CN111474153A - Paper chip for detecting mercury ions and method for detecting mercury ions by using paper chip - Google Patents
Paper chip for detecting mercury ions and method for detecting mercury ions by using paper chip Download PDFInfo
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- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 claims description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 9
- 239000002202 Polyethylene glycol Substances 0.000 claims description 7
- 229920001223 polyethylene glycol Polymers 0.000 claims description 7
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 claims description 7
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention uses paper as a carrier, the above conversion luminescent material as a signal molecule, and realizes the high-sensitivity detection of mercury ions by using a fluorescence colorimetric method. The method specifically comprises the following steps: (1) manufacturing a paper chip substrate; (2) covalently coupling single-stranded nucleic acid S1 on the surface of the paper base; (3) preparing an up-conversion luminescent material and coupling with single-stranded nucleic acid S2; (4) and (3) carrying out fluorescence colorimetric detection on mercury ions. The conversion luminescent material is used as a signal molecule, so that the interference of background fluorescence in a paper substrate and an actual sample can be effectively overcome, and the detection sensitivity is improved; the portable 980 nm laser is used as an excitation light source, the mobile phone is used as a signal collecting device, the detection of the mercury ions is realized through the color change of the fluorescent picture, the detection device is portable and easy to obtain, and a feasible new method is provided for the on-site rapid detection of the mercury ions.
Description
Technical Field
The invention belongs to the technical field of application of paper-based micro-fluidic chips, and particularly relates to an up-conversion fluorescent paper chip and a method for detecting mercury ions by using the same.
Background
As mercury ions can generate great harm to human bodies under extremely low concentration, the maximum limit of mercury ions in drinking water is limited in many countries and organizations, for example, the maximum allowable limit of mercury ions in drinking water does not exceed 6ng/m L (30nM) specified by the world health organization, and the maximum allowable limit of mercury ions does not exceed 1ng/m L (5nM) specified by the Ministry of health in China, the detection of trace mercury ions is a common concern all over the world, and the realization of accurate, sensitive and rapid detection of mercury ions is very important.
At present, a plurality of methods for detecting mercury ions exist, wherein the methods include atomic absorption spectrometry, atomic emission spectrometry, an ICP (inductively coupled plasma) method, fluorescence spectrometry, an electrochemical method, ion chromatography, a capillary electrophoresis method and the like, which have high accuracy and sensitivity, but all the methods need to use precise and complex instruments, need professional personnel to operate, have high analysis cost, and cannot meet the requirement of on-site detection of mercury ions. The ultraviolet-visible spectrophotometry has poor selectivity and unsatisfactory sensitivity. The colorimetric method is simple to operate, but the detection sensitivity is usually low, and the national standard detection requirement cannot be met. Although the heavy metal rapid detection method can meet the requirements of field detection, the sensitivity is low, the selectivity is poor, a sample pretreatment step is required, and certain limitations exist in practical use. The test paper detection technology has the advantages of simple operation, low detection cost, high speed, convenience in carrying and the like, and many chromatographic test paper strips for detecting mercury ions are developed at present, but most of the chromatographic test paper strips are only suitable for qualitative detection, and the sensitivity is yet to be further improved.
Paper-based analytical equipment (PADs), called paper chips for short, adopts paper as a substrate, has the advantages of easily available raw materials, low detection cost, good biocompatibility, no need of external driving force and the like, and provides powerful means for rapid diagnosis, environmental monitoring and food safety control. Therefore, paper chips are increasingly being used for rapid, simple, and low-cost detection of mercury ions. Chengwei et al (Chinese patent application No. 201610256924.7) disclose a colorimetric analysis system with paper chip for detecting mercury ions, which utilizes the property of platinum nanoparticles similar to catalase to rapidly catalyze TMB/H 2O2The substrate generates blue precipitate, and when mercury ions exist, the platinum nanoparticles can rapidly react with the substrate, the activity of the self enzyme is inhibited, and the mercury is realized through the color change after the reaction And (4) detecting ions. The lowest detection concentration of the mercury ions in the patent is 10nM, and the mercury ions in the actual water sample are not detected. Plum blossom and the like (Chinese patent application number: 201610043793.4) report a method for detecting mercury ions based on a fluorescence resonance energy transfer system of a paper chip, the concentration range of the detected mercury ions is 0.2-1000 mu M, the lowest concentration detected by naked eyes is 1 mu M, the direct detection of the highest limit quantity of the mercury ions in drinking water required by the Chinese Ministry of health cannot be realized, and the practical application of the method is limited.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a paper chip for detecting mercury ions aiming at the defects in the prior art, and the upconversion luminescent nano material is used as a fluorescent signal molecule, so that the interference of background fluorescence of a paper substrate and an actual sample is effectively avoided, and the detection sensitivity is obviously improved.
The technical scheme adopted by the invention for solving the problems is as follows:
A paper chip for detecting mercury ions uses cellulose filter paper as a carrier, up-conversion luminescent nano materials (UCNPs) as signal molecules, single-stranded nucleic acids (S1 and S2) rich in T basic groups are respectively modified on the surfaces of a paper substrate and the signal molecules, and the mercury ions and the T basic groups can form T-Hg 2+And the property of the T structure is used for fixing the signal molecules on the surface of the paper substrate, so that the detection of mercury ions is realized.
The preparation method of the paper chip for detecting mercury ions mainly comprises the following steps:
(1) Using a water-insoluble solvent as a hydrophobic barrier to define a detection area on the surface of the paper to obtain a paper substrate;
(2) Treating a paper substrate with sodium periodate and lithium chloride, and fixing single-chain nucleic acid S1 on the surface of the paper substrate through a covalent bond by using sodium cyanoborohydride to serve as a detection substrate;
(3) Taking up-conversion luminescent nano materials (UCNPs) with carboxyl on the surface as signal molecules, and fixing amino-modified single-stranded nucleic acid S2 on the surface of the UCNPs in a buffer solution by using EDC and NHS as coupling agents to serve as detection solutions;
(4) And (3) combining the detection substrate obtained in the step (2) with the detection solution obtained in the step (3) to obtain the paper chip for detecting mercury ions.
According to the scheme, in the step (1), cellulose filter paper is preferably selected as paper. Furthermore, the pattern of the paper can be designed through software, the pattern is directly printed on the surface of the paper, and a water-insoluble solvent (such as an oil pen) is used for drawing a hydrophobic barrier along the printed pattern to define the detection area.
According to the scheme, the steps (1) and (2) can also adopt the following processes: the paper is treated by sodium periodate and lithium chloride, then a water-insoluble solvent is used as a hydrophobic barrier to define a detection area on the surface of the paper, and sodium cyanoborohydride is used for fixing single-chain nucleic acid S1 on the surface of a paper substrate through a covalent bond to serve as a detection substrate.
According to the above scheme, the sequence of the single-stranded nucleic acid S1 is: 5' -NH 2-CAGTTTGGAC-3'; the sequence of the single-stranded nucleic acid S2 is: 5' -NH 2-AAAAAAAAAAGTCCTTTCTG-3’。
According to the scheme, in the step (2), the relation between the area of the detection area and the dosage of S1 is S10.11-0.42nmol per square centimeter.
According to the scheme, in the step (2), the sodium periodate and the lithium chloride are prepared into a solution to soak the paper substrate, the concentration of the sodium periodate is 25-30mM, and the concentration of the lithium chloride is 45-50 mM.
According to the above scheme, in the step (2), the single-stranded nucleic acid S1 is immobilized on the surface of the paper substrate by a covalent bond using sodium cyanoborohydride in a buffer solution, the concentration of the single-stranded nucleic acid S1 in the buffer solution is 2.5 to 10. mu.M, and the concentration of sodium cyanoborohydride is 150 to 250 mM.
according to the scheme, in the step (3), the dosage of the single-stranded nucleic acid S2 is 0.5-5.0nmol per mg of UCNPs, the dosage of EDC is 0.25-0.75mg per mg of UCNPs, the dosage of NHS is 0.25-0.75mg per mg of UCNPs, the concentration ratio of EDC to NHS is 1:1, and the UCNP concentration of the detection solution obtained in the step (3) is 0.5-4.0mg/m L.
According to the scheme, in the step (3), after fixing the amino-modified single-stranded nucleic acid S2 on the surface of the UCNPs by using EDC and NHS as coupling agents in a buffer solution, the method further comprises the step of blocking unreacted carboxyl on the surface of the UCNPs by using a tris (hydroxymethyl) aminomethane solid. Wherein the mass ratio of the tris (hydroxymethyl) aminomethane solid to the UCNPs is (5-15):1, the tris (hydroxymethyl) aminomethane solid is added to react for 15-25min generally, and then the UCNPs are washed.
According to the scheme, in the step (3), the upconversion luminescent nano material NaYF 4Yb and Er are spherical, the grain diameter is 30-100nm, and the surface is modified with carboxyl.
The invention relates to a using method of a paper chip for detecting mercury ions, namely a method for detecting mercury ions by using the paper chip, which mainly comprises the following steps:
1) Dripping mercury ion solutions with different concentrations into a detection area of the detection substrate, adding a polyethylene glycol 2000 solution, dripping a detection solution, and reacting for 30-50min under the condition of keeping the paper chip moist;
2) Placing the paper chip on water-absorbing filter paper, adding a small amount of high-purity water into the detection area, washing to remove unreacted UCNPs, and air-drying;
3) Placing the paper chip obtained in the step 2) in a dark room, taking a 980nm laser as an excitation light source, recording a fluorescence photo, qualitatively detecting mercury ions according to picture colors, and quantitatively detecting mercury ions according to picture signal intensity.
According to the scheme, the mass fraction of polyethylene glycol 2000 added into the detection area in the step 1) is 0.01-0.25%.
according to the scheme, the adding amount of the polyethylene glycol 2000 solution is 0.3-1.3 mu L per square millimeter of detection area, the adding amount of the mercury ion solution with different concentrations and the detecting solution is 0.3-1.3 mu L per square millimeter of detection area, the adding amount of the high-purity water is 0.4-1.5 mu L per square millimeter of detection area, for example, the detection area is circular and has a diameter of 2mm-3mm, the adding volume of the mercury ion solution is 2-4 mu L and has a concentration of 5nM-500 mu M, the adding volume of the detecting solution is 2-4 mu L, the UCNP concentration of the detecting solution is 0.5-4.0mg/M L, and the adding volume of the high-purity water is 3-5 mu L.
According to the scheme, the fluorescent photo can be acquired by a camera, a tablet personal computer, a mobile phone and other mobile communication equipment with a photographing function. In addition, a 500-550nm band-pass filter is additionally required for collecting the fluorescent photos.
The paper chip for detecting mercury ions can be used for detecting the concentration of mercury ions in actual samples (such as tap water, lake water, river water and the like).
Compared with the existing mercury ion detection method, the method has the beneficial effects of simple operation, low cost, rapidness, directness and the like, and specifically comprises the following steps:
Firstly, the upconversion luminescent nano material is adopted as a fluorescent signal molecule, so that the interference of the background fluorescence of the paper substrate and the actual sample can be effectively avoided, the detection sensitivity is improved, the detection sensitivity of naked eye observation can reach 5nM, and the direct detection of the maximum limit quantity of mercury ions in drinking water can be met.
Secondly, the upconversion luminescent nano material adopts 980nm near infrared light as an excitation light source, and interferents in an actual sample cannot be excited under the wavelength, so that the interference of autofluorescence of the sample can be avoided.
Thirdly, the detection signal of the paper chip can be collected through a camera, a mobile phone and the like, the detection device is portable, the detection can be carried out by directly observing the color of the picture through naked eyes, the operation is simple, and the realization is easy.
Drawings
Fig. 1 is a schematic diagram of the preparation process of the paper chip and the detection of mercury ions according to the present invention.
Fig. 2 is a paper chip pattern designed in the example.
FIG. 3 is a fluorescent photograph of the paper chip used in the example for detecting mercury ions.
FIG. 4 is a linear curve of the paper chip for detecting mercury ions in the example.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the content of the present invention, but the present invention is not limited to the following examples.
In the following examples, the upconversion luminescent nano-material NaYF with carboxyl on the surface 4Of Yb, Er the preparation method comprises weighing 900mg polyacrylic acid in a polytetrafluoroethylene reaction kettle, adding 2M L0.25.25M L N (NO) 3)3solution (wherein Y: Yb: Er ═ 78:20:2), stirring and dissolving, adding 8m L pure water and 18m L absolute ethyl alcohol, adding 8 ml of NaF solution (containing 0.21g of NaF) under the condition of vigorous stirring, stirring at room temperature for 20min, then reacting at 200 ℃ for 10h, after the reaction liquid is naturally cooled to room temperature, respectively washing the reaction liquid with ethanol and water for three times, and dispersing into 4m L high-purity water to obtain the up-conversion luminescent nano-material UCNPs with carboxyl on the surface, prepared by other methods, the up-conversion luminescent nano-material UCNPs with carboxyl on the surface can also be used in the invention as long as the shape is close to spherical and the particle size is less than 100 nm.
In the following examples, the sequence of the single-stranded nucleic acid S1 is: 5' -NH 2-CAGTTTGGAC-3'; the sequence of the single-stranded nucleic acid S2 is: 5' -NH 2-AAAAAAAAAAGTCCTTTCTG-3'. Other amino-modified single-stranded nucleic acids rich in T bases can also be used in the present invention.
Examples
A preparation method of a paper chip for detecting mercury ions and a method for detecting mercury ions comprise the following specific steps:
(1) designing a paper chip pattern (as shown in figure 2) by software, pasting filter paper on the surface of A4 printing paper, directly printing the paper chip pattern on the surface of the filter paper, drawing a hydrophobic barrier on the surface of the filter paper by using an oil pen to define a detection area, and naturally drying the detection area to be used as a paper substrate, wherein the specific size of the filter paper is about 1.5cm multiplied by 4.5cm, each detection area is circular and is arranged in a 3 × 9 array, and the diameter of each detection area is 3 mm;
(2) weighing sodium periodate and lithium chloride, dissolving the sodium periodate and the lithium chloride in 75m L high-purity water (wherein the concentration of the sodium periodate is 26mM, and the concentration of the lithium chloride is 47mM), weighing 1.5g of the prepared paper substrate (or soaking the paper firstly, then printing patterns and defining a detection area) and soaking the paper substrate in the prepared solution, reacting for 1h at the temperature of 55 ℃, then washing the paper substrate twice by pure water, and airing for later use;
(3) Adding each detection area in the paper substrate obtained in the step (2) into 3. mu. L HEPES (100mM, pH 7.2) buffer containing 5.0. mu.M of S1, NaCNBH 2Naturally airing the sample with the concentration of 200mM to obtain a detection substrate;
(4) preparing 1500 μ L of HEPES (100mM, pH 7.2) buffer solution containing 2mg of UCNPs, 1mg of EDC and 1mg of NHS, activating at room temperature for 1h, centrifuging at 12000rpm for 12min, washing the obtained precipitate with HEPES buffer for 2 times, adding 2nmo L S2 into the activated UCNPs, reacting at room temperature for 12h, adding 20mg of tris (hydroxymethyl) aminomethane solid, reacting for 20min to block unreacted carboxyl on the surface of the UCNPs, centrifuging at 12000rpm for 12min, washing the obtained precipitate with HEPES buffer for 2 times, dispersing into 1m L of HEPES buffer solution, wherein the concentration of UCNPs-S2 is about 2mg/m L, and the solution is used as a detection solution (namely UCNPs-S2 solution);
(5) adding 3 mu L of 0.02% polyethylene glycol 2000 into each detection area on a detection substrate, then respectively adding 3 mu L of mercury ion solutions with different concentrations (the concentrations are 0, 5nM, 50nM, 500nM, 5 mu M, 50 mu M and 500 mu M) into each detection area, then adding 3 mu L of UCNPs-S2 solution into each detection area, reacting at 37 ℃ for 35min under a humid condition, placing the paper chip on absorbent filter paper in a turned-over manner after the reaction is finished, then dropwise adding 3 mu L of pure water into each detection area, washing to remove unreacted UCNPs, and airing the paper chip;
(6) And (3) placing the paper chip obtained in the step (5) in a dark room, taking a 980nm laser as an excitation light source, and taking a fluorescent photo by adding a 500-550nm band-pass filter outside the mobile phone.
As can be seen from fig. 3: when the concentration of mercury ions reaches 5nM, macroscopic luminescence can be shot in the detection area, the concentration of mercury ions is in the range of 5nM-500 μ M, the higher the concentration of mercury ions is, the more the fixed amount of UCNPs in the paper chip detection area is, the green light intensity of the fluorescence image is gradually enhanced, and the concentration of mercury ions can be qualitatively judged according to the color intensity of the fluorescence image. Therefore, the sensitivity of the paper chip prepared by the invention to mercury ion detection can reach 5 nM.
As can be seen from fig. 4: the paper chip established by the invention can realize quantitative detection of mercury ion concentration in a certain concentration range, the green light intensity y of a fluorescence picture and the log value x of the mercury ion concentration present a good linear relation, the linear curve equation is that y is 24.91+9.46x, and the linear correlation coefficient R is 0.9950. The linear range of the paper chip for detecting mercury ions is 5nM-500 mu M, and the detection sensitivity can reach 5 nM.
The paper chip and the method of the embodiment are adopted to detect the concentration of mercury ions in an actual water sample, and the results are shown in table 1. As can be seen from Table 1: for actual water body samples containing mercury ions, the accuracy of the detection result of the paper chip established by the invention is 92.7-108.9%, and the relative standard deviation among multiple measurement results is less than 15%, which indicates that the detection method established by the invention has good accuracy and stability for the actual water body samples.
TABLE 1
| Kind of sample | Add concentration (nM) | Test concentration (nM) | Recovery (%) | Relative standard deviation (%) |
| Tap water | 100 | 108.9 | 108.9 | 11.5 |
| River water | 100 | 92.7 | 92.7 | 14.7 |
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and changes can be made without departing from the inventive concept of the present invention, and these modifications and changes are within the protection scope of the present invention.
Claims (10)
1. A paper chip for detecting mercury ions is characterized in that cellulose filter paper is used as a carrier, a conversion luminescent nano material is used as a signal molecule, single-stranded nucleic acid rich in T basic groups is respectively modified on the surfaces of a paper substrate and the signal molecule, and the mercury ions and the T basic groups can form T-Hg 2+And the property of the T structure is used for fixing the signal molecules on the surface of the paper substrate, so that the detection of mercury ions is realized.
2. A preparation method of a paper chip for detecting mercury ions is characterized by mainly comprising the following steps:
(1) Using a water-insoluble solvent as a hydrophobic barrier to define a detection area on the surface of the paper to obtain a paper substrate;
(2) Treating a paper substrate with sodium periodate and lithium chloride, and then fixing single-stranded nucleic acid S1 rich in T basic groups on the surface of the paper substrate through covalent bonds by using sodium cyanoborohydride to serve as a detection substrate;
(3) Taking UCNPs (u-channel nuclear fluorescent nanoparticles) with carboxyl on the surface as signal molecules, and fixing amino-modified single-stranded nucleic acid S2 rich in T base on the surface of the UCNPs in a solution by using EDC and NHS as coupling agents to serve as a detection solution;
(4) And (3) combining the detection substrate obtained in the step (2) with the detection solution obtained in the step (3) to obtain the paper chip for detecting mercury ions.
3. The method for preparing a paper chip for detecting mercury ions according to claim 2, wherein the paper is cellulose filter paper; the sequence of the single-stranded nucleic acid S1 is: 5' -NH 2-CAGTTTGGAC-3'; the sequence of the single-stranded nucleic acid S2 is: 5' -NH 2-AAAAAAAAAAGTCCTTTCTG-3’。
4. the method of claim 2, wherein in the step (2), the relationship between the area of the detection region and the amount of S1 is 10.11-0.42 nmol/cm, and the concentration of UCNP in the detection solution obtained in the step (3) is 0.5-4.0 mg/m L.
5. The method for preparing a paper chip for detecting mercury ions according to claim 2, wherein in the step (2), the solution prepared from sodium periodate and lithium chloride is used for soaking a paper substrate to be treated, wherein the concentration of the sodium periodate is 25-30 mM, and the concentration of the lithium chloride is 45-50 mM; the single-stranded nucleic acid S1 was immobilized on the surface of the paper substrate by covalent bond using sodium cyanoborohydride in a buffer solution at a concentration of 2.5-10. mu.M for single-stranded nucleic acid S1 and 150-250 mM for sodium cyanoborohydride.
6. The method of claim 2, wherein in the step (3), the amount of the single-stranded nucleic acid S2 is 0.5-5.0 nmol per mg of UCNPs, and the amount of EDC is 0.25-0.75 mg per mg of UCNPs; the amount of NHS is 0.25-0.75 mg per mg UCNPs; the concentration ratio of EDC to NHS was 1: 1.
7. The method for preparing a paper chip for detecting mercury ions according to claim 2, wherein in the step (3), the upconversion luminescent nano material NaYF is adopted 4Yb and Er are spherical, the grain diameter is 30-100 nm, and the surface is modified with carboxyl.
8. The method for detecting mercury ions by using the paper chip of any one of claims 2 to 7 is characterized by mainly comprising the following steps:
1) Dripping mercury ion solutions with different concentrations into a detection area of the detection substrate, adding a polyethylene glycol 2000 solution, dripping a detection solution, and reacting for 30-50 min under the condition of keeping the paper chip moist;
2) Placing the paper chip on water-absorbing filter paper, adding a small amount of high-purity water into the detection area, washing to remove unreacted UCNPs, and air-drying;
3) Placing the paper chip obtained in the step 2) in a dark room, taking a 980 nm laser as an excitation light source, recording a fluorescence photo, qualitatively detecting mercury ions according to picture colors, and quantitatively detecting mercury ions according to picture signal intensity.
9. the method for detecting mercury ions by using the paper chip as claimed in claim 8, wherein the mass fraction of the polyethylene glycol 2000 added into the detection area in the step 1) is 0.01-0.25%, the addition amount of the polyethylene glycol 2000 solution is 0.3-1.3 μ L per square millimeter of the detection area, the addition amount of mercury ion solutions with different concentrations and the addition amount of detection solutions are 0.3-1.3 μ L per square millimeter of the detection area, and the addition amount of high purity water is 0.4-1.5 μ L per square millimeter of the detection area.
10. The method for detecting mercury ions by using the paper chip as claimed in claim 8, wherein a 500-550 nm band-pass filter is additionally arranged during fluorescent photograph acquisition.
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