CN102645420A - Method for detecting multiple harmful substance residual components in food based on surface plasma resonance technique - Google Patents
Method for detecting multiple harmful substance residual components in food based on surface plasma resonance technique Download PDFInfo
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Abstract
The invention relates to a method for detecting multiple harmful substance residual components in food based on a surface plasma resonance (SPR) technique. The method comprises four steps of sensing surface aptamer fixing, sample marking, sample detecting and sensing element regenerating. The surface of a multi-probe optical fiber sensor is connected with the aptamer corresponding to different residual components; a molecular probe marker corresponding to different residual components is added and introduced on the sensing surface; unmarked and marked residual components carry out specificity competitive identification on the corresponding aptamer; molecular probe aptamer solution is introduced and combined with the marked sample connected on the surface of the optical fiber sensor for SPR detection by using the multi-probe optical fiber sensor; and the multi-probe optical fiber sensor can be used for regenerating to realize repeated use of the sensing element. Based on the SPR technique and the aptamer specificity competitive identification, multiple harmful substance residual components in one sample can be simultaneously detected, and the method provided by the invention is particularly suitable for detecting substances with small molecular weight.
Description
Technical field
The present invention relates to the how residual while detection technique of objectionable impurities in a kind of food.Particularly, carry out specificity competition identification, adopt the Fibre Optical Sensors of popping one's head in to detect the method for multiple harmful substances in the food simultaneously more through aptamer and unmarked, mark sample based on surface plasma body resonant vibration technology.
Background technology
In the food the residual serious harm of objectionable impurities the public healthy, in the face of the significant challenge of food-safety problem, objectionable impurities is the important step that ensures food safety in the analyzing and testing food.Wherein, (Surface Plasmon Resonance SPR), as a kind of hypersensitive fast detecting means, is one of ideal tools of food safety detection to surface plasma resonance.But still there are deficiencies such as signal to noise ratio (S/N ratio) is big, response is little, sensitivity is low in SPR when detecting low molecular weight substance, and Acetamiprid, kanamycins, terramycin (agricultural chemicals veterinary drug), ochracin (biotoxin), malachite green (chemical pollutant) etc. are the low-molecular-weight objectionable impurities in the food.In addition, often have multiple harmful substances in the food simultaneously, make that the testing process complicacy is loaded down with trivial details, the single material that generally adopts at present detects and can't meet the demands.Therefore, press for the novel detection method of exploitation, realize the hypersensitive of multiple low-molecular-weight objectionable impurities in the food, fast, detect simultaneously based on SPR technology.
In recent years, the detection method based on aptamers identification demonstrated the huge applications prospect in food safety detection.Aptamers is nucleic acid (DNA or the RNA) fragment that can combine with the target molecules high specific.Because aptamers has high specific and high-affinity, therefore be called " chemical antibody ".Compare with antibody, aptamers has higher chemical stability, is difficult for inactivation, therefore has more application potential.In addition, the aptamers preparation does not rely on animal or cell, can pass through polymerase chain reaction (PCR) technology external a large amount of synthetic.Therefore, based on the aptamers specific recognition, multiple harmful substances provides the foundation and foundation in the food in order to detect simultaneously.Mating surface plasma resonance technology is expected to realize that the hypersensitive of multiple harmful substances detects simultaneously in the food.
Summary of the invention
For solving the problem that exists in the prior art; The present invention provides a kind of surface plasma body resonant vibration spectral technology that adopts to detect the residual method of multiple low-molecular-weight objectionable impurities in the food, solves the problem that is difficult to detect simultaneously multiple low molecular weight substance in the existing surface plasma body resonant vibration technology.
Technical scheme of the present invention is: the how residual while detection method of objectionable impurities in a kind of food of application surface plasma resonance technology, the content of the multiple harmful substances residual component in the same sample is detected simultaneously, and it is characterized in that comprising the steps:
(1) the sensor surface aptamers is fixed: many probes Fibre Optical Sensor of selecting Streptavidin SA to modify; Each detecting head surface connects a kind of aptamers; Through feeding 5-50 μ L concentration is the 1-50 μ g/L corresponding aptamer of residual component of mark biotin, on many probe Fibre Optical Sensors, fixes different residual component corresponding nucleic acids aptamers;
(2) residual component to be measured is carried out the molecular probe mark, labeling method is different according to the determinand group, selects the crosslinked carboxyl of EDC/NHS method and amino or crosslinked two amino of glutaraldehyde method;
(3) standard solution preparation: the standard model storing solution that disposes residual component with the phosphate buffer of 0.01-0.1mol/L (pH=7.4); Storing solution concentration is 0.1ng/mL to 1mg/mL; Equivalent is measured the standard reserving solution of residual component respectively; Preparation contains the hybrid standard stoste of objectionable impurities residual component; With phosphate buffer hybrid standard stoste is mixed with the standard solution of variable concentrations, gets the standard solution of the unmarked residual component to be measured of variable concentrations and the residual component to be measured of molecular probe mark and fully mix;
(4) set up typical curve: adopt the surface plasma resonance spectrometer to measure; Phosphate buffer with 0.02mol/L is a measuring basis; Feed 5-100 μ L biased sample to be measured through Micropump; Feeding 5-100 μ L concentration subsequently is the aptamers solution of the molecular probe of 5-100ng/mL, notes surface plasma body resonant vibration appearance spectrogram; Get the surface plasma body resonant vibration spectrogram stationary value of variable concentrations testing sample, the drawing curve, and carry out polynomial curve fitting, obtain the equation of regression curve; Record the working curve of multiple harmful substances residual component successively, carry out polynomial curve fitting, obtain typical curve separately;
(5) detection by quantitative: the multiple residual component sample for unknown content detects, and carries out according to step (three) equally; Feed an amount of molecular probe aptamers solution again, adopt the residual spectrogram of surface plasma resonance spectrometer record multiple harmful substances; With the corresponding regression curve equation of the stationary value substitution of different residual components in the spectrogram, calculate the concentration value of residual component in the different food products;
(6) glycocoll-hydrochloric acid buffer solution of feeding 0.01-0.05mol/L (pH is 2-3), the many probes of flushing Fibre Optical Sensors surface is carried out sensing element and is regenerated, and is used for measuring next time.
Said molecular probe comprises fibrin ferment or lysozyme or immunoglobulin E, and labeling method comprises EDC/NHS method or glutaraldehyde method.
Can adopt aptamer is the identification molecule, and the aptamer sequence is directly related with determinand.
Can adopt many probe Fibre Optical Sensors is the sensing element of surface plasma resonance instrument, and each probe connects a kind of aptamers, connects 4 ~ 16 kinds of different aptamers altogether.
Said step (one) sensor surface aptamers is fixed, and comprises the steps:
(1) optical fiber sensing probe of Streptavidin SA being modified inserts in the surface plasma body resonant vibration detector, in service aisle, carries out online sensing surface and modifies;
(2) phosphate buffer of feeding 0.02mol/L (pH=7.4);
(3) feeding 5-50 μ L concentration is the labeled molecule aptamers solution of 1-50 μ g/L biotin modification, carries out the online baseline stability that is coupled to;
(4) repeating step (2) obtains the optical fiber sensing probe that the labeled molecule aptamers is modified to (3).
Can adopt molecular probe mark determinand, with unmarked sample and aptamer generation specificity competition identification.
Feed an amount of molecular probe aptamers solution among said step (four) and (five), improve surface plasma body resonant vibration spectrometer response, be applicable to and measure the less small-molecular weight objectionable impurities residual component of response signal.
Molecular weight < 1000 Da of described small-molecular weight objectionable impurities residual component.
The surface plasma body resonant vibration that adopts in the inventive method Fibre Optical Sensor of popping one's head in the fixing multiple different aptamers of sensor surface, based on the aptamers specific recognition, can detect multiple material simultaneously.
The inventive method is introduced big molecular marked compound fibrin ferment or lysozyme or immunoglobulin E; Discern target molecule unmarked and mark based on the aptamers competition of surface plasma resonance sensing surface; If target molecule is few in the testing sample; Then the labeled molecule of surface plasma body resonant vibration surface adsorption is many, and the surface plasma resonance response that cause this moment is big more, and anti regular is more little.Because the molecular probe molecular weight is big, after the absorption of molecular probe aptamers specific recognition, the surface plasma resonance response value further increases, and therefore, can realize the super sensitivity detection of small-molecule substance.
Provided by the invention based on the surface plasma body resonant vibration spectrometer; Adopt many probe Fibre Optical Sensors; Detect the residual method of multiple low-molecular-weight objectionable impurities in the food, compared with prior art have the following advantages: (1) adopts the aptamer of high stability to be the identification molecule, compares antibody-antigen recognizing and detects; Improve the serviceable life of sensing element greatly, reduced the detection cost; (2) adopt the surface plasma body resonant vibration Fibre Optical Sensor of popping one's head in more, can detect multiple harmful substances simultaneously, compare single material detection technique, improved sample detection efficient greatly, shortened the sample detection time; Problems such as (3) introducing has the molecular probe of macromolecule, based on competition identification, can realize the super sensitivity detection of micromolecule objectionable impurities, and the response that has solved surface plasma body resonant vibration technology for detection small molecular weight material is little, sensitivity is low.
Description of drawings
Fig. 1 is that the multiple micromolecule objectionable impurities based on surface plasma resonance according to the invention detects schematic diagram simultaneously; Wherein
is the fibrin ferment aptamers.
Embodiment
Below in conjunction with specific embodiment the present invention is done to describe in detail.
Embodiment: the selection fibrin ferment is a molecular probe, and fibrin ferment aptamers sequence is: GGTTGGTGTGGTTGG.
Select that the micromolecule objectionable impurities is a detected object in five kinds of typical food, be respectively Acetamiprid, kanamycins, terramycin, ochracin, malachite green, corresponding aptamers sequence is:
Acetamiprid aptamers---CCTGCCACGCTCCGCAAGCTTTGTAATTTGTCTGCAGCGGTTCTT
GATCGCTGACACCATATTATGAAGATAAGCTTGGCACCCGCATCGT
Kanamycins aptamers---CACCTAATACGACTCSCTATAGCGGATCCGAAGATGGGGGTTG
AGGCTAAGCCGACCGTAAGTTGGGCCGTCTGGCTCGAACAAGCTTGC
Terramycin aptamers---CGTACGGAATTCGCTAGCCGAGTTGAGCCGGGCGCGGTACGGGT
ACTGGTATGTGTGGGGATCCGAGCTCCACGTGGGATCCGAGCTCCACGTG
Ochracin aptamers---GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA
Malachite green aptamers---GGAUCCCGACUGGCGAGAGCCAGGUAACGAAUGGAUCC.
The concrete operations step is following:
(1) the sensor surface aptamers is fixed: many probes Fibre Optical Sensor of selecting Streptavidin (SA) to modify; Each detecting head surface connects a kind of aptamers; Through feeding 10 μ L concentration is the 30 μ g/L corresponding aptamers of residual component of mark biotin, on many probe Fibre Optical Sensors, fixes different residual component corresponding nucleic acids aptamers;
(2) molecular marked compound coupling: five kinds of determinands are carried out the coupling mark through glutaraldehyde method and fibrin ferment; Labeling method comprises EDC/NHS method, glutaraldehyde method etc., like kanamycins according to determinand group different choice; Can adopt glutaraldehyde method to be connected with fibrin ferment, for use;
(3) standard solution preparation: take by weighing Acetamiprid, kanamycins, terramycin, ochracin, 5 kinds of standard items 10mg of malachite green respectively, be dissolved in 0.02mol/L (pH=7.4) phosphate buffer and be settled to 10mL, be the standard stock solution of 1mg/mL; Equivalent is measured 5 kinds of standard stock solutions respectively, and preparation contains the hybrid standard stoste 10 μ g/mL of 5 kinds of objectionable impurities residual components; With phosphate buffer hybrid standard stoste is mixed with the standard solution of variable concentrations, concentration is followed successively by 0 in pg/mL ~ ng/mL level, 1ng/mL, 2ng/mL, 4ng/mL, 6ng/mL, 8ng/mL;
(4) typical curve is drawn: with above-mentioned concentration is that the unlabelled standard solution of 0 ng/mL is marked with harmful substances solution with an amount of fibrin ferment and fully mixes, and 5 kinds of objectionable impurities residual component content are also equal in this label solution; Adopt the surface plasma resonance spectrometer to measure; Feed 0.02mol/L (pH=7.4) phosphate buffer as measuring basis; Pump into biased sample to be measured then; The stable back of response feeds fibrin ferment aptamers solution, treat the surface plasma resonance response value stabilization after, recording surface plasma resonance optical spectrum figure;
(5) feed 0.02mol/L (pH=2.4) glycocoll-hydrochloric acid solution, the combination of aptamers and determinand is destroyed on the many probes of flushing Fibre Optical Sensor surface, accomplishes sensing element regeneration;
(6) concentration feeds the standard model measuring method to be measured same (4) of other concentration (1-8ng/mL) from low to high successively, obtains respective surfaces plasma resonance optical spectrum figure; Get the surface plasma spectrogram stationary value of each objectionable impurities residual component, the drawing standard curve, and carry out polynomial curve fitting, obtain concentration-stationary value and concern the regression curve equation;
(7) after the sensing chip regeneration, with actual unknown concentration testing sample set by step (4) be marked with harmful substances solution with fibrin ferment in right amount and fully mix; In the surface plasma body resonant vibration spectrometer that pumps into, write down the spectrogram of different objectionable impurities residual components simultaneously, confirm corresponding stationary value, substitution is typical curve separately, confirms each component concentration, and detectability can reach pg/mL ~ ng/mL level;
(8) feed 0.02mol/L (pH=2.4) glycocoll-hydrochloric acid solution once more, the flushing surface plasma body resonant vibration Fibre Optical Sensor of popping one's head in more, the regeneration of popping one's head in is used for measurement next time.
The present invention is accurate and convenient for what narrate; Be that example is described in detail with Acetamiprid, kanamycins, terramycin, ochracin, malachite green in an embodiment; But the present invention is equally applicable to the mensuration of other objectionable impuritiess in the food; Like the accurate detection and the qualitative analysis of botulin toxin, estradiol, TCs etc., so foregoing is all within protection domain of the present invention.In addition; Method according to embodiment is carried out test sample, compares with existing surface plasma detection technique, and accuracy improves 2 ~ 10 times; Shorten detection time more than 5 times; The probe life-span prolongs 20%, and the small-molecular weights such as Acetamiprid that existing surface plasma detection technique can not accurately detect (1000Da) material, can adopt method of the present invention to obtain testing result accurately.
Claims (8)
1. the how residual while detection method of objectionable impurities in the food of an application surface plasma resonance instrument detects the content of the multiple harmful substances residual component in the same sample simultaneously, it is characterized in that comprising the steps:
(1) the sensor surface aptamers is fixed: many probes Fibre Optical Sensor of selecting Streptavidin SA to modify; Each detecting head surface connects a kind of aptamers; Through feeding 5-50 μ L concentration is the 1-50 μ g/L corresponding aptamer of residual component of mark biotin, on many probe Fibre Optical Sensors, fixes different residual component corresponding nucleic acids aptamers;
(2) residual component to be measured is carried out the molecular probe mark, labeling method is different according to the determinand group, selects the crosslinked carboxyl of EDC/NHS method and amino or crosslinked two amino of glutaraldehyde method;
(3) standard solution preparation: the standard model storing solution that disposes residual component with the phosphate buffer of 0.01-0.1mol/L (pH=7.4); Storing solution concentration is 0.1ng/mL to 1mg/mL; Equivalent is measured the standard reserving solution of residual component respectively; Preparation contains the hybrid standard stoste of objectionable impurities residual component; With phosphate buffer hybrid standard stoste is mixed with the standard solution of variable concentrations, gets the standard solution of the unmarked residual component to be measured of variable concentrations and the residual component to be measured of molecular probe mark and fully mix;
(4) set up typical curve: adopt the surface plasma resonance spectrometer to measure; Phosphate buffer with 0.02mol/L is a measuring basis; Feed 5-100 μ L biased sample to be measured through Micropump; Feeding 5-100 μ L concentration subsequently is the aptamers solution of the molecular probe of 5-100ng/mL, notes surface plasma body resonant vibration appearance spectrogram; Get the surface plasma body resonant vibration spectrogram stationary value of variable concentrations testing sample, the drawing curve, and carry out polynomial curve fitting, obtain the equation of regression curve; Record the working curve of multiple harmful substances residual component successively, carry out polynomial curve fitting, obtain typical curve separately;
(5) detection by quantitative: the multiple residual component sample for unknown content detects, and carries out according to step (three) equally; Feed an amount of molecular probe aptamers solution again, adopt the residual spectrogram of surface plasma resonance spectrometer record multiple harmful substances; With the corresponding regression curve equation of the stationary value substitution of different residual components in the spectrogram, calculate the concentration value of residual component in the different food products;
(6) glycocoll-hydrochloric acid buffer solution of feeding 0.01-0.05mol/L (pH is 2-3), the many probes of flushing Fibre Optical Sensors surface is carried out sensing element and is regenerated, and is used for measuring next time.
2. method according to claim 1 is characterized in that said molecular probe comprises fibrin ferment or lysozyme or immunoglobulin E, and labeling method comprises EDC/NHS method or glutaraldehyde method.
3. method according to claim 1, it is characterized in that adopting aptamer is the identification molecule, the aptamer sequence is directly related with determinand.
4. method according to claim 1, it is characterized in that adopting many probe Fibre Optical Sensors is the sensing element of surface plasma resonance instrument, each probe connects a kind of aptamers, connects 4～16 kinds of different aptamers altogether.
5. method according to claim 1 is characterized in that said step () sensor surface aptamers fixes, and comprises the steps:
(1) optical fiber sensing probe of Streptavidin SA being modified inserts in the surface plasma body resonant vibration detector, in service aisle, carries out online sensing surface and modifies;
(2) phosphate buffer of feeding 0.02mol/L (pH=7.4);
(3) feeding 5-50 μ L concentration is the labeled molecule aptamers solution of 1-50 μ g/L biotin modification, carries out the online baseline stability that is coupled to;
(4) repeating step (2) obtains the optical fiber sensing probe that the labeled molecule aptamers is modified to (3).
6. method according to claim 1 is characterized in that adopting molecular probe mark determinand, with unmarked sample and aptamer generation specificity competition identification.
7. method according to claim 1; It is characterized in that feeding among said step (four) and (five) an amount of molecular probe aptamers solution; Improve surface plasma body resonant vibration spectrometer response, be applicable to and measure the less small-molecular weight objectionable impurities residual component of response signal.
8. method according to claim 1 is characterized in that, molecular weight < 1000 Da of described small-molecular weight objectionable impurities residual component.
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| CN104178568A (en) * | 2014-07-25 | 2014-12-03 | 清华大学 | Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe |
| CN105138820A (en) * | 2015-07-24 | 2015-12-09 | 天津大学 | Calculation method suitable for resonant wavelength of surface Plasmon resonance signal under multi-mode optical fiber |
| CN106596927A (en) * | 2016-12-26 | 2017-04-26 | 中国科学院长春光学精密机械与物理研究所 | Optical fiber bioprobe, and preparation method and whole-blood detection method thereof |
| CN106970048A (en) * | 2017-02-17 | 2017-07-21 | 丁利 | The method for detecting a variety of agricultural and veterinary chemicals residuals in food simultaneously based on the surface plasma resonance technology that regenerated liquid optimizes |
| CN106996923A (en) * | 2017-02-17 | 2017-08-01 | 丁利 | The method that a variety of environmental hormones are detected based on the surface plasma resonance technology that regenerated liquid optimizes simultaneously |
| CN109540847A (en) * | 2018-12-13 | 2019-03-29 | 山东师范大学 | A kind of graphene/gold/D plastic optical fiber SPR sensor and preparation method |
| CN110501506A (en) * | 2018-07-05 | 2019-11-26 | 东莞东阳光医疗智能器件研发有限公司 | A kind of biosensor and its application |
| CN113933281A (en) * | 2021-12-14 | 2022-01-14 | 中国农业大学 | Exosome detection method based on optical fiber evanescent wave fluorescence biosensor |
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| CN103558154A (en) * | 2013-11-18 | 2014-02-05 | 广州赛宝计量检测中心服务有限公司 | Rotary telescopic type analytic sample box and analytic sample |
| CN104178568B (en) * | 2014-07-25 | 2016-03-30 | 清华大学 | A kind of method based on the target substance in nucleic acid aptamer probe fluorescence sense analyzing and testing sample to be tested |
| CN104178568A (en) * | 2014-07-25 | 2014-12-03 | 清华大学 | Method for detecting target substance in to-be-detected sample based on fluorescent sensing analysis of aptamer probe |
| CN105138820B (en) * | 2015-07-24 | 2019-01-15 | 天津大学 | A kind of calculation method suitable for the surface plasma resonance signal resonant wavelength under multimode fibre |
| CN105138820A (en) * | 2015-07-24 | 2015-12-09 | 天津大学 | Calculation method suitable for resonant wavelength of surface Plasmon resonance signal under multi-mode optical fiber |
| CN106596927A (en) * | 2016-12-26 | 2017-04-26 | 中国科学院长春光学精密机械与物理研究所 | Optical fiber bioprobe, and preparation method and whole-blood detection method thereof |
| CN106596927B (en) * | 2016-12-26 | 2018-10-19 | 中国科学院长春光学精密机械与物理研究所 | A kind of optical fiber bio probe and preparation method thereof and whole blood test method |
| CN106970048A (en) * | 2017-02-17 | 2017-07-21 | 丁利 | The method for detecting a variety of agricultural and veterinary chemicals residuals in food simultaneously based on the surface plasma resonance technology that regenerated liquid optimizes |
| CN106996923A (en) * | 2017-02-17 | 2017-08-01 | 丁利 | The method that a variety of environmental hormones are detected based on the surface plasma resonance technology that regenerated liquid optimizes simultaneously |
| CN110501506A (en) * | 2018-07-05 | 2019-11-26 | 东莞东阳光医疗智能器件研发有限公司 | A kind of biosensor and its application |
| CN109540847A (en) * | 2018-12-13 | 2019-03-29 | 山东师范大学 | A kind of graphene/gold/D plastic optical fiber SPR sensor and preparation method |
| CN113933281A (en) * | 2021-12-14 | 2022-01-14 | 中国农业大学 | Exosome detection method based on optical fiber evanescent wave fluorescence biosensor |
| CN113933281B (en) * | 2021-12-14 | 2022-03-18 | 中国农业大学 | Exosome detection method based on optical fiber evanescent wave fluorescence biosensor |
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