cannot be identified by naked eyes, but the high sensitivity characteristic of surface enhanced Raman is relied on, and the improvement of sensitivity and quantitative detection are realized by collecting Raman signals of 4-MPY on the array.
It should be noted that the key technical means of the present invention lies in the coordination of the nucleic acid macro-array and the signal probe (including the bifunctional molecule and the gold nanoparticles therein), and further the enhancement of signal intensity by silver sensitization, and the preparation method of the above nucleic acid macro-array and the specific preparation method of the signal probe are only used as the contents for facilitating the understanding of the present invention by those skilled in the art and exemplarily illustrated by the implementation of the present invention, but not the key technical means of the present invention, and should not be construed as the limitation of the protection scope of the present invention.
Accordingly, in order to implement the detection method, the embodiment of the present invention may further provide a kit corresponding to the reagents required in the method.
The types and concentrations of the reagents related to the kit correspond to those of the above methods one to one, and are not described herein again.
It is understood that the skilled person can fully refer to the various preparation methods disclosed in the prior art, and can adapt the preparation methods different from the above even directly commercially available to obtain the nucleic acid macro-array and/or bifunctional molecule with the same function, based on the knowledge of the structure determining function, as long as the structure of the nucleic acid macro-array and/or bifunctional molecule is formed as in the technical scheme provided by the present invention, and can have the same function, without being limited to the specific preparation method.
The technical scheme of the invention is further explained in detail by a plurality of embodiments and the accompanying drawings. However, the examples are chosen only for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
Example 1
This example illustrates a process for mercury ion detection based on nucleic acid macroarrays and bifunctional molecules, which includes the preparation of nucleic acid macroarrays and signaling probes, as follows:
(1) Immobilization of capture probes on NC membrane:
the purchased nitrocellulose membrane (NC membrane) was cut into sheets of about 1.5 × 1.5 cm size for use. The 5' end of the immobilized probe is modified by biotin, and the coupling process with Streptavidin (SAV) is as follows: 3 mu L of 100 mu M immobilized probe is added with 3 mu L of 5 mg/mL streptavidin and 14 mu L of 10mMPBS (pH value is 7.4) and mixed evenly, then at room temperature (room temperature usually refers to 25-35 ℃, the same applies below), after 2 h is incubated by a shaking table, and then the mixture is fully shaken and mixed evenly, thus being used for preparing the nucleic acid macroarray. And (3) spotting the prepared mixed solution of the immobilized probe and the streptavidin on the prepared NC membrane by using a pipette with the amount of 0.3 mu L of each spot, uniformly and symmetrically spotting five spots on each plate to form an array form, and then putting the spotted array into a 37 ℃ oven to incubate 3 h so as to stably immobilize the immobilized probe on the array. Then, the mixture is taken out and placed at room temperature for standby.
The sequences of the immobilized probes used were:
5’（Bio）-ttcgcctctctttgtgtttttgctttgtt-3’。
(2) Preparing colloidal gold dispersion liquid:
soaking all the used glass instruments in strong acid, cleaning the glass instruments by double distilled water, and drying the glass instruments in an oven for later use; filling about two thirds of deionized water and a stirrer into a conical flask, boiling on a heating magnetic stirrer, pouring out boiling water inside, and repeating for several times after the flask is cooled to prepare the gold nanoparticles. During preparation, 150 mL double distilled water and 2.55 mL chloroauric acid solution with the mass fraction of 0.5% are added into a clean conical flask, stirring is carried out at medium speed, trisodium citrate solution 3 mL with the mass concentration of 1% is added when the mixture is heated to be slightly boiled, stirring is carried out while heating and boiling is maintained, the color of the solution is finally changed into wine red, and heating and stirring are stopped for 5 min after the color is not changed any more, so that the reaction is fully completed; finally, colloidal gold dispersion is prepared and preserved at 4 ℃.
(3) Preparation of a signal probe:
adding Jin Fensan liquid colloid prepared by 1 mL and 30 mu L of potassium carbonate solution with the concentration of 0.1mol/L into each 1.5 mL centrifuge tube respectively, uniformly mixing, adding 10 mu L of 4-mercaptopyridine solution with the concentration of 0.1mol/L, uniformly stirring again, carrying out rotary incubation on an incubator for 15 min, taking down, placing in a refrigerator at 4 ℃ and aging for 6 h, and fully coupling; after the coupling is finished, the mixture is centrifuged for 10min at 9000 rpm, the supernatant is discarded, and the volume of the supernatant is recovered to 1/10 of the original volume by using sterile water to form the gold nanoparticle-4-mercaptopyridine compound, and the compound is preserved at 4 ℃ for later use.
(4) Detection of heavy metal mercury ions:
Hg 2+ preparation of a standard sample: accurately measuring 10mL concentrated sample water mercury from an ampoule bottle into a 250 mL volumetric flask, diluting with 3% nitric acid to a constant volume to scale to obtain 11.7 ng/mL Hg 2+ Diluting the standard solution with water to various concentrations to be detected for detection; 100 mu L of Hg with each concentration is respectively dripped on the surface of the nucleic acid macro array nucleic acid array 2+ The solution, so that the immobilized probe captures mercury ions, after reacting fully for half an hour, using eluent (10 mM Tris-HCl +1 PEG +1% Tween-20) to elute and remove free mercury ions in the solution, adding 100 μ l of gold nanoparticle-4-mercaptopyridine complex to react for 10min, and washing the nucleic acid array with double distilled water three times to observe the color development effect with naked eyes, wherein the color development effect is shown as the first line in FIG. 2.
Irradiating the color developing array on the array by using a laser, acquiring a Raman signal spectrum corresponding to 4-MPY, analyzing the relationship between the Raman signal intensity at the characteristic peak and the corresponding mercury ion concentration as shown in figure 3, establishing a linear relationship, and completing quantitative detection. The intensity of the characteristic peak of 4-mercaptopyridine is mainly detected, a standard curve is drawn, as shown in figure 4, then the sample to be detected is tested by adopting the same method in the step (4), and then the content of mercury ions in the sample can be calculated based on the standard curve.
Example 2
This example further illustrates a method for detecting mercury ions with higher sensitivity based on example 1, which is specifically as follows:
after the step (4) of example 1, the following steps are further added:
(5) Silver staining sensitization:
in a light-proof environment. Silver staining is carried out on the detection system obtained after the reaction, and three solutions are used:
a.0.5 gAgNO 3 /2 mL H 2 O;
b.1.7 g Hydroquinone/30 mL H 2 O;
c.2.55 g citric acid/2.35/g trisodium citrate/10 mL H 2 O。
The two solutions should be used in situ to ensure no crystal precipitation and no color, and solution b and solution c should be mixed in volume (3000 μ L:1000 μ L), and incubated at 37 deg.C 30 min before silver staining, and 120 μ LAgNO should be added during silver staining 3 And (3) rapidly mixing the mixture evenly, immediately adding the mixture into each culture dish, namely 2 mL of each culture dish, reacting for 150 seconds, then adding ultrapure water, terminating the reaction, and repeatedly washing with the ultrapure water to finally obtain the detection system after silver staining sensitization treatment.
(6) Raman detection:
and irradiating a color development area on a detection system by using a laser, collecting a Raman signal spectrum corresponding to 4-MPY, analyzing the relationship between the Raman signal intensity at the characteristic peak and the corresponding mercury ion concentration, and establishing a linear relationship to complete quantitative detection.
Moreover, in the embodiment, the macroscopic color change effect is as shown in the second row in fig. 2, and it can be obviously known that after silver staining sensitization treatment, not only the sensitivity of the raman test is significantly improved, but also the macroscopic color characteristics are significantly improved.
Example 3
This embodiment is substantially the same as embodiment 1 except that:
the bifunctional molecule is replaced by 2-mercapto-3-pyridinol.
The sequence of the immobilized probe was replaced with 5 '(Bio) -ttctcctctctttgtgttattgctttgtt-3'.
The nitrocellulose membrane was replaced with Whatman qualitative filter paper.
The nucleic acid macroarray and the signaling probe thus prepared still have the same color-changing ability and raman spectrum responsiveness as the detection of mercury ions by the same method as in step (4) in example 1, but the shape of raman spectrum curve is changed and characteristic peaks are shifted due to the substitution of bifunctional molecules.
Example 4
This example further illustrates a method for detecting mercury ions with higher sensitivity based on example 3, which is specifically as follows:
the silver staining sensitization treatment was further continued on the basis of example 3 by using the same steps (5) and (4) as in example 2.
Similar to example 2, after further silver-staining sensitization treatment is performed on the basis of example 3, the color change is more obvious, and the intensity of the characteristic peak of the raman spectrum is higher, which means that even if fixed probes and bifunctional molecules with different molecular structures are adopted, the detection sensitivity after silver-staining sensitization treatment can be improved as long as the fixed probes and the bifunctional molecules have consistent structural characteristics and functional characteristics.
Example 5
This embodiment also illustrates a method for detecting mercury ions, which is substantially the same as embodiment 3, except that:
during the preparation of the signal probe, the concentration of the bifunctional molecule is increased, and the concentration is 0.2 mol/L.
The signal probe prepared by the method has limited surface active sites of the colloidal gold, and even if bifunctional molecules with higher concentration are used, the bifunctional molecules marked on the surface of the colloidal gold are not obviously increased, the detection of mercury ions is carried out by continuously adopting the same method as the step (4) in the embodiment 3, the bifunctional molecules still have the same color change capability and response of Raman spectrum, and under the same condition, raman signals are not obviously enhanced, which indicates that the signal probe with the concentration of 0.1mol/L completely meets the requirements, and the technical effect of the invention can be realized by the higher concentration, but certain waste is caused.
Example 6
This example further illustrates a method for detecting mercury ions with higher sensitivity based on example 5, which is specifically as follows:
the silver staining sensitization treatment was further continued on the basis of example 5 by using the same steps (5) and (4) as in example 4.
Similar to example 4, after further silver sensitization treatment based on example 5, the color change is more obvious, and the intensity of the characteristic peak of the raman spectrum is higher, which means that even if the concentration of the bifunctional molecule is increased, the detection sensitivity after silver sensitization treatment is not affected.
Example 7
This embodiment still illustrates a method for detecting mercury ions, which is substantially the same as embodiment 6, except that:
in the step (4), the mercury ion standard substance is replaced by a tap water sample containing mercury ions with the same concentration.
Under the detection environment, due to the specific combination of the mercury ions, the T basic groups and the bifunctional signal molecules, the interference of other components in tap water is avoided, and the same color change capability and Raman spectrum responsiveness can be still realized.
Example 8
This example further illustrates a method for detecting mercury ions with higher sensitivity based on example 7, which is specifically as follows:
further silver sensitization treatment was continued on the basis of example 7 by using the same steps (5) and (4) as in example 4.
Similar to example 4, after further silver-sensitized sensitization treatment is performed on the basis of example 7, the color change is more obvious, and the intensity of the characteristic peak of the raman spectrum is higher, which means that even if the detected sample is a mercury ion sample with a complex component, the detection sensitivity after silver-sensitized sensitization treatment can be improved.
Comparative example 1
This comparative example is substantially the same as example 1, differing primarily in that:
in the step (2) and the step (3), the signal probe does not participate in the reaction by the colloidal gold dispersion liquid, but directly takes the 4-mercaptopyridine solution with the same concentration as the solution of the signal probe.
Namely: this example lacks the enhancement effect of gold nanoparticles on raman signal intensity.
Meanwhile, the color development effect of the gold nanoparticles is not achieved, and obviously, whether mercury ions are contained or not can not be determined by observing color change through naked eyes.
In addition, according to the same method and under the same mercury ion concentration, the characteristic peak intensity of the Raman signal measured by the comparative example is only 1/10 of that of the example 1 5 The signal intensity is significantly weaker than in example 1, which indicates that the gold nanoparticles have a very important role in enhancing the raman signal.
Comparative example 2
This comparative example is substantially the same as example 1, differing primarily in that: the surface of the gold nano particle is not provided with bifunctional molecules.
Under the condition, the bridge function of the bifunctional molecules is not existed, the colloidal gold can not be captured to the membrane surface, the membrane surface can not develop color, and obviously, the color change can not be observed by naked eyes to determine whether the membrane contains mercury ions.
In addition, according to the same method, under the same mercury ion concentration, no signal molecule exists in the comparative example, so that a corresponding Raman spectrum cannot be detected, and the fact that the bifunctional molecule plays a key role in signal generation is shown.
Comparative example 3
This comparative example is substantially the same as example 1, differing primarily in that: no immobilized probe was included.
Under the condition that no probe is fixed, the membrane surface loses the capability of capturing mercury ions, so that colloidal gold modified with bifunctional molecules cannot be captured, the membrane surface cannot develop color, whether a detected sample contains mercury ions or not can not be judged through visual observation, and a Raman spectrum cannot be detected, so that the fixed probe is very important for detecting the mercury ions in the scheme.
Comparative example 4
This comparative example is substantially the same as example 1, differing primarily in that: silver nanoparticles are used to replace colloidal gold to bind bifunctional molecules.
In the step (2), after the glassware is washed clean, 30 mL of 1mmol/L silver nitrate aqueous solution, 0.04 g sodium citrate, 0.02 g polyvinylpyrrolidone and 30 mL polyethylene glycol are added into a glassware provided with a magnetic stirrer, and the glassware is heated and stirred in a water bath at 40 ℃ for 30 min, so that the medicaments are fully mixed. Then, 30 mL of 2 mmol/L sodium borohydride is slowly dropped while stirring, and finally the solution gradually changes from milky white to earthy yellow to obtain the required silver nanoparticle solution which is stored at 4 ℃ for later use. The same steps (3) and (4) as in example 1 were then employed, and the colloidal gold was replaced with the prepared silver nanoparticles in step (3).
Under the condition of directly utilizing the silver nano particles, the color can be developed, and the same color changing capability and Raman spectrum responsiveness can be realized. However, silver nanoparticles are easy to oxidize, and irregular particles are easily formed in the preparation process, so that the preparation uniformity of the signal probe is influenced; in addition, the prepared silver nanoparticles are easy to influence the repeatability of detection due to the instability of the silver nanoparticles, so stable gold nanoparticles are preferred to prepare the signal probe.
Comparative example 5
This comparative example is substantially the same as example 1, except that the system required for silver-staining sensitization in step (5) is replaced with a conventional silver mirror reaction system:
liquid a: silver nitrate 3.5 g
Proper amount of ammonium hydroxide (the solution is dripped from turbid to transparent)
Sodium hydroxide 2.5 g/100 Ml
Distilled water 60 mL
b, liquid: glucose 45 g
Tartaric acid 4 g
Ethanol 100 mL
When the detection system is used, mixing according to a: b =1:1, reacting at the temperature of 10-15 ℃ for 150 seconds, adding ultrapure water to terminate the reaction, and repeatedly washing with the ultrapure water to finally obtain the detection system after silver staining sensitization treatment.
Using this conventional electroless silver plating process, there was no solution c (2.55 g citric acid/2.35 g trisodium citrate/10 mL H) as in example 1 during the reaction 2 O) provides a stable buffer system, the reduction reaction is relatively fast due to the strong reducibility of glucose, the specificity of the reduction reaction cannot be guaranteed to occur on the surface of the colloidal gold with high activity, and the film matrix has strong adsorption performance, so that a silver layer is non-specifically plated on the whole film, but is non-specifically deposited on an array, a silver layer is deposited on the whole film, but the silver layer is not in an array form, and the accurate detection of mercury ions is difficult finally.
Test example 1
The mercury-containing wastewater was examined by the methods of examples 1 to 8 and comparative examples 1 to 5, and the results are shown in table 1 below.
Table 1 test results of the methods of examples 1 to 8 and comparative examples 1 to 5 on mercury-containing wastewater
Based on the above embodiments and comparative examples, it is clear that the method for detecting mercury ions provided by the embodiments of the present invention utilizes the characteristics that the bifunctional molecules modified on the surface of the gold nanoparticles can combine with mercury ions and generate raman signals, and performs color development and/or raman detection by identifying and capturing mercury ions through the nucleic acid immobilized probe and the bifunctional molecules, thereby avoiding the conventional detection method based on T-Hg 2+ The modification of gold nanoparticle surface nucleic acid in the method for detecting mercury ions by the-T structure is more convenient and sensitive to the detection of mercury ions.
It should be understood that the above-mentioned embodiments are merely illustrative of the technical concepts and features of the present invention, which are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and therefore, the protection scope of the present invention is not limited thereby. All equivalent changes and modifications made according to the spirit of the present invention should be covered in the protection scope of the present invention.
Claims (10)
1. A method for detecting mercury ions based on a nucleic acid macroarray and bifunctional molecules, comprising:
providing a nucleic acid macro array and a signal probe, wherein the nucleic acid macro array comprises a plurality of fixed probes arranged in an array form, the fixed probes are oligonucleotide chains capable of specifically capturing mercury ions, the signal probe comprises gold nanoparticles and bifunctional molecules modified on the surfaces of the gold nanoparticles, and the bifunctional molecules are Raman signal molecules and can be specifically combined with the mercury ions;
contacting the nucleic acid macroarray with a sample to be detected so that the immobilized probe captures mercury ions in the sample to be detected;
contacting the nucleic acid macro array capturing mercury ions with the signal probe so that the mercury ions captured by the nucleic acid macro array are specifically combined with the signal probe to form a mercury ion detection system;
and detecting the mercury ions in the sample to be detected by testing the color and/or Raman spectrum of the mercury ion detection system.
2. The mercury ion detection method according to claim 1, characterized in that: the immobilized probe comprises a plurality of T bases; and/or the signal probe is obtained by performing a coupling reaction on a coupling reaction system containing the gold nanoparticles and the bifunctional molecules at 2-8 ℃ for 5.5-6.5-h, wherein the mass ratio of the gold nanoparticles to the bifunctional molecules is (0.4); and/or the particle size of the gold nanoparticles is 25-30 nm; and/or the bifunctional molecule comprises any one or the combination of more than two of 4-mercaptopyridine, 2-mercaptopyridine and 2-mercapto-3-pyridinol.
3. The mercury ion detection method according to claim 1, further comprising: eluting free mercury ions not captured by the immobilized probes with an eluent before contacting the nucleic acid macroarray capturing mercury ions with the signaling probes.
4. The mercury ion detection method of claim 1, further comprising: before performing color and/or Raman spectrum test on the mercury ion detection system, performing silver staining sensitization treatment on the mercury ion detection system; wherein, the silver staining sensitization treatment specifically comprises the following steps:
providing a silver staining solution, wherein the silver staining solution contains 1.2-1.8 mol/L silver ions and 0.1-0.3 g/mL reducing agent;
and contacting the silver staining solution with the mercury ion detection system and carrying out silver deposition reaction, thereby depositing a silver nano material on the surface of the mercury ion detection system.
5. The mercury ion detection method according to claim 4, wherein; the silver deposition reaction is carried out at the temperature of 15-40 ℃ for 2-3 min.
6. The mercury ion detection method according to claim 4, comprising:
providing a reducing agent solution containing hydroquinone at a concentration of 0.05-0.06-g/mL and a reducing buffer solution containing citric acid at a concentration of 0.25-0.30 g/mL and sodium citrate at a concentration of 2.3-2.4 g/L;
within 25-35 min before the silver-staining sensitization treatment, the reducing agent solution and the reducing buffer solution are mixed in a ratio of (2.5-3.5): 1, and then keeping the temperature of 35-40 ℃ for incubation to obtain a mixed solution;
and when the silver staining sensitization treatment is required, fully mixing the mixed solution with a silver nitrate solution to form the silver staining solution.
7. The mercury ion detection method according to claim 1, specifically comprising:
if the mercury ions in the sample to be detected are detected by testing the color of the mercury ion detection system, the corresponding qualitative detection limit is 0.4-0.6 nmol/L;
and if the mercury ions in the sample to be detected are detected by testing the Raman spectrum of the mercury ion detection system, the corresponding qualitative detection limit is 0.04-0.06 nmol/L and the corresponding quantitative detection limit is 0.02-0.03 nmol/L.
8. A nucleic acid macroarray and bifunctional molecule-based detection kit, comprising:
a nucleic acid macroarray comprising a plurality of immobilized probes arranged in an array on a surface of a solid support, the immobilized probes being oligonucleotide chains capable of specifically capturing mercury ions;
the signal probe comprises gold nanoparticles and bifunctional molecules modified on the surfaces of the gold nanoparticles, wherein the bifunctional molecules are Raman signal molecules and can be specifically combined with mercury ions.
9. The detection kit according to claim 8, characterized in that: the immobilized probe comprises a plurality of T basic groups and/or is obtained by carrying out coupling reaction on a coupling reaction system containing gold nanoparticles and bifunctional molecules at 2-8 ℃ for 5.5-6.5 h, wherein the mass ratio of the gold nanoparticles to the bifunctional molecules is 0.4; and/or the particle size of the gold nanoparticles is 25-30 nm; and/or the bifunctional molecule comprises any one or the combination of more than two of 4-mercaptopyridine, 2-mercaptopyridine and 2-mercapto-3-pyridinol.
10. The detection kit according to claim 8, characterized in that: the kit also comprises a silver staining solution, wherein the silver staining solution contains 1.2-1.8 mol/L silver ions and 0.1-0.3 g/mL reducing agent.
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