CN102207502B - Mercury ion test paper and preparation method thereof - Google Patents

Mercury ion test paper and preparation method thereof Download PDF

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Publication number
CN102207502B
CN102207502B CN201110073819.7A CN201110073819A CN102207502B CN 102207502 B CN102207502 B CN 102207502B CN 201110073819 A CN201110073819 A CN 201110073819A CN 102207502 B CN102207502 B CN 102207502B
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dna
described
probing needle
solution
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CN201110073819.7A
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CN102207502A (en
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杨飞
郭智勇
段静
李敏
郝婷婷
王邃
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宁波大学
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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

A kind of mercury ion test paper and preparation method thereof

Technical field

The present invention relates to a kind of detection of heavy metal ion technical field, especially relate to a kind of mercury ion test paper and preparation method thereof.

Background technology

Mercury (Hg), is liquid metal unique under normal temperature, belongs to heavy metal classification.In recent years, the mankind are increasing to the exploitation of heavy metal Hg, smelting, processing and business manufacturing activities, and a large amount of mercury enters in atmosphere, water, soil and retains, accumulates and move.The toxicity of mercury is large, in vivo with environment in the residence time long, serious threat human health, therefore strict restriction has all been done to mercury content in food, daily necessities, 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 accurate, sensitive, Site Detection is extremely important fast.

The method that detects at present mercury ion 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 to use specific instrument, expensive, complex operation, and require testing staff to possess certain professional knowledge, analysis cost is high, cannot be applied to Site Detection, be difficult to popularize; Heavy metal rapid detector method can be applicable to Site Detection, but sensitivity is not high, poor selectivity, and sample pretreatment is complicated; Colourimetry based on 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, test strips method is quick, easy and other tool advantage in detecting at the scene with it, but the test strips based on traditional chelating developer, selectivity is poor, sensitivity is low, detect lower limit and be about 0.1~1mg/L, the testing requirement that cannot be up to state standards (as: potable water <0.01 mg/L).

Summary of the invention

Technical matters to be solved by this invention is to provide a kind of mercury ion test paper based on collaurum and specific DNA and preparation method thereof, and this test strips can high sensitivity, highly selective detects the mercury ion in sample.

The present invention solves the problems of the technologies described above adopted technical scheme: a kind of mercury ion test paper, described test strips by bar shaped bottom plate and on bar shaped bottom plate successively overlap joint sample pad, apply the glass fiber conjugate pad of specificity DNA probing needle, nitrocellulose membrane and the adsorptive pads of specified packet quilt form, described specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, on described nitrocellulose membrane, useful b-DNA is coated with orthoscopic detection line T line and is coated with orthoscopic nature controlling line C line with streptavidin, between the base of the base of described b-DNA and described specificity DNA probing needle, can form T-Hg 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 Tris-HCl damping fluid is that 1~10%, pH value is 5.0~8.0 containing the mass percent of sucrose.

The quantity for spray of described specificity DNA probing needle solution in described glass fiber conjugate pad is 2.0~10.0 μ L/cm 2described specificity DNA probing needle solution is that after DNA that 20~70 μ g sequences are HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin is combined with the collaurum of 0.5~2 μ mol, the concentration that is dissolved in 50~200 μ L is gained in 0.01~0.05 mol/L phosphate buffer.

The coated consumption of described b-DNA is 0.0003~0.003 μ mol/mm.

The coated consumption of described streptavidin is 0.01~0.06 μ g/mm.

A preparation method for mercury ion test paper, comprises the following steps:

(1) preparation of sample pad

After all-glass paper is wetting with 0.01~0.05 mol/L Tris-HCl damping fluid immersion, the dry sample pad that obtains for 4~12 hours at 20 ℃~37 ℃, wherein Tris-HCl damping fluid is that 1~10%, pH value is 5.0~8.0 containing the mass percent of sucrose;

(2) apply the preparation of the glass fiber conjugate pad of specificity DNA probing needle

After all-glass paper is wetting with 0.01~0.05 mol/L Tris-HCl damping fluid immersion, dry after 4~12 hours at 20 ℃~37 ℃, on the all-glass paper of every square centimeter, evenly spray the specificity DNA probing needle solution of 2.0~10.0 μ L, dry after 2~5 hours at 20 ℃~37 ℃, 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 Tris-HCl damping fluid is 1~10% containing the mass percent of sucrose, pH value is 5.0~8.0,

(3) preparation of the nitrocellulose membrane of specified packet quilt

With the b-DNA solution that spray sample instrument is 30~100 μ mol/L by concentration, be evenly sprayed on nitrocellulose membrane, at 25 ℃~37 ℃, be dried 2~8 hours, form orthoscopic detection line T line, with the streptavidin solution edge that spray sample instrument is 1~2 mg/L by concentration, be evenly sprayed on nitrocellulose membrane with the direction of detection line T line parallel, at 25 ℃~37 ℃, be dried 2~8 hours, form orthoscopic nature controlling line C line, obtain the nitrocellulose membrane of specified packet quilt, between the base of wherein said b-DNA and the base of described specificity DNA probing needle, can form T-Hg 2+-T mispairing, the structure of described b-DNA is 5 '-GTTTCTTCTTTGGTTTGATT-3 ';

(4) sample pad step (1) being 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 base plate, be cut into the wide slice of 3~10 mm, obtain mercury ion test paper.

The preparation method of described specificity DNA probing needle solution is as follows: the DNA that is HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin by 20~70 μ g sequences is that 0.01~0.05 mol/L Tris-HCl damping fluid dissolves by the concentration of 1~5 mL, after vibration mixes, joining 1 mL concentration is in the colloidal gold solution of 0.5~2 mmol/L, vibration mixes, under room temperature, incubation is after 5~24 hours, centrifugal abandoning supernatant, the concentration that adds 50~200 μ L is after 0.01~0.05 mol/L phosphate buffer dissolves, obtain specificity DNA probing needle solution, wherein said Tris-HCl damping fluid is 1~10% containing the mass percent of sucrose, described Tris-HCl pH of cushioning fluid is 5.0~8.0, in described phosphate buffer, containing NaCl mass percent, be 0.5~1%, containing lauryl sodium sulfate mass percent, be 0.01~0.05%, described phosphate buffer pH value is 5.0~8.0.

The coated consumption of described b-DNA solution is 1.0~3.0 μ L/cm, the solvent of described b-DNA solution is concentration 0.01~0.05 mol/L phosphate buffer, in described phosphate buffer, containing NaCl mass percent, be 0.5~1%, containing lauryl sodium sulfate mass percent, be 0.01~0.05%, described phosphate buffer pH value is 5.0~8.0.

The coated consumption of described streptavidin solution is 1.0~3.0 μ L/cm, the solvent of described streptavidin solution is concentration 0.01~0.05 mol/L phosphate buffer, in described phosphate buffer, containing NaCl mass percent, be 0.5~1%, containing lauryl sodium sulfate mass percent, be 0.01~0.05%, described phosphate buffer pH value is 5.0~8.0.

The mercury ion test paper that the present invention proposes: apply b-DNA on detection line, apply Streptavidin on nature controlling line, apply the specificity DNA probing needle that two ends are combined with respectively collaurum and biotin in glass fiber conjugate pad; When mercury ion exists, there is T-Hg in the b-DNA on specificity DNA probing needle and 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 object that detects mercury ion; The Streptavidin generation specific binding applying on the biotin energy that the specificity DNA probing needle chain other end is modified and nature controlling line, therefore being hunted down, excessive specificity DNA probing needle rests on nature controlling line, the colloidal gold aggregation of its end of the chain combination develops the color, and reaches the Quality Control object of detection.By this test strips, sample liquid is detected: if the ion concentration of mercury in sample liquid during higher than detectability, detection line and nature controlling line show redness simultaneously; If while not containing mercury ion or ion concentration of mercury lower than detectability in sample liquid, detection line does not develop the color, nature controlling line shows red; If nature controlling line does not develop the color, it is invalid to detect.

Compared with prior art, the invention has the advantages that:

(1) high sensitivity, detectability reaches 0.005 mg/L, and reason is: first, 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; Again, detecting and carry out in test strips, is a colour developing under white background, and people's naked eyes are more prone to identification; Finally, test strips, by filtering and expansion effect, can effectively be eliminated the impact of coloured interfering material on colour developing;

(2) high selectivity, common metal ion is 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, the interference of other metallic ion can be ignored;

(3) sample is without complicated pre-service.Reason is: specific DNA can specific recognition mercury ion, and can, by the filtration capacity of expansion effect and test strips itself, eliminate sample matrices and disturb;

(4) simple to operate, detection time is short, only needs several minutes, and reason is: only need through simple immersion, expansion, i.e. observable testing result;

(5) testing cost is low, approximately 3 yuan of each test strips costs of manufacture, and reason is: reagent and material usage are few, and production stage is simple.

In sum, the mercury ion test paper based on collaurum and specific DNA, this test strips can high sensitivity, highly selective detects the mercury ion in sample, and the preparation method of this test strips is simple, easy to operate.

Accompanying drawing explanation

Fig. 1 is the structural representation of test strips of the present invention;

Fig. 2 is that the present invention detects effect schematic diagram;

A: contain the mercury ion higher than detectability in sample liquid;

B: in sample liquid, not mercurous or mercury ion content is lower than detectability;

C: it is invalid to detect;

Fig. 3 is the result figure that the present invention detects actual sample.

Embodiment

Below in conjunction with accompanying drawing, embodiment is described in further detail the present invention.

Embodiment 1

A kind of mercury ion test paper of the present invention, as shown in Figure 1, the sample pad 2 that comprises bar shaped bottom plate 1 and overlap successively on bar shaped bottom plate, the coating glass fiber conjugate pad 3 of specificity DNA probing needle, the nitrocellulose membrane 4 of specified packet quilt and adsorptive pads 5 form, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, on this nitrocellulose membrane, useful b-DNA is coated with orthoscopic detection line T line 6 and with the coated orthoscopic nature controlling line C line 7 of streptavidin, between the base of described b-DNA and the base of described specificity DNA probing needle, can forms T-Hg 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 Tris-HCl damping fluid is that 1~10%, pH value is 5.0~8.0 containing the mass percent of sucrose; The quantity for spray of specificity DNA probing needle solution in described glass fiber conjugate pad is 2.0~10.0 μ L/cm 2this specificity DNA probing needle solution is that after DNA that 20~70 μ g sequences are HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin is combined with the collaurum of 0.5~2 μ mol, the concentration that is dissolved in 50~200 μ L is gained in 0.01~0.05 mol/L phosphate buffer; The coated consumption of b-DNA is 0.0003~0.003 μ mol/mm; The coated consumption of streptavidin is 0.01~0.06 μ g/mm; Adsorptive pads is a kind of thieving paper (model is SX27 or CH27), and thieving paper is attached to the end of bar shaped bottom plate, and adsorptive pads can be also glass fibre membrane SB08 or BT50.

Embodiment 2

Mercury ion test paper of the present invention detects mercury ion based on collaurum and specific DNA, and when mercury ion exists, T-Hg occurs the b-DNA on specificity DNA probing needle and 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 object that detects mercury ion; The Streptavidin generation specific binding applying on the biotin energy that the specificity DNA probing needle chain other end is modified and nature controlling line, therefore being hunted down, excessive specificity DNA probing needle rests on nature controlling line, the colloidal gold aggregation colour developing of its end of the chain combination, reach the Quality Control object of detection, as shown in Figure 2, a: contain the mercury ion higher than detectability in sample liquid; B: in sample liquid, not mercurous or mercury ion content is lower than detectability; C: it is invalid to detect; Its concrete preparation method is as follows:

1, solution preparation

The preparation of 1.1 0.5~2mmol/L colloidal gold solution

A. 0.5~2mmol/L HAuCl 4the preparation of solution: the HAuCl that takes 0.0205~0.0822 g 44H 2o, is settled in 100mL volumetric flask with distilled water, stand-by;

B. the preparation of 38.8mmol/L sodium citrate solution: take 1.1407 g sodium citrates, with distilled water, be settled in 100 mL volumetric flasks, stand-by;

C. use chloroazotic acid (hydrochloric acid and the nitric acid by volume ratio of 3:1 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 two neck flasks, and a mouth of flask connects condenser pipe, and another mouthful clogs with stopper, stirs, and heating, refluxes.When solution starts to reflux, open stopper, after adding fast the sodium citrate solution of 38.8 mmol/L of 10~50 mL, clog stopper, continue vigorous stirring, add hot reflux 20 minutes, stop heating, continue stirring and be cooled to room temperature, make colloidal gold solution, under 4 ℃ of conditions, preserve the colloidal gold solution preparing;

1.2 Tris-HCl damping fluid preparations

The preparation method of 0.01~0.05 mol/L Tris-HCl damping fluid: weigh 0.7880~3.9398 g Tris-HCl and be placed in 1 L beaker, the deionized water dissolving that adds 400mL, add again 0.234~2.34 g NaCl and 0.030~0.298 g KCl, the concentration that makes NaCl is that the concentration of 0.01~0.1 mol/L, KCl is 0.001~0.01 mol/L, after dissolving mixes, add appropriate sucrose, the mass percent concentration that makes sucrose is 1~10%, with acid-base solution, regulates Tris-HCl pH of buffer to 5.0~8.0;

The preparation of 1.3 0.01~0.05 mol/L phosphate buffer

By amount of substance than taking for 1:1: 0.3~1.5 g NaH 2pO 42H 2o and 0.355~1.775 g Na 2hPO 42H 2o is placed in 1 L beaker, the deionized water dissolving that adds 400mL, add again NaCl and lauryl sodium sulfate, making in this phosphate buffer is 0.5~1% containing NaCl mass percent, containing lauryl sodium sulfate mass percent, be 0.01~0.05%, with acid-base solution, regulating phosphate buffer pH value is 5.0~8.0;

2, the preparation of sample pad 2

After all-glass paper is soaked with above-mentioned Tris-HCl damping fluid immersion for 0.5~1 hour, the dry sample pad 2 that obtains for 4~12 hours at 20 ℃~37 ℃;

3, apply the preparation of the glass fiber conjugate pad 3 of specificity DNA probing needle

A. after all-glass paper being soaked with above-mentioned Tris-HCl damping fluid immersion for 0.5~1 hour, at 20 ℃~37 ℃, be dried 4~12 hours, must seal the all-glass paper of processing;

B. the DNA that is HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin by 20~70 μ g sequences dissolves with the above-mentioned Tris-HCl damping fluid of 1~5mL, after vibration mixes, join in the colloidal gold solution of the above-mentioned preparation of 1mL, vibration mixes, under room temperature, incubation is after 5~24 hours, centrifugal abandoning supernatant, add after the above-mentioned phosphate buffer dissolving of 50~200 μ L, obtain specificity DNA probing needle solution, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, the structure of this specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin,

C. with spray sample instrument, get above-mentioned gained specificity DNA probing needle solution, on all-glass paper every square centimeter, that sealing was processed, evenly spray 2.0~10.0 μ L, dry after 2~5 hours at 20 ℃~37 ℃, must apply the glass fiber conjugate pad 3 of specificity DNA probing needle;

4, the preparation of the nitrocellulose membrane 4 of specified packet quilt

With the b-DNA solution that spray sample instrument is 30~100 μ mol/L by concentration, be evenly sprayed on nitrocellulose membrane, at 25 ℃~37 ℃, be dried 2~8 hours, form orthoscopic detection line T line 6, with the streptavidin solution that spray sample instrument is 1~2 mg/L by concentration, along the direction parallel with detection line T line 6, be evenly sprayed on nitrocellulose membrane, at 25 ℃~37 ℃, be dried 2~8 hours, form orthoscopic nature controlling line C line 7, obtain the nitrocellulose membrane 4 of specified packet quilt; Wherein the coated consumption of b-DNA solution is 1.0~3.0 μ L/cm, the solvent of this b-DNA solution is the phosphate buffer of above-mentioned steps 1 preparation, the coated consumption of streptavidin solution is 1.0~3.0 μ L/cm, the solvent of this streptavidin solution is the phosphate buffer of above-mentioned steps 1 preparation, wherein between the base of b-DNA and the base of specificity DNA probing needle, can form T-Hg 2+-T mispairing, the structure of this b-DNA is 5 '-GTTTCTTCTTTGGTTTGATT-3 ';

5, mercury ion test paper preparation

On bar shaped bottom plate 1, overlap successively sample pad 2, 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 bar shaped bottom plate are stained with respectively self-adhesive paper, wherein the self-adhesive paper with arrow is attached on the junction of sample pad and pad, arrow has been indicated the depth capacity immersing, finally test paper is cut into the wide slice of 3~10 mm, obtain mercury ion test paper, one end of during detection, arrow being indicated is immersed in 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, reach the object of detection.

Embodiment 3

A kind of mercury ion test paper of the present invention, as shown in Figure 1, the sample pad 2 that comprises bar shaped bottom plate 1 and overlap successively on bar shaped bottom plate 1, the coating glass fiber conjugate pad 3 of specificity DNA probing needle, the nitrocellulose membrane 4 of specified packet quilt and adsorptive pads 5 form, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, on this nitrocellulose membrane, useful b-DNA is coated with orthoscopic detection line T line 6 and with the coated orthoscopic nature controlling line C line 7 of streptavidin, wherein between the base of b-DNA and the base of specificity DNA probing needle, can forms T-Hg 2+-T mispairing, its preparation method specifically comprises the following steps:

1, solution preparation: with embodiment 2

2, the preparation of sample pad 2

After all-glass paper is soaked with above-mentioned Tris-HCl damping fluid immersion for 0.7 hour, the dry sample pad 2 that obtains for 8 hours at 30 ℃;

3, apply the preparation of the glass fiber conjugate pad 3 of specificity DNA probing needle

A. after all-glass paper being soaked with above-mentioned Tris-HCl damping fluid immersion for 0.7 hour, at 30 ℃, be dried 8 hours, must seal the all-glass paper of processing;

B. the DNA that is HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin by 50 μ g sequences dissolves with the above-mentioned Tris-HCl damping fluid of 3 mL, after vibration mixes, join in the colloidal gold solution of the above-mentioned preparation of 1 mL, vibration mixes, under room temperature, incubation is after 15 hours, centrifugal abandoning supernatant, add after the above-mentioned phosphate buffer dissolving of 100 μ L, obtain specificity DNA probing needle solution, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, the structure of this specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin,

C. with spray sample instrument, get above-mentioned gained specificity DNA probing needle solution, on the all-glass paper of processing the sealing of every square centimeter, evenly spray 6.0 μ L, be dried after 3 hours at 30 ℃, must apply the glass fiber conjugate pad 3 of specificity DNA probing needle;

4, the preparation of the nitrocellulose membrane 4 of specified packet quilt

With the b-DNA solution that spray sample instrument is 60 μ mol/L by concentration, be evenly sprayed on nitrocellulose membrane, at 30 ℃, be dried 6 hours, form orthoscopic detection line T line 6, with the streptavidin solution that spray sample instrument is 1.5 mg/L by concentration, along the direction parallel with detection line T line 6, be evenly sprayed on nitrocellulose membrane, at 30 ℃, be dried 6 hours, form orthoscopic nature controlling line C line 7, obtain the nitrocellulose membrane 4 of specified packet quilt; Wherein the coated consumption of b-DNA solution is 2.0 μ L/cm, the solvent of this b-DNA solution is the phosphate buffer of above-mentioned steps 1 preparation, the coated consumption of streptavidin solution is 2.0 μ L/cm, the solvent of this streptavidin solution is the phosphate buffer of above-mentioned steps 1 preparation, and wherein the base of b-DNA and the base between specificity DNA probing needle can form T-Hg 2+-T mispairing, the structure of this b-DNA is 5 '-GTTTCTTCTTTGGTTTGATT-3 ';

5, mercury ion test paper preparation

On bar shaped bottom plate 1, overlap successively sample pad 2, the coating glass fiber conjugate pad 3 of specificity DNA probing needle, the nitrocellulose membrane 4 of specified packet quilt, adsorptive pads 5, two ends at bar shaped bottom plate are stained with respectively self-adhesive paper, wherein the self-adhesive paper with arrow is attached on the junction of sample pad 2 and glass fiber conjugate pad 3, arrow has been indicated the depth capacity immersing, finally test paper is cut into the wide slice of 7 mm, obtains mercury ion test paper.

Embodiment 4

A kind of mercury ion test paper of the present invention, as shown in Figure 1, the sample pad 2 that comprises bar shaped bottom plate 1 and overlap successively on bar shaped bottom plate, the coating glass fiber conjugate pad 3 of specificity DNA probing needle, the nitrocellulose membrane 4 of specified packet quilt and adsorptive pads 5 form, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, on this nitrocellulose membrane, useful b-DNA is coated with orthoscopic detection line T line 6 and with the coated orthoscopic nature controlling line C line 7 of streptavidin, wherein between the base of b-DNA and the base of specificity DNA probing needle, can forms 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

After all-glass paper is soaked with above-mentioned Tris-HCl damping fluid immersion for 1 hour, the dry sample pad 2 that obtains for 12 hours at 37 ℃;

3, apply the preparation of the glass fiber conjugate pad 3 of specificity DNA probing needle

A. after all-glass paper being soaked with above-mentioned Tris-HCl damping fluid immersion for 1 hour, at 37 ℃, be dried 4~12 hours, must seal the all-glass paper of processing;

B. the DNA that is HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin by 70 μ g sequences dissolves with the above-mentioned Tris-HCl damping fluid of 5 mL, after vibration mixes, join in the colloidal gold solution of the above-mentioned preparation of 1 mL, vibration mixes, under room temperature, incubation is after 24 hours, centrifugal abandoning supernatant, add after the above-mentioned phosphate buffer dissolving of 200 μ L, obtain specificity DNA probing needle solution, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, the structure of this specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin,

C. with spray sample instrument, get above-mentioned gained specificity DNA probing needle solution, on all-glass paper every square centimeter, that sealing was processed, evenly spray 10.0 μ L, be dried after 5 hours at 37 ℃, must apply the glass fiber conjugate pad 3 of specificity DNA probing needle;

4, the preparation of the nitrocellulose membrane 4 of specified packet quilt

With the b-DNA solution that spray sample instrument is 100 μ mol/L by concentration, be evenly sprayed on nitrocellulose membrane, at 37 ℃, be dried 8 hours, form orthoscopic detection line T line 6, with the streptavidin solution that spray sample instrument is 2 mg/L by concentration, along the direction parallel with detection line T line 6, be evenly sprayed on nitrocellulose membrane, at 37 ℃, be dried 8 hours, form orthoscopic nature controlling line C line 7, obtain the nitrocellulose membrane 4 of specified packet quilt; Wherein the coated consumption of b-DNA solution is 3.0 μ L/cm, the solvent of this b-DNA solution is the phosphate buffer of above-mentioned steps 1 preparation, the coated consumption of streptavidin solution is 3.0 μ L/cm, the solvent of this streptavidin solution is the phosphate buffer of above-mentioned steps 1 preparation, and wherein the base of b-DNA and the base between specificity DNA probing needle can form T-Hg 2+-T mispairing, the structure of this b-DNA is 5 '-GTTTCTTCTTTGGTTTGATT-3 ';

5, mercury ion test paper preparation

On bar shaped bottom plate 1, overlap successively sample pad 2, the coating glass fiber conjugate pad 3 of specificity DNA probing needle, the nitrocellulose membrane 4 of specified packet quilt, adsorptive pads 5, two ends at bar shaped bottom plate are stained with respectively self-adhesive paper, wherein the self-adhesive paper with arrow is attached on the junction of sample pad 2 and glass fiber conjugate pad 3, arrow has been indicated the depth capacity immersing, finally test paper is cut into the wide slice of 10 mm, obtains mercury ion test paper.

Embodiment 5

A kind of mercury ion test paper of the present invention, as shown in Figure 1, the sample pad 2 that comprises bar shaped bottom plate 1 and overlap successively on bar shaped bottom plate, the coating glass fiber conjugate pad 3 of specificity DNA probing needle, the nitrocellulose membrane 4 of specified packet quilt and adsorptive pads 5 form, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, on this nitrocellulose membrane, useful b-DNA is coated with orthoscopic detection line T line 6 and is coated with orthoscopic nature controlling line C line 7 with streptavidin, and wherein the base of b-DNA and the base between 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

After all-glass paper is soaked with above-mentioned Tris-HCl damping fluid immersion for 0.5 hour, the dry sample pad that obtains for 4 hours at 20 ℃;

3, apply the preparation of the glass fiber conjugate pad 3 of specificity DNA probing needle

A. after all-glass paper being soaked with above-mentioned Tris-HCl damping fluid immersion for 0.5 hour, at 20 ℃, be dried 4 hours, must seal the all-glass paper of processing;

B. the DNA that is HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin by 20 μ g sequences dissolves with the above-mentioned Tris-HCl damping fluid of 1 mL, after vibration mixes, join in the colloidal gold solution of the above-mentioned preparation of 1 mL, vibration mixes, under room temperature, incubation is after 5 hours, centrifugal abandoning supernatant, add after the above-mentioned phosphate buffer dissolving of 50 μ L, obtain specificity DNA probing needle solution, this specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, the structure of this specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin,

C. with spray sample instrument, get above-mentioned gained specificity DNA probing needle solution, on all-glass paper every square centimeter, that sealing was processed, evenly spray 2.0 μ L, be dried after 2 hours at 20 ℃, must apply the glass fiber conjugate pad 3 of specificity DNA probing needle;

4, the preparation of the nitrocellulose membrane 4 of specified packet quilt

With the b-DNA solution that spray sample instrument is 30 μ mol/L by concentration, be evenly sprayed on nitrocellulose membrane, at 25 ℃, be dried 2 hours, form orthoscopic detection line T line 6, with the streptavidin solution that spray sample instrument is 1 mg/L by concentration, along the direction parallel with detection line T line 6, be evenly sprayed on nitrocellulose membrane, at 25 ℃, be dried 2 hours, form orthoscopic nature controlling line C line 7, obtain the nitrocellulose membrane 4 of specified packet quilt; Wherein the coated consumption of b-DNA solution is 1.0 μ L/cm, the solvent of this b-DNA solution is the phosphate buffer of above-mentioned steps 1 preparation, the coated consumption of streptavidin solution is 1.0 μ L/cm, the solvent of this streptavidin solution is the phosphate buffer of above-mentioned steps 1 preparation, and wherein the base of b-DNA and the base between specificity DNA probing needle can form T-Hg 2+-T mispairing, the structure of this b-DNA is 5 '-GTTTCTTCTTTGGTTTGATT-3 ';

5, mercury ion test paper preparation

On bar shaped bottom plate 1, overlap successively sample pad 2, the coating glass fiber conjugate pad 3 of specificity DNA probing needle, the nitrocellulose membrane 4 of specified packet quilt, adsorptive pads 5, two ends at bar shaped bottom plate are stained with respectively self-adhesive paper, wherein the self-adhesive paper with arrow is attached on the junction of sample pad 2 and glass fiber conjugate pad 3, arrow has been indicated the depth capacity immersing, finally test paper is cut into the wide slice of 3mm, obtains mercury ion test paper.

Embodiment 6

According to embodiment 2, prepare mercury ion test paper, detect respectively mixed solution (containing 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, respectively as shown in Figure 3, a is mixed solution testing result schematic diagram to result, b is 0.01 mg/L Hg 2+solution testing result schematic diagram, illustrates that mercury ion test paper of the present invention has high sensitivity and high selectivity.

Embodiment 7

The application of test strips in preparing mercury ion test card, test card comprises plastic clip and mercury ion test paper, this plastic clip be one with the standby card of plastics, by upper and lower two getting stuck of can being entrenched togather, formed, upper slice except having a hole that drips sample liquid, 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 specimen liquid, this process operation is simple, and detection time is short, only needs several minutes, and testing cost is low, approximately 3 yuan of each test strips costs of manufacture, and sample is also without complicated pre-service.

Above-mentioned is that embodiments of the invention are elaborated: the present embodiment is implemented take technical solution of the present invention under prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited only to above-described embodiment.

<110> University Of Ningbo

<120> mercury ion test paper

<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 (4)

1. a mercury ion test paper, it is characterized in that: described test strips by bar shaped bottom plate and on bar shaped bottom plate successively overlap joint sample pad, apply the glass fiber conjugate pad of specificity DNA probing needle, nitrocellulose membrane and the adsorptive pads of specified packet quilt form, described specificity DNA probing needle one end is combined with collaurum, its other end is combined with biotin, on described nitrocellulose membrane, useful b-DNA is coated with orthoscopic detection line T line and is coated with orthoscopic nature controlling line C line with streptavidin, between the base of the base of described b-DNA and described specificity DNA probing needle, can form T-Hg 2+-T mispairing, the structure of described specificity DNA probing needle is AuNPs-HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin, the structure of described b-DNA is 5 '-GTTTCTTCTTTGGTTTGATT-3 '.
2. a kind of mercury ion test paper according to claim 1, 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 Tris-HCl damping fluid is that 1~10%, pH value is 5.0~8.0 containing the mass percent of sucrose.
3. a kind of mercury ion test paper according to claim 2, is characterized in that: the quantity for spray of described specificity DNA probing needle solution in described glass fiber conjugate pad is 2.0~10.0 μ L/cm 2described specificity DNA probing needle solution is after DNA that 20~70 μ g sequences are HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin is combined with the collaurum of 0.5~2 μ mol, the concentration that is dissolved in 50~200 μ L is gained in 0.01~0.05mol/L phosphate buffer, the coated consumption of described b-DNA is 0.0003~0.003 μ mol/mm, and the coated consumption of described streptavidin is 0.01~0.06 μ g/mm.
4. a preparation method for mercury ion test paper, is characterized in that comprising the following steps:
(1) preparation of sample pad
After all-glass paper is wetting with the immersion of 0.01~0.05mol/L Tris-HCl damping fluid, the dry sample pad that obtains for 4~12 hours at 20 ℃~37 ℃, wherein Tris-HCl damping fluid is that 1~10%, pH value is 5.0~8.0 containing the mass percent of sucrose;
(2) apply the preparation of the glass fiber conjugate pad of specificity DNA probing needle
After all-glass paper is wetting with the immersion of 0.01~0.05mol/L Tris-HCl damping fluid, dry after 4~12 hours at 20 ℃~37 ℃, on the all-glass paper of every square centimeter, evenly spray the specificity DNA probing needle solution of 2.0~10.0 μ L, dry after 2~5 hours at 20 ℃~37 ℃, 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 Tris-HCl damping fluid is 1~10% containing the mass percent of sucrose, pH value is 5.0~8.0, the preparation method of wherein said specificity DNA probing needle solution is as follows: the DNA that is HS-5 '-TTTCATTCCTTTGTTGATTC-3 '-biotin by 20~70 μ g sequences is that 0.01~0.05mol/L Tris-HCl damping fluid dissolves by the concentration of 1~5mL, after vibration mixes, join in the colloidal gold solution that 1mL concentration is 0.5~2mmol/L, vibration mixes, under room temperature, incubation is after 5~24 hours, centrifugal abandoning supernatant, the concentration that adds 50~200 μ L is after 0.01~0.05mol/L phosphate buffer dissolves, obtain specificity DNA probing needle solution, wherein said Tris-HCl damping fluid is 1~10% containing the mass percent of sucrose, described Tris-HCl pH of cushioning fluid is 5.0~8.0, in described phosphate buffer, containing NaCl mass percent, be 0.5~1%, containing lauryl sodium sulfate mass percent, be 0.01~0.05%, described phosphate buffer pH value is 5.0~8.0,
(3) preparation of the nitrocellulose membrane of specified packet quilt
With the b-DNA solution that spray sample instrument is 30~100 μ mol/L by concentration, be evenly sprayed on nitrocellulose membrane, at 25 ℃~37 ℃, be dried 2~8 hours, form orthoscopic detection line T line, the streptavidin solution that is 1~2mg/L by concentration with spray sample instrument is along being evenly sprayed on nitrocellulose membrane with the direction of detection line T line parallel, at 25 ℃~37 ℃, be dried 2~8 hours, form orthoscopic nature controlling line C line, obtain the nitrocellulose membrane of specified packet quilt, between the base of wherein said b-DNA and the base of described specificity DNA probing needle, can form T-Hg 2+-T mispairing, the structure of described b-DNA is 5 '-GTTTCTTCTTTGGTTTGATT-3 '; The coated consumption of wherein said b-DNA solution is 1.0~3.0 μ L/cm, the solvent of described b-DNA solution is concentration 0.01~0.05mol/L phosphate buffer, in described phosphate buffer, containing NaCl mass percent, be 0.5~1%, containing lauryl sodium sulfate mass percent, be 0.01~0.05%, described phosphate buffer pH value is 5.0~8.0; The coated consumption of described streptavidin solution is 1.0~3.0 μ L/cm, the solvent of described streptavidin solution is concentration 0.01~0.05mol/L phosphate buffer, in described phosphate buffer, containing NaCl mass percent, be 0.5~1%, containing lauryl sodium sulfate mass percent, be 0.01~0.05%, described phosphate buffer pH value is 5.0~8.0;
(4) sample pad step (1) being 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 base plate, be cut into the wide slice of 3~10mm, obtain mercury ion test paper.
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