CN105334258A - Method for determining melamine by nano-fluorescence probe - Google Patents
Method for determining melamine by nano-fluorescence probe Download PDFInfo
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- CN105334258A CN105334258A CN201410404446.0A CN201410404446A CN105334258A CN 105334258 A CN105334258 A CN 105334258A CN 201410404446 A CN201410404446 A CN 201410404446A CN 105334258 A CN105334258 A CN 105334258A
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Abstract
The invention discloses a method for determining melamine by a nano-fluorescence probe. The method comprises the following steps: 1) preparation of gold nanoparticles; 2) preparation of a gold nanoparticles probe; 3) treatment of a gold electrode; 4) determination of melamine; and 5) determination of fluorescence intensity. According to the invention, the method uses specific binding between ferrocene as a signal molecule and melamine/thymidine, then detachment is carried out through a fluorescence dye and then obvious activation of the fluorescence probe can be realized, so that determination for melamine content and fluorescence degree can be realized.
Description
Technical field
The invention belongs to detection technique field, be specifically related to a kind of method that namo fluorescence probe measures melamine.
Background technology
Food industry generally adopts, what be decided to be national standard is Kjeldahl's method.Principle is: protein contains nitrogen element, uses strong acid treatment sample, allows nitrogen Element release in protein out, measures the content of nitrogen, just can calculate the content of protein.So in fact Kjeldahl's method survey is not protein content, but calculate protein content by surveying nitrogen content, obviously, if also have other to contain nitrogen compound in sample, this method is just inaccurate.
Melamine is a kind of poisonous material, if human body takes in melamine for a long time, can cause the infringement of reproduction, urinary system, makes bladder, kidney portion generation calculus, and can bring out carcinoma of urinary bladder further.Detection method of the prior art respectively has its shortcoming.Gold nano fluorescence probe efficiently can be combined with disease organism marker, when add to detection system compounds containing thiol groups as cysteamine after, the efficient rich poly-fluorescent dye in golden nanometer particle surface occurs to depart from and causes the remarkable activation of fluorescence probe, the concentration of the biological marker in fluorescence intensity and sample is proportional, thus reach the object of high-sensitivity detection disease organism marker, but the method is not for the detection field of melamine, therefore, how to invent a kind of method that namo fluorescence probe measures melamine, the method is made to have simply, cost is low, highly sensitive feature, it is the technical problem to be solved in the present invention.
Summary of the invention
The invention provides a kind of method that namo fluorescence probe measures melamine, utilize ferrocene as signaling molecule and the specific binding between melamine and thymine, occur to depart from by fluorescent dye and cause the remarkable activation of fluorescence probe, achieving the mensuration to content of melamine and fluorescence.
The technical scheme that the present invention takes is:
Namo fluorescence probe measures a method for melamine, it is characterized in that, specifically comprises the steps:
(1) preparation of golden nanometer particle: be add sodium phytate solution after 0.003-0.005mol/L liquor argenti nitratis ophthalmicus is heated to boiling and continuous heating extremely boiling by concentration, and to add concentration after keeping 15-20min be 0.003-0.005mol/L sodium phytate solution, with vigorous stirring, after being heated to boil, adding mass concentration is fast the citric acid three sodium solution that 1.5-1.8% newly prepares, boil continuously, treat that solution colour becomes redness from dark blue, red golden nanometer particle (Au) solution can be obtained, 100mL is diluted to after being cooled to room temperature, shake up, be placed in 4 ° of C refrigerators to preserve,
(2) preparation of gold nanoparticle probe: 5mg ferrocenecarboxylic acid is joined in the phosphate buffer solution solution of 10mL0.1mol/L, add the fluorescent dye containing 3-5% rhodamine B isothiocyanates again, by this mixed solution at room temperature vigorous stirring reaction 2h, excessive fluorescent dye is removed in centrifuging, with bovine serum albumin(BSA), the avtive spot on gold nano grain surface is carried out closed half an hour, acquisition can activate gold nano fluorescence probe, and 4 DEG C save backup;
(3) process of gold electrode: first gold electrode is used alumina powder polishing respectively on chamois leather, successively with ethanol and distilled water ultrasonic 8min respectively, finally at 0.1mol/LH
2sO
4cyclic voltammetry scan is carried out until i-v curve is stablized in solution; Drip 2 μMs of DNA1 in the gold electrode surfaces of cleaning, under 25 ° of C, assemble 120min, the gold electrode surfaces of having modified DNA1, after fully cleaning, dries up with high pure nitrogen, is then immersed by gold electrode in 1mM sulfydryl hexanol, closes 50min under 25 ° of C;
(4) mensuration of melamine: the melamine standard solution of 10.0 μ L variable concentrations or sample solution and 10.0 μ LAu-Fc-DNA2 complex solutions are added drop-wise to the gold electrode surfaces of having modified DNA1, at room temperature react 60min.Then, repeatedly electrode surface is rinsed with phosphate buffer solution, remove unconjugated melamine and Au-Fc-DNA2 compound, utilize the sweep speed of differential pulse voltammetry (DPV) with 100mV/s in phosphate buffer solution to scan, electric potential scanning scope is-0.7 ~ 0.1V.According to electrochemical signals, melamine is carried out quantitatively.
(5) mensuration of fluorescence intensity: join in 96 orifice bores by standard antigen (1pg/mL to 100ng/mL) and sample to be tested solution, and at 37 DEG C incubation 1 hour, gold electrode after process is joined in 96 orifice bores, add appropriate cysteamine (1mM), lucifuge tests fluorescence intensity after jolting 1 minute.
Beneficial effect of the present invention is:
In the present invention, the preparation technology of golden nanometer particle is simple, convenient, adds sodium phytate, makes to form avtive spot between golden nanometer particle, make it have stronger Raman enhancement effect; The testing result accuracy rate of golden nanometer particle is high, and stability is strong.
Efficient for fluorescent dye richness is gathered in golden nanometer particle surface, further modification detects antibody aptamers, gold nano fluorescence probe efficiently can be combined with disease organism marker, the efficient rich poly-fluorescent dye in golden nanometer particle surface occurs to depart from and causes the remarkable activation of fluorescence probe, reaches the object of high-sensitivity detection disease organism marker.
The present invention carries out fluorescent dye and test antibodies or aptamer modified on nanogold particle surface, has glimmering
Light can activate performance.
When the concentration of melamine is at 0.3ngmL
-1-700ngmL
-1between time, along with the change of melamine concentration, DPV intensity has significant change, and calculating and detecting the linear equation of melamine is I
pa(Ipa is DPV peak current intensity to=54.45C-302.8, unit nA; C is the concentration of melamine, unit ngmL
-1; R
2=0.9993, n=7).Detectability is 0.1ngmL
-1(3 σ).By being 3ngmL to concentration
-1melamine carry out 11 replicate determinations and calculate, relative standard deviation is respectively 4.2%, shows that assay method of the present invention has good reappearance.
The surface energy efficiently concentrating luminescent dye molecule of nanogold particle of the present invention also makes the effective cancellation of fluorescent dye, improve detection sensitivity, the minimum biomarker (melamine) that 1pg/mL level can be detected, highly sensitive, simple and easy to do, be conducive to applying.
Embodiment
Namo fluorescence probe measures a method for melamine, it is characterized in that, specifically comprises the steps:
(1) preparation of golden nanometer particle: be add sodium phytate solution after 0.003-0.005mol/L liquor argenti nitratis ophthalmicus is heated to boiling and continuous heating extremely boiling by concentration, and to add concentration after keeping 15-20min be 0.003-0.005mol/L sodium phytate solution, with vigorous stirring, after being heated to boil, adding mass concentration is fast the citric acid three sodium solution that 1.5-1.8% newly prepares, boil continuously, treat that solution colour becomes redness from dark blue, red golden nanometer particle (Au) solution can be obtained, 100mL is diluted to after being cooled to room temperature, shake up, be placed in 4 ° of C refrigerators to preserve,
(2) preparation of gold nanoparticle probe: 5mg ferrocenecarboxylic acid is joined in the phosphate buffer solution solution of 10mL0.1mol/L, add the fluorescent dye containing 3-5% rhodamine B isothiocyanates again, by this mixed solution at room temperature vigorous stirring reaction 2h, excessive fluorescent dye is removed in centrifuging, with bovine serum albumin(BSA), the avtive spot on gold nano grain surface is carried out closed half an hour, acquisition can activate gold nano fluorescence probe, and 4 DEG C save backup;
(3) process of gold electrode: first gold electrode is used alumina powder polishing respectively on chamois leather, successively with ethanol and distilled water ultrasonic 8min respectively, finally at 0.1mol/LH
2sO
4cyclic voltammetry scan is carried out until i-v curve is stablized in solution; Drip 2 μMs of DNA1 in the gold electrode surfaces of cleaning, under 25 ° of C, assemble 120min, the gold electrode surfaces of having modified DNA1, after fully cleaning, dries up with high pure nitrogen, is then immersed by gold electrode in 1mM sulfydryl hexanol, closes 50min under 25 ° of C;
(4) mensuration of melamine: the melamine standard solution of 10.0 μ L variable concentrations or sample solution and 10.0 μ LAu-Fc-DNA2 complex solutions are added drop-wise to the gold electrode surfaces of having modified DNA1, at room temperature react 60min.Then, repeatedly electrode surface is rinsed with phosphate buffer solution, remove unconjugated melamine and Au-Fc-DNA2 compound, utilize the sweep speed of differential pulse voltammetry (DPV) with 100mV/s in phosphate buffer solution to scan, electric potential scanning scope is-0.7 ~ 0.1V.According to electrochemical signals, melamine is carried out quantitatively.
Preferably, described DNA1 sequence is: 5 '-HS-CCGCTGCCGGTTTTGGTTCGG-3 '.
Preferably, described DNA2 sequence is: 5 '-CCGTTCCTTTTCCGG-HS-3 '.
Method according to invention measures content of melamine in milk sample, and adopt standard addition method to evaluate method, the sample determination recovery is 96.0 – 102.6%, and measurement result is in table 1, and method of the present invention has the high feature of precision in melamine detects.
The measurement result of melamine in table 1 milk sample.
| Numbering | Content a, b | Addition | Measured amount | The recovery (%) |
| 1 | ND c | 1.00 | 0.96 | 96.0 |
| 2 | ND | 5.00 | 5.11 | 102.2 |
| 3 | ND | 50.00 | 51.08 | 102.6 |
a7 measurement results
bunit: ngmL
-1
cnD: do not detect.
(5) mensuration of fluorescence intensity: join in 96 orifice bores by standard antigen (1pg/mL to 100ng/mL) and sample to be tested solution, and at 37 DEG C incubation 1 hour, gold electrode after process is joined in 96 orifice bores, add appropriate cysteamine (1mM), lucifuge tests fluorescence intensity after jolting 1 minute.The detection limit that result display can activate gold nano fluorescence probe is low to moderate 1pg/mL's, conventional fluorescent probe (1ng/mL) 3 orders of magnitude that this limit detects are exceeded, melamine specific antigen in energy efficient detection milk sample, the key that the susceptibility of this probe improves is luminescent dye molecule efficiently concentrating and cancellation and efficiently hightail golden nanometer particle and recover fluorescence, and background signal is low.Of the present inventionly activate that the preparation of gold nano fluorescence probe is simple, performance efficiency is stablized.
Claims (1)
1. namo fluorescence probe measures a method for melamine, it is characterized in that, specifically comprises the steps:
(1) preparation of golden nanometer particle: be add sodium phytate solution after 0.003-0.005mol/L liquor argenti nitratis ophthalmicus is heated to boiling and continuous heating extremely boiling by concentration, and to add concentration after keeping 15-20min be 0.003-0.005mol/L sodium phytate solution, with vigorous stirring, after being heated to boil, adding mass concentration is fast the citric acid three sodium solution that 1.5-1.8% newly prepares, boil continuously, treat that solution colour becomes redness from dark blue, red golden nanometer particle (Au) solution can be obtained, 100mL is diluted to after being cooled to room temperature, shake up, be placed in 4 ° of C refrigerators to preserve,
(2) preparation of gold nanoparticle probe: 5mg ferrocenecarboxylic acid is joined in the phosphate buffer solution solution of 10mL0.1mol/L, add the fluorescent dye containing 3-5% rhodamine B isothiocyanates again, by this mixed solution at room temperature vigorous stirring reaction 2h, excessive fluorescent dye is removed in centrifuging, with bovine serum albumin(BSA), the avtive spot on gold nano grain surface is carried out closed half an hour, acquisition can activate gold nano fluorescence probe, and 4 DEG C save backup;
(3) process of gold electrode: first gold electrode is used alumina powder polishing respectively on chamois leather, successively with ethanol and distilled water ultrasonic 8min respectively, finally at 0.1mol/LH
2sO
4cyclic voltammetry scan is carried out until i-v curve is stablized in solution; Drip 2 μMs of DNA1 in the gold electrode surfaces of cleaning, under 25 ° of C, assemble 120min, the gold electrode surfaces of having modified DNA1, after fully cleaning, dries up with high pure nitrogen, is then immersed by gold electrode in 1mM sulfydryl hexanol, closes 50min under 25 ° of C;
(4) mensuration of melamine: the melamine standard solution of 10.0 μ L variable concentrations or sample solution and 10.0 μ LAu-Fc-DNA2 complex solutions are added drop-wise to the gold electrode surfaces of having modified DNA1, at room temperature react 60min, then, repeatedly electrode surface is rinsed with phosphate buffer solution, remove unconjugated melamine and Au-Fc-DNA2 compound, the sweep speed of differential pulse voltammetry (DPV) with 100mV/s in phosphate buffer solution is utilized to scan, electric potential scanning scope is-0.7 ~ 0.1V, according to electrochemical signals, melamine is carried out quantitatively
(5) mensuration of fluorescence intensity: join in 96 orifice bores by standard antigen (1pg/mL to 100ng/mL) and sample to be tested solution, and at 37 DEG C incubation 1 hour, gold electrode after process is joined in 96 orifice bores, add appropriate cysteamine (1mM), lucifuge tests fluorescence intensity after jolting 1 minute.
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| CN201410404446.0A CN105334258A (en) | 2014-08-16 | 2014-08-16 | Method for determining melamine by nano-fluorescence probe |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106018295A (en) * | 2016-05-11 | 2016-10-12 | 上海应用技术学院 | Measuring method for positive-negative ion pairs needed by surfaces of gold nano particles in phase extraction method |
| CN106053419A (en) * | 2016-08-02 | 2016-10-26 | 东北林业大学 | Water content detection fluorescent probe and device applying same |
| CN110078750A (en) * | 2019-04-24 | 2019-08-02 | 上海市质量监督检验技术研究院 | Asymmetric terpyridyl complex compound and its preparation method and application |
| WO2019237769A1 (en) * | 2018-06-13 | 2019-12-19 | 青岛大学 | Method for preparing melamine ratiometric fluorescent probe based on silver nanocluster composite |
-
2014
- 2014-08-16 CN CN201410404446.0A patent/CN105334258A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106018295A (en) * | 2016-05-11 | 2016-10-12 | 上海应用技术学院 | Measuring method for positive-negative ion pairs needed by surfaces of gold nano particles in phase extraction method |
| CN106053419A (en) * | 2016-08-02 | 2016-10-26 | 东北林业大学 | Water content detection fluorescent probe and device applying same |
| WO2019237769A1 (en) * | 2018-06-13 | 2019-12-19 | 青岛大学 | Method for preparing melamine ratiometric fluorescent probe based on silver nanocluster composite |
| US10913892B1 (en) | 2018-06-13 | 2021-02-09 | Qingdao University | Method for preparing ratiometric fluorescent probe for melamine based on silver nanocluster complex |
| CN110078750A (en) * | 2019-04-24 | 2019-08-02 | 上海市质量监督检验技术研究院 | Asymmetric terpyridyl complex compound and its preparation method and application |
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Application publication date: 20160217 |