CN102207502A - Mercury ion test paper and preparation method thereof - Google Patents
Mercury ion test paper and preparation method thereof Download PDFInfo
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- CN102207502A CN102207502A CN2011100738197A CN201110073819A CN102207502A CN 102207502 A CN102207502 A CN 102207502A CN 2011100738197 A CN2011100738197 A CN 2011100738197A CN 201110073819 A CN201110073819 A CN 201110073819A CN 102207502 A CN102207502 A CN 102207502A
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Abstract
The invention provides a mercury ion test paper and a preparation method thereof. The test paper is characterized by comprising a strip base plate and a sample pad, a glass fiber combination pad coated with specific DNA probes, a specifically coated nitrocellulose membrane and an absorbent pad that are successively overlapped on the strip base plate. One end of each specific DNA probe is bonded with colloidal gold, and the other end is bonded with biotin; b-DNA coated linear detection line T line and streptavidin coated linear quality control line C line are arranged on the nitrocellulose membrane; a base of the b-DNA and a base of the DNA probe can form a T-Hg<2+>-T mismatching. The preparation method comprises the following steps: overlapping the successively prepared sample pad, glass fiber combination pad coated with specific DNA probes, specifically coated nitrocellulose membrane and an absorbent pad to shape up, pasting on the base plate, and cutting into fine strips of 3-10mm wide to obtain the mercury ion test paper. The mercury ion test paper has the advantages of high sensitivity and high selectivity for sample mercury ion detection.
Description
Technical field
The present invention relates to a kind of heavy metal ion detection technique field, especially relate to a kind of mercury ion detecting test strips and preparation method thereof.
Background technology
Mercury (Hg) is liquid metal unique under the normal temperature, belongs to the heavy metal classification.In recent years, human increasing to exploitation, smelting, processing and the commercial manufacturing activities of heavy metal Hg, a large amount of mercury enters in atmosphere, water, the soil and retains, accumulates and move.The toxicity of mercury is big, in vivo with environment in the residence time long, the serious threat human health, so strict qualification has all been done to mercury content in food, daily necessities, the environment in many countries and regions such as the U.S., European Union, Canada and China, the monitoring of mercury pollution has become the problem of paying close attention to.The mercury of occurring in nature is mainly with ionic species (Hg
2+) exist, historical facts or anecdotes existing to mercury ion accurately, sensitive, fast on-the-spot detect extremely important.
The method that detects mercury ion at present mainly contains: ultraviolet-visible spectrophotometry, atomic absorption spectrography (AAS), atomic emission spectrometry, ICP method, fluorescent spectrometry, electrochemical process, the chromatography of ions, capillary electrophoresis, heavy metal rapid detector method, colourimetry, test strips method etc.Ultraviolet-visible spectrophotometry poor selectivity detects limit for height; Atomic absorption spectrography (AAS), atomic emission spectrometry, ICP method, fluorescent spectrometry, electrochemical process, the chromatography of ions, capillary electrophoresis, accuracy and highly sensitive, but all need use specific instrument, cost an arm and a leg, complex operation, and require the testing staff to possess certain professional knowledge, the analysis cost height, can't be applied to on-the-spot the detection, be difficult to popularize; The heavy metal rapid detector method can be applicable to on-the-spot the detection, but sensitivity is not high, poor selectivity, the sample pretreatment complexity; Based on the colourimetry of collaurum (AuNPs) and specific DNA, this method sensitivity still can, selectivity is good, but it is comparatively serious disturbed by sample matrices.Compare with said method, the test strips method is quick, easy and other tool advantage in detecting at the scene with it, but test strips based on traditional chelating developer, selectivity is relatively poor, sensitivity is low, detect lower limit and be about 0.1～1mg/L, can't be up to state standards to detect requires (as: potable water＜0.01 mg/L).
Summary of the invention
Technical matters to be solved by this invention provides a kind of mercury ion detecting test strips based on collaurum and specific DNA and preparation method thereof, and this test strips can high sensitivity, the mercury ion in the highly selective test sample.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of mercury ion detecting test strips, described test strips by the bar shaped base plate and on the bar shaped base plate successively the overlap joint sample pad, apply the glass fiber conjugate pad of specificity DNA probing needle, the nitrocellulose membrane of specified packet quilt and adsorptive pads are formed, described specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, useful b-DNA bag wraps by orthoscopic nature controlling line C line by orthoscopic detection line T line with the strepto-affinity is plain on the described nitrocellulose membrane, can form T-Hg between the base of described b-DNA and the base of described specificity DNA probing needle
2+-T mispairing.
The structure of described specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin, and the structure of described b-DNA is 5 '-GTTTCTTCTTTGGTTTGATT-3 '.
Described sample pad is the all-glass paper through 0.01～0.05 mol/L Tris-HCl damping fluid immersion treatment, and wherein to contain the mass percent of sucrose be 1～10% to the Tris-HCl damping fluid, and the pH value is 5.0～8.0.
The quantity for spray of described specificity DNA probing needle solution on described glass fiber conjugate pad is 2.0～10.0 μ L/cm
2Described specificity DNA probing needle solution be 20～70 μ g sequences be the DNA of HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin with after the collaurum of 0.5～2 μ mol combines, the concentration that is dissolved in 50～200 μ L is gained in 0.01～0.05 mol/L phosphate buffer.
Described b-DNA bag is 0.0003～0.003 μ mol/mm by consumption.
The plain bag of described strepto-affinity is 0.01～0.06 μ g/mm by consumption.
A kind of preparation method of mercury ion detecting test strips may further comprise the steps:
(1) preparation of sample pad
With all-glass paper with 0.01～0.05 mol/L Tris-HCl damping fluid soak wetting after, promptly obtained sample pad down in dry 4～12 hours at 20 ℃～37 ℃, wherein to contain the mass percent of sucrose be 1～10% to the Tris-HCl damping fluid, the pH value is 5.0～8.0;
(2) preparation of the glass fiber conjugate pad of coating specificity DNA probing needle
With all-glass paper with 0.01～0.05 mol/L Tris-HCl damping fluid soak wetting after, after under 20 ℃～37 ℃ dry 4～12 hours, on every square centimeter all-glass paper, evenly spray the specificity DNA probing needle solution of 2.0～10.0 μ L, after under 20 ℃～37 ℃ dry 2～5 hours, promptly obtain applying the glass fiber conjugate pad of specificity DNA probing needle, described specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, the structure of described specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin, wherein to contain the mass percent of sucrose be 1～10% to the Tris-HCl damping fluid, and the pH value is 5.0～8.0;
(3) preparation of the nitrocellulose membrane of specified packet quilt
Is that the b-DNA solution of 30～100 μ mol/L evenly is sprayed on the nitrocellulose membrane with spray sample instrument with concentration, drying is 2～8 hours under 25 ℃～37 ℃, form orthoscopic detection line T line, with spray sample instrument is that the strepto-affinity cellulose solution of 1～2 mg/L evenly is sprayed on the nitrocellulose membrane along the direction with detection line T line parallel with concentration, drying is 2～8 hours under 25 ℃～37 ℃, form orthoscopic nature controlling line C line, promptly obtain the nitrocellulose membrane of specified packet quilt, can form T-Hg between the base of wherein said b-DNA and the base of described specificity DNA probing needle
2+-T mispairing, the structure of described b-DNA are 5 '-GTTTCTTCTTTGGTTTGATT-3 ';
(4) sample pad that step (1) is obtained, the glass fiber conjugate pad of the coating specificity DNA probing needle that step (2) obtains, the nitrocellulose membrane of the specified packet quilt that step (3) obtains and adsorptive pads overlap according to the order of sequence and are pasted on the base plate, be cut into the wide slice of 3～10 mm, promptly get the mercury ion detecting test strips.
The preparation method of described specificity DNA probing needle solution is as follows: the DNA that with 20～70 μ g sequences is HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin is the dissolving of 0.01～0.05 mol/L Tris-HCl damping fluid with the concentration of 1～5 mL, behind the vibration mixing, joining 1 mL concentration is in the colloidal gold solution of 0.5～2 mmol/L, the vibration mixing, incubation is after 5～24 hours under the room temperature, centrifugal abandoning supernatant, after the concentration that adds 50～200 μ L is the dissolving of 0.01～0.05 mol/L phosphate buffer, promptly get specificity DNA probing needle solution, the mass percent that wherein said Tris-HCl damping fluid contains sucrose is 1～10%, described Tris-HCl pH of buffer value is 5.0～8.0, containing the NaCl mass percent in the described phosphate buffer is 0.5～1%, containing the lauryl sodium sulfate mass percent is 0.01～0.05%, and described phosphate buffer pH value is 5.0～8.0.
Described b-DNA solution bag is 1.0～3.0 μ L/cm by consumption, the solvent of described b-DNA solution is concentration 0.01～0.05 a mol/L phosphate buffer, containing the NaCl mass percent in the described phosphate buffer is 0.5～1%, containing the lauryl sodium sulfate mass percent is 0.01～0.05%, and described phosphate buffer pH value is 5.0～8.0.
Described strepto-affinity cellulose solution bag is 1.0～3.0 μ L/cm by consumption, the solvent of described strepto-affinity cellulose solution is concentration 0.01～0.05 a mol/L phosphate buffer, containing the NaCl mass percent in the described phosphate buffer is 0.5～1%, containing the lauryl sodium sulfate mass percent is 0.01～0.05%, and described phosphate buffer pH value is 5.0～8.0.
The mercury ion detecting test strips that the present invention proposes: apply b-DNA on the detection line, apply Streptavidin on the nature controlling line, apply the specificity DNA probing needle that two ends are combined with collaurum and biotin respectively on the glass fiber conjugate pad; When mercury ion existed, T-Hg took place in the b-DNA on specificity DNA probing needle and the detection line
2+-T mispairing is stranded and be hunted down, and the colloidal gold aggregation colour developing of its end of the chain combination reaches the purpose that detects mercury ion; The Streptavidin generation specificity combination that applies on specificity DNA probing needle chain other end modified biotin energy and the nature controlling line, so excessive specificity DNA probing needle is hunted down and rests on the nature controlling line, the colloidal gold aggregation of its end of the chain combination develops the color, and reaches the Quality Control purpose of detection.With this test strips sample liquid is detected: if when the ion concentration of mercury in the sample liquid is higher than detectability, detection line and nature controlling line show redness simultaneously; If when not containing mercury ion or ion concentration of mercury in the sample liquid and being lower than detectability, detection line does not develop the color, nature controlling line shows red; If nature controlling line does not develop the color, it is invalid then to detect.
Compared with prior art, the invention has the advantages that:
(1) high sensitivity, detectability reach 0.005 mg/L, and reason is: at first, and the T-Hg of specific DNA
2+-T mispairing is very sensitive to mercury ion; Secondly, the molar absorptivity of collaurum is very high, and colour developing is distinct; Once more, detecting and carry out on test strips, is a colour developing under white background, and people's naked eyes are more prone to identification; At last, test strips can effectively be eliminated the influence of coloured interfering material to colour developing by filtering and the expansion effect;
(2) high selectivity, common metal ion such as Pb
2+, Cd
2+, Mg
2+, Ca
2+, Fe
2+, Cu
2+, Ni
2+, Co
2+, Mn
2+, Zn
2+All noiseless to detecting.Reason is: the T-Hg of specific DNA
2+-T mispairing has the recognition capability of high specific to mercury ion, and the interference of other metallic ion can be ignored;
(3) sample need not complicated pre-service.Reason is: but specific DNA specific recognition mercury ion, and can eliminate sample matrices and disturb by the filtration capacity of expansion effect and test strips itself;
(4) simple to operate, detection time is short, only needs several minutes, and reason is: only need can observe testing result through simple immersion, expansion;
(5) the detection cost is low, about 3 yuan of each test strips cost of manufacture, and reason is: reagent and material usage are few, and production stage is simple.
In sum, based on the mercury ion detecting test strips of collaurum and specific DNA, this test strips can high sensitivity, the mercury ion in the highly selective test sample, and the preparation method of this test strips is simple, easy to operate.
Description of drawings
Fig. 1 is the structural representation of test strips of the present invention;
Fig. 2 detects the effect synoptic diagram for the present invention;
A: contain the mercury ion that is higher than detectability in the sample liquid;
B: not mercurous or mercury ion content is lower than detectability in the sample liquid;
C: it is invalid to detect;
Fig. 3 detects the figure as a result of actual sample for the present invention.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
A kind of mercury ion detecting test strips of the present invention, as shown in Figure 1, the nitrocellulose membrane 4 of the sample pad 2 that comprises bar shaped base plate 1 and overlap successively on the bar shaped base plate, the glass fiber conjugate pad 3 that applies specificity DNA probing needle, specified packet quilt and adsorptive pads 5 are formed, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, useful b-DNA bag wraps by orthoscopic nature controlling line C line 7 by orthoscopic detection line T line 6 with the strepto-affinity is plain on this nitrocellulose membrane, can form T-Hg between the base of described b-DNA and the base of described specificity DNA probing needle
2+-T mispairing.
In this specific embodiment, the structure of specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin, and the structure of b-DNA is 5 '-GTTTCTTCTTTGGTTTGATT-3 '; Sample pad is the all-glass paper through 0.01～0.05mol/L Tris-HCl damping fluid immersion treatment, and wherein to contain the mass percent of sucrose be 1～10% to the Tris-HCl damping fluid, and the pH value is 5.0～8.0; The quantity for spray of specificity DNA probing needle solution on described glass fiber conjugate pad is 2.0～10.0 μ L/cm
2This specificity DNA probing needle solution be 20～70 μ g sequences be the DNA of HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin with after the collaurum of 0.5～2 μ mol combines, the concentration that is dissolved in 50～200 μ L is gained in 0.01～0.05 mol/L phosphate buffer; The b-DNA bag is 0.0003～0.003 μ mol/mm by consumption; The plain bag of strepto-affinity is 0.01～0.06 μ g/mm by consumption; Adsorptive pads is a kind of thieving paper (model is SX27 or CH27), and thieving paper is attached to the end of bar shaped base plate, and adsorptive pads also can be glass fibre membrane SB08 or BT50.
Mercury ion detecting test strips of the present invention detects mercury ion based on collaurum and specific DNA, and when mercury ion existed, T-Hg took place the b-DNA on specificity DNA probing needle and the detection line
2+-T mispairing is stranded and be hunted down, and the colloidal gold aggregation colour developing of its end of the chain combination reaches the purpose that detects mercury ion; The Streptavidin generation specificity combination that applies on specificity DNA probing needle chain other end modified biotin energy and the nature controlling line, so excessive specificity DNA probing needle is hunted down and rests on the nature controlling line, the colloidal gold aggregation colour developing of its end of the chain combination, reach the Quality Control purpose of detection, as shown in Figure 2, a: contain the mercury ion that is higher than detectability in the sample liquid; B: not mercurous or mercury ion content is lower than detectability in the sample liquid; C: it is invalid to detect; Its concrete preparation method is as follows:
1, solution preparation
1.1 the preparation of 0.5～2mmol/L colloidal gold solution
A. 0.5～2mmol/L HAuCl
4The preparation of solution: the HAuCl that takes by weighing 0.0205～0.0822 g
44H
2O is settled in the 100mL volumetric flask with distilled water, and is stand-by;
B. the preparation of 38.8mmol/L sodium citrate solution: take by weighing 1.1407 g sodium citrates, be settled in the 100 mL volumetric flasks with distilled water, stand-by;
C. use chloroazotic acid (hydrochloric acid and the nitric acid ratio of 3:1 by volume mix) to soak 200 mL, two neck flasks, stirrer, glass stopper and condenser pipe after 10～25 minutes, clean standby;
D. get the HAuCl of 0.5～2mmol/L of 100mL
4Solution adds in the two neck flasks, and a mouth of flask connects condenser pipe, and another mouthful clogs with stopper, stirs, and heating refluxes.When solution begins to reflux, open stopper, clog stopper behind the sodium citrate solution of 38.8 mmol/L of adding 10～50 mL fast, continued vigorous stirring, reflux 20 minutes, stop heating, continue stirring and be cooled to room temperature, promptly make colloidal gold solution, preserve the colloidal gold solution for preparing under 4 ℃ of conditions;
1.2 Tris-HCl damping fluid preparation
0.01 the preparation method of～0.05 mol/L Tris-HCl damping fluid: weighing 0.7880～3.9398 g Tris-HCl places 1 L beaker, the deionized water dissolving that adds 400mL, add 0.234～2.34 g NaCl and 0.030～0.298 g KCl again, the concentration that makes NaCl is that the concentration of 0.01～0.1 mol/L, KCl is 0.001～0.01 mol/L, behind the dissolving mixing, add suitable amount of sucrose, the mass percent concentration that makes sucrose is 1～10%, regulates Tris-HCl pH of buffer to 5.0～8.0 with acid-base solution;
1.3 the preparation of 0.01～0.05 mol/L phosphate buffer
Take by weighing for 1:1 by the amount of substance ratio: 0.3～1.5 g NaH
2PO
42H
2O and 0.355～1.775 g Na
2HPO
42H
2O places 1 L beaker, the deionized water dissolving that adds 400mL, add NaCl and lauryl sodium sulfate again, make that to contain the NaCl mass percent in this phosphate buffer be 0.5～1%, containing the lauryl sodium sulfate mass percent is 0.01～0.05%, and regulating phosphate buffer pH value with acid-base solution is 5.0～8.0;
2, the preparation of sample pad 2
With all-glass paper with above-mentioned Tris-HCl damping fluid soak 0.5～1 hour wetting after, promptly obtained sample pad 2 down in dry 4～12 hours at 20 ℃～37 ℃;
3, apply the preparation of the glass fiber conjugate pad 3 of specificity DNA probing needle
A. with all-glass paper with above-mentioned Tris-HCl damping fluid soak 0.5～1 hour wetting after, 20 ℃～37 ℃ dry 4～12 hours down, promptly get and seal the all-glass paper of handling;
B. with 20～70 μ g sequences the above-mentioned Tris-HCl damping fluid dissolving of the DNA of HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin with 1～5mL, behind the vibration mixing, join in the colloidal gold solution of the above-mentioned preparation of 1mL, the vibration mixing, incubation is after 5～24 hours under the room temperature, centrifugal abandoning supernatant, after adding the above-mentioned phosphate buffer dissolving of 50～200 μ L, promptly get specificity DNA probing needle solution, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, and the structure of this specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin;
C. get above-mentioned gained specificity DNA probing needle solution with spray sample instrument, on all-glass paper every square centimeter, that sealing was handled, evenly spray 2.0～10.0 μ L, after dry 2～5 hours, promptly get the glass fiber conjugate pad 3 that applies specificity DNA probing needle under 20 ℃～37 ℃;
4, the preparation of the nitrocellulose membrane 4 of specified packet quilt
Is that the b-DNA solution of 30～100 μ mol/L evenly is sprayed on the nitrocellulose membrane with spray sample instrument with concentration, drying is 2～8 hours under 25 ℃～37 ℃, form orthoscopic detection line T line 6, the strepto-affinity cellulose solution that with spray sample instrument with concentration is 1～2 mg/L evenly is sprayed on the nitrocellulose membrane along the direction parallel with detection line T line 6, drying is 2～8 hours under 25 ℃～37 ℃, form orthoscopic nature controlling line C line 7, promptly obtain the nitrocellulose membrane 4 of specified packet quilt; Wherein b-DNA solution bag is 1.0～3.0 μ L/cm by consumption, the solvent of this b-DNA solution is the phosphate buffer of above-mentioned steps 1 preparation, strepto-affinity cellulose solution bag is 1.0～3.0 μ L/cm by consumption, the solvent of this strepto-affinity cellulose solution is the phosphate buffer of above-mentioned steps 1 preparation, wherein can form T-Hg between the base of the base of b-DNA and specificity DNA probing needle
2+-T mispairing, the structure of this b-DNA are 5 '-GTTTCTTCTTTGGTTTGATT-3 ';
5, mercury ion detecting test strips preparation
On bar shaped base plate 1, overlap sample pad 2 successively, apply the glass fiber conjugate pad 3 of specificity DNA probing needle, the nitrocellulose membrane 4 of specified packet quilt, adsorptive pads 5, two ends at the bar shaped base plate are stained with self-adhesive paper respectively, the self-adhesive paper that wherein has arrow is attached on the junction of sample pad and pad, arrow has been indicated the depth capacity that immerses, at last test paper is cut into the wide slice of 3～10 mm, promptly get the mercury ion detecting test strips, an end of during detection arrow being indicated immerses in the sample solution, water sorption by adsorptive pads, make testing sample successively by applying the glass fiber conjugate pad 3 of specificity DNA probing needle, the nitrocellulose membrane 4 of specified packet quilt reaches the purpose of detection.
A kind of mercury ion detecting test strips of the present invention, as shown in Figure 1, the nitrocellulose membrane 4 of the sample pad 2 that comprises bar shaped base plate 1 and overlap successively on bar shaped base plate 1, the glass fiber conjugate pad 3 that applies specificity DNA probing needle, specified packet quilt and adsorptive pads 5 are formed, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, useful b-DNA bag wraps by orthoscopic nature controlling line C line 7 by orthoscopic detection line T line 6 with the strepto-affinity is plain on this nitrocellulose membrane, wherein can form T-Hg between the base of the base of b-DNA and specificity DNA probing needle
2+-T mispairing, its preparation method specifically may further comprise the steps:
1, solution preparation: with embodiment 2
2, the preparation of sample pad 2
With all-glass paper with above-mentioned Tris-HCl damping fluid soak 0.7 hour wetting after, promptly obtained sample pad 2 down in dry 8 hours at 30 ℃;
3, apply the preparation of the glass fiber conjugate pad 3 of specificity DNA probing needle
A. with all-glass paper with above-mentioned Tris-HCl damping fluid soak 0.7 hour wetting after, 30 ℃ dry 8 hours down, promptly get and seal the all-glass paper of handling;
B. with 50 μ g sequences the above-mentioned Tris-HCl damping fluid dissolving of the DNA of HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin with 3 mL, behind the vibration mixing, join in the colloidal gold solution of the above-mentioned preparation of 1 mL, the vibration mixing, incubation is after 15 hours under the room temperature, centrifugal abandoning supernatant, after adding the above-mentioned phosphate buffer dissolving of 100 μ L, promptly get specificity DNA probing needle solution, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, and the structure of this specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin;
C. get above-mentioned gained specificity DNA probing needle solution with spray sample instrument, even spraying 6.0 μ L on the all-glass paper that every square centimeter sealing was handled, drying promptly got the glass fiber conjugate pad 3 that applies specificity DNA probing needle after 3 hours under 30 ℃;
4, the preparation of the nitrocellulose membrane 4 of specified packet quilt
Is that the b-DNA solution of 60 μ mol/L evenly is sprayed on the nitrocellulose membrane with spray sample instrument with concentration, drying is 6 hours under 30 ℃, form orthoscopic detection line T line 6, the strepto-affinity cellulose solution that with spray sample instrument with concentration is 1.5 mg/L evenly is sprayed on the nitrocellulose membrane along the direction parallel with detection line T line 6, drying is 6 hours under 30 ℃, form orthoscopic nature controlling line C line 7, promptly obtain the nitrocellulose membrane 4 of specified packet quilt; Wherein b-DNA solution bag is 2.0 μ L/cm by consumption, the solvent of this b-DNA solution is the phosphate buffer of above-mentioned steps 1 preparation, strepto-affinity cellulose solution bag is 2.0 μ L/cm by consumption, the solvent of this strepto-affinity cellulose solution is the phosphate buffer of above-mentioned steps 1 preparation, and wherein the base of b-DNA and the base between the specificity DNA probing needle can form T-Hg
2+-T mispairing, the structure of this b-DNA are 5 '-GTTTCTTCTTTGGTTTGATT-3 ';
5, mercury ion detecting test strips preparation
On bar shaped base plate 1, overlap glass fiber conjugate pad 3, the nitrocellulose membrane 4 of specified packet quilt, the adsorptive pads 5 of sample pad 2, coating specificity DNA probing needle successively, two ends at the bar shaped base plate are stained with self-adhesive paper respectively, the self-adhesive paper that wherein has arrow is attached on the junction of sample pad 2 and glass fiber conjugate pad 3, arrow has been indicated the depth capacity that immerses, at last test paper is cut into the wide slice of 7 mm, promptly gets the mercury ion detecting test strips.
A kind of mercury ion detecting test strips of the present invention, as shown in Figure 1, the nitrocellulose membrane 4 of the sample pad 2 that comprises bar shaped base plate 1 and overlap successively on the bar shaped base plate, the glass fiber conjugate pad 3 that applies specificity DNA probing needle, specified packet quilt and adsorptive pads 5 are formed, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, useful b-DNA bag wraps by orthoscopic nature controlling line C line 7 by orthoscopic detection line T line 6 with the strepto-affinity is plain on this nitrocellulose membrane, wherein can form T-Hg between the base of the base of b-DNA and specificity DNA probing needle
2+-T mispairing, its preparation method specifically comprises the steps:
1, solution preparation: with embodiment 2
2, the preparation of sample pad 2
With all-glass paper with above-mentioned Tris-HCl damping fluid soak 1 hour wetting after, promptly obtained sample pad 2 down in dry 12 hours at 37 ℃;
3, apply the preparation of the glass fiber conjugate pad 3 of specificity DNA probing needle
A. with all-glass paper with above-mentioned Tris-HCl damping fluid soak 1 hour wetting after, 37 ℃ dry 4～12 hours down, promptly get and seal the all-glass paper of handling;
B. with 70 μ g sequences the above-mentioned Tris-HCl damping fluid dissolving of the DNA of HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin with 5 mL, behind the vibration mixing, join in the colloidal gold solution of the above-mentioned preparation of 1 mL, the vibration mixing, incubation is after 24 hours under the room temperature, centrifugal abandoning supernatant, after adding the above-mentioned phosphate buffer dissolving of 200 μ L, promptly get specificity DNA probing needle solution, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, and the structure of this specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin;
C. get above-mentioned gained specificity DNA probing needle solution with spray sample instrument, even spraying 10.0 μ L on all-glass paper every square centimeter, that sealing was handled, drying promptly got the glass fiber conjugate pad 3 that applies specificity DNA probing needle after 5 hours under 37 ℃;
4, the preparation of the nitrocellulose membrane 4 of specified packet quilt
Is that the b-DNA solution of 100 μ mol/L evenly is sprayed on the nitrocellulose membrane with spray sample instrument with concentration, drying is 8 hours under 37 ℃, form orthoscopic detection line T line 6, the strepto-affinity cellulose solution that with spray sample instrument with concentration is 2 mg/L evenly is sprayed on the nitrocellulose membrane along the direction parallel with detection line T line 6, drying is 8 hours under 37 ℃, form orthoscopic nature controlling line C line 7, promptly obtain the nitrocellulose membrane 4 of specified packet quilt; Wherein b-DNA solution bag is 3.0 μ L/cm by consumption, the solvent of this b-DNA solution is the phosphate buffer of above-mentioned steps 1 preparation, strepto-affinity cellulose solution bag is 3.0 μ L/cm by consumption, the solvent of this strepto-affinity cellulose solution is the phosphate buffer of above-mentioned steps 1 preparation, and wherein the base of b-DNA and the base between the specificity DNA probing needle can form T-Hg
2+-T mispairing, the structure of this b-DNA are 5 '-GTTTCTTCTTTGGTTTGATT-3 ';
5, mercury ion detecting test strips preparation
On bar shaped base plate 1, overlap glass fiber conjugate pad 3, the nitrocellulose membrane 4 of specified packet quilt, the adsorptive pads 5 of sample pad 2, coating specificity DNA probing needle successively, two ends at the bar shaped base plate are stained with self-adhesive paper respectively, the self-adhesive paper that wherein has arrow is attached on the junction of sample pad 2 and glass fiber conjugate pad 3, arrow has been indicated the depth capacity that immerses, at last test paper is cut into the wide slice of 10 mm, promptly gets the mercury ion detecting test strips.
Embodiment 5
A kind of mercury ion detecting test strips of the present invention, as shown in Figure 1, the nitrocellulose membrane 4 of the sample pad 2 that comprises bar shaped base plate 1 and overlap successively on the bar shaped base plate, the glass fiber conjugate pad 3 that applies specificity DNA probing needle, specified packet quilt and adsorptive pads 5 are formed, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, useful b-DNA bag wraps by orthoscopic nature controlling line C line 7 by orthoscopic detection line T line 6 with the strepto-affinity is plain on this nitrocellulose membrane, and wherein the base of b-DNA and the base between the specificity DNA probing needle can form T-Hg
2+-T mispairing, its preparation method specifically comprises the steps:
1, solution preparation: with embodiment 2
2, the preparation of sample pad 2
With all-glass paper with above-mentioned Tris-HCl damping fluid soak 0.5 hour wetting after, promptly obtained sample pad down in dry 4 hours at 20 ℃;
3, apply the preparation of the glass fiber conjugate pad 3 of specificity DNA probing needle
A. with all-glass paper with above-mentioned Tris-HCl damping fluid soak 0.5 hour wetting after, 20 ℃ dry 4 hours down, promptly get and seal the all-glass paper of handling;
B. with 20 μ g sequences the above-mentioned Tris-HCl damping fluid dissolving of the DNA of HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin with 1 mL, behind the vibration mixing, join in the colloidal gold solution of the above-mentioned preparation of 1 mL, the vibration mixing, incubation is after 5 hours under the room temperature, centrifugal abandoning supernatant, after adding the above-mentioned phosphate buffer dissolving of 50 μ L, promptly get specificity DNA probing needle solution, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, and the structure of this specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin;
C. get above-mentioned gained specificity DNA probing needle solution with spray sample instrument, even spraying 2.0 μ L on all-glass paper every square centimeter, that sealing was handled, drying promptly got the glass fiber conjugate pad 3 that applies specificity DNA probing needle after 2 hours under 20 ℃;
4, the preparation of the nitrocellulose membrane 4 of specified packet quilt
Is that the b-DNA solution of 30 μ mol/L evenly is sprayed on the nitrocellulose membrane with spray sample instrument with concentration, drying is 2 hours under 25 ℃, form orthoscopic detection line T line 6, the strepto-affinity cellulose solution that with spray sample instrument with concentration is 1 mg/L evenly is sprayed on the nitrocellulose membrane along the direction parallel with detection line T line 6, drying is 2 hours under 25 ℃, form orthoscopic nature controlling line C line 7, promptly obtain the nitrocellulose membrane 4 of specified packet quilt; Wherein b-DNA solution bag is 1.0 μ L/cm by consumption, the solvent of this b-DNA solution is the phosphate buffer of above-mentioned steps 1 preparation, strepto-affinity cellulose solution bag is 1.0 μ L/cm by consumption, the solvent of this strepto-affinity cellulose solution is the phosphate buffer of above-mentioned steps 1 preparation, and wherein the base of b-DNA and the base between the specificity DNA probing needle can form T-Hg
2+-T mispairing, the structure of this b-DNA are 5 '-GTTTCTTCTTTGGTTTGATT-3 ';
5, mercury ion detecting test strips preparation
On bar shaped base plate 1, overlap glass fiber conjugate pad 3, the nitrocellulose membrane 4 of specified packet quilt, the adsorptive pads 5 of sample pad 2, coating specificity DNA probing needle successively, two ends at the bar shaped base plate are stained with self-adhesive paper respectively, the self-adhesive paper that wherein has arrow is attached on the junction of sample pad 2 and glass fiber conjugate pad 3, arrow has been indicated the depth capacity that immerses, at last test paper is cut into the wide slice of 3mm, promptly gets the mercury ion detecting test strips.
According to embodiment 2 preparation mercury ion detecting test strips, detect mixed solution respectively and (contain Pb
2+, Cd
2+, Mg
2+, Ca
2+, Fe
2+, Cu
2+, Ni
2+, Co
2+, Mn
2+, Zn
2+, each ion concentration is 1 mg/L), 0.01 mg/L Hg
2+Solution, the result distinguishes as shown in Figure 3, and a is a mixed solution testing result synoptic diagram, and b is 0.01 mg/L Hg
2+Solution testing result synoptic diagram illustrates that mercury ion detecting test strips of the present invention has high sensitivity and high selectivity.
The application of test strips in preparation mercury ion test card, test card comprises plastic clip and mercury ion detecting test strips, this plastic clip is a card with the plastics preparation, form by two getting stuck of can being entrenched togather up and down, last slice except a hole that drips sample liquid is arranged, also indicate C, T, T represents the position of detection line, and C represents the position of nature controlling line.Use this mercury ion test card only need be inserted in the specimen liquid, this process operation is simple, and detection time is short, only needs several minutes, and it is low to detect cost, about 3 yuan of each test strips cost of manufacture, and sample also need not complicated pre-service.
Above-mentioned is that embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited only to the foregoing description.
＜110〉University Of Ningbo
＜120〉a kind of mercury ion detecting test strips
<160>?2
<170>?PatentIn?version?3.1
<210>?1
<211>?20
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉specificity DNA probing needle
<400>?1
AuNPs-HS-5’-TTTCA?TTCCT?TTGTT?GATTC-3’-biotin 20
<210>?2
<211>?20
<212>?DNA
＜213〉artificial sequence
<220>
<223>?b-DNA
<400>?2
5’-GTTTC?TTCTT?TGGTT?TGATT-3’ 20
Claims (11)
1. mercury ion detecting test strips, it is characterized in that: described test strips by the bar shaped base plate and on the bar shaped base plate successively the overlap joint sample pad, apply the glass fiber conjugate pad of specificity DNA probing needle, the nitrocellulose membrane of specified packet quilt and adsorptive pads are formed, described specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, useful b-DNA bag wraps by orthoscopic nature controlling line C line by orthoscopic detection line T line with the strepto-affinity is plain on the described nitrocellulose membrane, can form T-Hg between the base of described b-DNA and the base of described specificity DNA probing needle
2+-T mispairing.
2. a kind of mercury ion detecting test strips according to claim 1, it is characterized in that: the structure of described specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin, and the structure of described b-DNA is 5 '-GTTTCTTCTTTGGTTTGATT-3 '.
3. a kind of mercury ion detecting test strips according to claim 2, it is characterized in that: described sample pad is the all-glass paper through 0.01～0.05mol/L Tris-HCl damping fluid immersion treatment, wherein to contain the mass percent of sucrose be 1～10% to the Tris-HCl damping fluid, and the pH value is 5.0～8.0.
4. a kind of mercury ion detecting test strips according to claim 2 is characterized in that: the quantity for spray of described specificity DNA probing needle solution on described glass fiber conjugate pad is 2.0～10.0 μ L/cm
2Described specificity DNA probing needle solution be 20～70 μ g sequences be the DNA of HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin with after the collaurum of 0.5～2 μ mol combines, the concentration that is dissolved in 50～200 μ L is gained in 0.01～0.05 mol/L phosphate buffer.
5. a kind of mercury ion detecting test strips according to claim 2 is characterized in that: described b-DNA bag is 0.0003～0.003 μ mol/mm by consumption.
6. a kind of mercury ion detecting test strips according to claim 2 is characterized in that: the plain bag of described strepto-affinity is 0.01～0.06 μ g/mm by consumption.
7. the preparation method of a mercury ion detecting test strips is characterized in that may further comprise the steps:
(1) preparation of sample pad
With all-glass paper with 0.01～0.05 mol/L Tris-HCl damping fluid soak wetting after, promptly obtained sample pad down in dry 4～12 hours at 20 ℃～37 ℃, wherein to contain the mass percent of sucrose be 1～10% to the Tris-HCl damping fluid, the pH value is 5.0～8.0;
(2) preparation of the glass fiber conjugate pad of coating specificity DNA probing needle
With all-glass paper with 0.01～0.05 mol/L Tris-HCl damping fluid soak wetting after, after under 20 ℃～37 ℃ dry 4～12 hours, on every square centimeter all-glass paper, evenly spray the specificity DNA probing needle solution of 2.0～10.0 μ L, after under 20 ℃～37 ℃ dry 2～5 hours, promptly obtain applying the glass fiber conjugate pad of specificity DNA probing needle, described specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, the structure of described specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin, wherein to contain the mass percent of sucrose be 1～10% to the Tris-HCl damping fluid, and the pH value is 5.0～8.0;
(3) preparation of the nitrocellulose membrane of specified packet quilt
Is that the b-DNA solution of 30～100 μ mol/L evenly is sprayed on the nitrocellulose membrane with spray sample instrument with concentration, drying is 2～8 hours under 25 ℃～37 ℃, form orthoscopic detection line T line, with spray sample instrument is that the strepto-affinity cellulose solution of 1～2mg/L evenly is sprayed on the nitrocellulose membrane along the direction with detection line T line parallel with concentration, drying is 2～8 hours under 25 ℃～37 ℃, form orthoscopic nature controlling line C line, promptly obtain the nitrocellulose membrane of specified packet quilt, can form T-Hg between the base of wherein said b-DNA and the base of described specificity DNA probing needle
2+-T mispairing, the structure of described b-DNA are 5 '-GTTTCTTCTTTGGTTTGATT-3 '.
8.(4) sample pad that step (1) is obtained, the glass fiber conjugate pad of the coating specificity DNA probing needle that step (2) obtains, the nitrocellulose membrane of the specified packet quilt that step (3) obtains and adsorptive pads overlap according to the order of sequence and are pasted on the base plate, be cut into the wide slice of 3～10 mm, promptly get the mercury ion detecting test strips.
9. the preparation method of a kind of mercury ion detecting test strips according to claim 7, the preparation method who it is characterized in that described specificity DNA probing needle solution is as follows: the DNA that with 20～70 μ g sequences is HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin is the dissolving of 0.01～0.05 mol/L Tris-HCl damping fluid with the concentration of 1～5mL, behind the vibration mixing, join in the colloidal gold solution that 1mL concentration is 0.5～2mmol/L, the vibration mixing, incubation is after 5～24 hours under the room temperature, centrifugal abandoning supernatant, after the concentration that adds 50～200 μ L is the dissolving of 0.01～0.05 mol/L phosphate buffer, promptly get specificity DNA probing needle solution, the mass percent that wherein said Tris-HCl damping fluid contains sucrose is 1～10%, described Tris-HCl pH of buffer value is 5.0～8.0, containing the NaCl mass percent in the described phosphate buffer is 0.5～1%, containing the lauryl sodium sulfate mass percent is 0.01～0.05%, and described phosphate buffer pH value is 5.0～8.0.
10. the preparation method of a kind of mercury ion detecting test strips according to claim 7, it is characterized in that: described b-DNA solution bag is 1.0～3.0 μ L/cm by consumption, the solvent of described b-DNA solution is concentration 0.01～0.05 a mol/L phosphate buffer, containing the NaCl mass percent in the described phosphate buffer is 0.5～1%, containing the lauryl sodium sulfate mass percent is 0.01～0.05 %, and described phosphate buffer pH value is 5.0～8.0.
11. the preparation method of a kind of mercury ion detecting test strips according to claim 7, it is characterized in that: described strepto-affinity cellulose solution bag is 1.0～3.0 μ L/cm by consumption, the solvent of described strepto-affinity cellulose solution is concentration 0.01～0.05 a mol/L phosphate buffer, containing the NaCl mass percent in the described phosphate buffer is 0.5～1%, containing the lauryl sodium sulfate mass percent is 0.01～0.05 %, and described phosphate buffer pH value is 5.0～8.0.
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