CN106124475B - A kind of trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer - Google Patents
A kind of trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer Download PDFInfo
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The present invention relates to a kind of trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer, belongs to field of pesticide detection.The present invention is using the gold nano grain of Raman active dye marker as enhancing substrate, using the specific binding of pesticide and aptamers, aggregation difference of the gold nano grain for causing reactive dye to mark in salting liquid；By Portable Raman spectrometer, the SERS spectrogram of corresponding reactive dye is obtained, realizes the quantitative detection to trace pesticide, and carries out the verifying of method feasibility in tealeaves actual sample.Detection method provided by the invention, the conventional thinking of raman characteristic peak as rational judgment in traditional Raman technology to detect object can be changed into the quantitative criterion using the raman characteristic peak of reactive dye marker as target analyte detection, not only increase detection sensitivity, complicated enhancing substrate preparation flow is simplified, more trace pesticide residue is provided convenience in the research, detection, supervision etc. of food and industrial circle.
Description
Technical field
The present invention relates to a kind of Raman spectra detection process for pesticide residue, refer in particular to the trace based on aptamer
Pesticide residue Surface enhanced Raman spectroscopy (SERS) detection method, belongs to field of pesticide detection.
Background technique
Pesticide, with its outstanding contributions in terms of crop and the prevention and control of plant diseases, pest control, so that its agricultural life at home and abroad
It produces and is widely used with cultivation.However, when pesticide is ensureing that vegetables increase income, while Food Production, pesticide residue phenomenon also with
?.Extensive and serious Pesticide Residue, bring is not only the worry in terms of ecological environment, more seriously residual
Pesticide is stayed to be transferred to the food safety hazards caused in vivo from nature by way of passively taking in or being enriched with.Currently, I
State's Pesticides Testing method is mainly traditional detection mode, comprising: the methods of gas phase, liquid phase and its mass spectrometry, they can be with
It realizes the accurate residue detection of pesticide, but needs instrument and equipment and the professional operator of complex and expensive, in addition sample pre-treatments
It is cumbersome it is time-consuming, testing cost is high, it is difficult to meet scene, low cost detection needs.And the immunoassay side based on antigen-antibody
Although method specificity is high, there is false positive, the high disadvantage of price.
Raman spectroscopy, which is one, has high sensitivity, ease for operation, the spectrum skill of a variety of advantages such as low cost detection
Art, and it is widely used in the multiple fields such as medicine, biology, environment, archaeology.Wherein, Surface enhanced Raman spectroscopy (SERS) technology
Discovery, even more pushed Raman spectroscopy individual molecule detection, trace materials context of detection application and development.Mesh
Before, single nanogold/silver colloid is very extensive as the application of enhancing substrate, and the Raman with marker enhances substrate
It is but rarely reported in conjunction with the trace Detecting Pesticide mode of aptamer.The present invention is residual as a kind of novel trace pesticide
Raman detection method is stayed, not only improves surface enhanced Raman technique detection efficiency to a certain extent, while having widened trace
The detection thinking of pesticide residue.
Summary of the invention
The trace pesticide residue Surface enhanced Raman spectroscopy inspection based on aptamer that the object of the present invention is to provide a kind of
Survey method, base material synthesis is simple, detection sensitivity is high, easy to operate, low in cost, high specificity, is widely used in
Research, detection, supervision of food and industrial circle etc..
To achieve the goals above, the present invention uses Surface enhanced Raman spectroscopy technology, and this method utilizes label substance markers
Nano material as enhancing substrate, through pesticide with the specific recognition of aptamers ining conjunction with, influence nano material detecting body
Distribution of charges balance in system, it is poor in the different aggregations detected under pesticide concentrations so as to cause the nano material of label substance markers
It is different；The enhancing substrate of different aggregation extents can carry out different degrees of amplification, the mark according to different raman scattering intensities to Raman signal
Remember object raman characteristic peak map, realizes the purpose for carrying out sensitive indirect quantification detection to trace pesticide.Meanwhile this method is applied to
The detection of Tea Samples, to verify the reliability of its method in practical applications.
The above-mentioned trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer, the Raman
Enhancing substrate is the gold nano grain of Raman active Crystal Violet absorption.
The above-mentioned trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer, the trace
The characterization of pesticide aptamers specific binding corresponding to its is using circular dichroism spectra.
The above-mentioned trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer, the trace
Pesticide is Acetamiprid, and the sequence of aptamers is: 5 '-TGT AAT TTG TCT GCA GCG GTT CTT GAT CGC TGA
CAC CAT ATT ATG AAG A-3’。
The above-mentioned trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer, specific steps
It is as follows:
1) enhance the preparation of substrate: carrying out the preparation of gold nano grain using trisodium citrate heating reduction method.It is preparing
In the process, keep concentration be 0.02% (M/V) chlorauric acid solution mixed with the citric acid three sodium solution of 1% (M/V) of 2.3mL with
Prepare the gold nano grain of particle size suitable (about 30nm)；And by 0.8 μM of 20 μ L of Raman active Crystal Violet solution
Resulting gold nano grain mixing is prepared with 100 μ L, so that Raman active Crystal Violet is adsorbed in the form of Electrostatic Absorption
The surface of gold nano grain.
2) characterization of detection method: here, the specific binding of pesticide and aptamer is characterized using circular dichroism spectra: using
The core of 10mM Tris buffer preparation containing 10mM sodium chloride and 5mM magnesium chloride configuration equivalent in centrifuge tube (1.5mL)
Acid adaptation liquid solution；Then, 5 μM of Acetamiprid pesticides are added in a wherein pipe, the ultrapure water of equivalent is added in another pipe, finally in
The detection of circular dichroism spectrogram is carried out under 230nm-300nm wave-length coverage.The enhancing substrate of different coherent conditions uses transmission electron microscope picture
As shooting: drawing the mixed solution of a certain amount of different detection architectures, be added dropwise on copper mesh, carry out transmission image shooting.
3) detection of Raman spectrum: firstly, the Acetamiprid solution of 20 μ L various concentrations is adapted in 4 μ L nucleic acid of same matrix
Body hybrid reaction 30 minutes.Then, enhancing substrate liquid is added, and this mixed system whirlpool is uniform, is placed in and continues at room temperature instead
It answers 8 minutes.In this stage, its nucleic acid structure changes with the aptamer of Acetamiprid specific recognition, not with Acetamiprid
The aptamer of specific recognition is adsorbed in the surface of gold nano grain in the form of Electrostatic Absorption.Finally, 20 μ L are added
Sodium chloride (NaCl) solution of 350mM and 20 μ L ultrapure waters make the 200 μ L of total volume of its detection architecture.The detection architecture is filled
After dividing mixing, it is placed in progress Raman spectrum detection in microcolorimetric ware.Here, the optical maser wavelength of Raman spectrum is set as 785nm.
4) detection of actual sample: the detection of practical Tea Samples trace pesticide residue uses standard addition method.Firstly, quasi-
5g really is weighed through the baking oven Tea Samples that drying to constant weight, and by itself and 150mL methylene chloride (CH2Cl2) mixing ultrasonic extraction
45min；Then, mixed liquor is filtered, leaves and takes filtrate, and prepare certain density Acetamiprid with clear extracting solution and mix
Liquid；Then, it takes the above-mentioned Acetamiprid mixed liquor of half to carry out rotary evaporation concentration, and gained concentrate is used and contains a certain amount of methanol
(CH3OH ultrapure water) is diluted it；Finally, Raman spectrum detection is carried out according to step 3), to realize to practical sample
The quantitative detection of trace pesticide residue in product.
Beneficial effects of the present invention:
The gold nano grain substrate of Raman active Crystal Violet absorption provided by the invention, on the one hand can utilize nanometer
The reinforcement of intergranular phasmon coupled electromagnetic field adjusts Surface enhanced Raman scattering signal；On the other hand the drawing of absorption is utilized
Graceful reactive dye crystal violet, can by traditional Raman technology to detect the raman characteristic peak of object as the routine of rational judgment
Thinking is changed into the quantitative criterion using the raman characteristic peak of marker as target analyte detection, this replaces substance special using marker peak
The step of opening one's minds for peak of sign not only increases detection sensitivity, simplifies substrate preparation and chemical modification, and avoid
The fluorescence interference of background material under complex detection environment.
The present invention, can be straight using spectrum is obtained using the specific binding of circular dichroism spectra characterization pesticide and aptamer
Obtain the specific binding for taking pesticide and aptamer and nucleic acid conformation change information；Different inspections are shot using transmission electron microscope
The image of survey system can intuitively obtain the enhancing substrate information of different coherent conditions using image is obtained.Two kinds of characterizing methods
It provides for the feasibility analysis of experiment and facilitates efficient observation method.
The present invention quotes aptamer as specific recognition probe, on the one hand can stablize nano material in detection system
Dispersity in system；On the other hand can recognition detection object with high specificity, synthesis cost is low, and chemical property is stablized,
And it is applicable in the detection for extending to other objects.
The trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer that the present invention designs, operation
Process is simple, and detection confidence is strong, can be widely applied in directions such as the researchs, detection, supervision of food and industrial circle.
Detailed description of the invention
Fig. 1 is that the aptamer of embodiment and Acetamiprid specific binding characterize；
Fig. 2 is the Acetamiprid Raman standard curve of embodiment；
Fig. 3 is the surface-enhanced Raman curve graph of embodiment with corresponding transmission electron microscope testing principle phenogram: (A) enhances base
Bottom a and the corresponding transmission electron microscope testing principle of enhancing substrate-sodium chloride b solution system surface-enhanced Raman curve graph characterize
Figure；(B) aptamer-enhancing substrate-sodium chloride c and Acetamiprid-aptamer-enhancing substrate-sodium chloride d solution system
The corresponding transmission electron microscope testing principle phenogram of surface-enhanced Raman curve graph.
Specific embodiment
The present invention has versatility to trace pesticide residue, but since pesticide variety is more, the present invention has only selected mainstream
For the most widely used Acetamiprid as embodiment, the detection of other pesticides can be with reference to the embodiment in pesticide classification
Method.
Example implementation steps combination attached drawing is described in detail.
(1) preparation of gold nano grain
The preparation of gold nano grain is carried out using trisodium citrate heating reduction method.Firstly, with newly prepare chloroazotic acid (1:
3HNO3/ HCl) the required glassware used in preparation is impregnated, and be rinsed using preceding with distilled water, it is done in air
It is used after dry.Secondly, taking 0.0200g gold chloride that 100ml ultrapure water is added, it is configured to 0.02% (M/V) solution and is placed in three necks burning
In bottle, and it is placed under 550rpm revolving speed using electric heating and carries out condensing reflux heating；After solution is heated to boiling, it is rapidly added
The citric acid three sodium solution of 1% (M/V) of 2.3ml；After a few minutes, it can be observed that be changed into wine red by faint yellow for solution colour
Color；It is further continued for being heated at reflux 30min.Finally, obtaining the gold nano grain solution of 30nm particle size, this solution is placed in room temperature
Lower natural cooling, and be stored in spare at 4 DEG C.
(2) characterization of aptamer and detection object specific recognition
It will be added in two 1.5mL centrifuge tubes containing 10mM sodium chloride and the 10mM Tris buffer solution of 5mM magnesium chloride
The aptamer of equivalent (5ml, 5 μM)；The Acetamiprid and ultrapure water of equivalent (5ml, 5 μM) is added into centrifuge tube respectively again,
After reaction 30 minutes, circular dichroism spectra measurement is carried out.The result shows that can clearly be seen that at feature peak position (279nm, 251nm)
Aptamer and Acetamiprid-aptamer peak value difference.This phenomenon illustrates that Acetamiprid and aptamer can be special
Property combine, and change the space structure of aptamer, it caused to be unable to maintain that the gold nano grain of crystal violet label in salt
Dispersity under solution；The Acetamiprid of various concentration leads to the aggregation of different degrees of gold nano grain.
(3) detection of Raman spectrum
The 4 μ L aptamers of 20 μ L are added in the Acetamiprid solution of 20 μ L various concentrations, and hybrid reaction is added after 30 minutes
The Raman active Crystal Violet of 20 μ L0.8 μM and 100 μ L prepare resulting gold nano grain, after continueing to mix reaction 8 minutes,
The sodium chloride solution and 20 μ L ultrapure waters of 20 μ L 350mM is added, makes the 200 μ L of total volume of its detection architecture, finally by the inspection
After survey system is sufficiently mixed, it is placed in progress Raman spectrum detection in microcolorimetric ware.The testing conditions of Raman spectrum are as follows: laser wave
A length of 785nm, laser power 0.3W-0.5W, time of integration 5-10s, times of collection are 2-5 times, detection range 500-
1700cm-1。
It is established and is marked according to the peak value of the Raman spectrum 1175nm feature peak position of the Acetamiprid of various concentration system corresponding to its
Directrix curve carries out analytical calculation.As a result as Fig. 2 shows that the linear relationship between Acetamiprid concentration and 1175nm peak intensity is good, pyridine
The detection of worm amidine is limited to 1.76 × 10-8M, the range of linearity are 3 × 10-8M-4×10-6M。
(4) acquisition of images of transmissive electron microscope
30 μ L mixed solutions are drawn from above-mentioned detection architecture, are added dropwise on copper mesh, are carried out images of transmissive electron microscope and are adopted
Collection.Fig. 3 shows Raman spectrum and its corresponding transmission electron microscope picture under different testing conditions.Fig. 3 (A) a, which is shown, to be illustrated to prepare
Gained monodisperse gold particle, favorable dispersibility, substantially without Raman signal；Fig. 3 (A) b display explanation, which is worked as, salt ion
When addition, the monodisperse gold particle Severe aggregation of crystal violet label, and generate powerful Raman signal；Fig. 3 (B) c is shown
Illustrate, in aptamer-crystal violet label gold nano grain-sodium chloride system, aptamer can protect knot
The gold nano grain of crystalviolet label generates aggregation under salting liquid, and interdependent than Fig. 3 (A) a, observes apparent crystal violet Raman
Characteristic peak illustrates that Raman signal can be expanded using marker；Fig. 3 (B) d display explanation, in the presence of Acetamiprid pesticide, nucleic acid
Aptamers generate specific binding with object and aptamer conformation are made to change, accordingly the gold nano of crystal violet label
Particle generates under salting liquid generates aggregation again, and generates stronger Raman signal.Therefore, from the images of transmissive electron microscope of acquisition
In as can be seen that Acetamiprid persticide residue number directly determine crystal violet mark gold nano grain under NaCl solution
Coherent condition, and the characteristic peak for being proportional to marker crystal violet is strong and weak.
(5) detection of practical Tea Samples
Tealeaves actual sample trace pesticide residue is detected using standard addition method.5g is accurately weighed to dry through baking oven
To the Tea Samples and 150mL dichloromethane solution ultrasonic mixing 45min of constant weight；Then, tea grounds is filtered, supernatant is retained；Its
It is secondary, the Acetamiprid pesticide of various concentration is prepared with clear extracting solution, and half mixed liquor is taken to carry out rotation steaming after evenly mixing
Hair concentration；Then, mixed liquor is diluted with the ultrapure water containing a certain amount of methanol；Finally, carrying out Raman light according to step (3)
Spectrum detection.According to the peak value of the Raman spectrum 1175nm feature peak position of the Acetamiprid of various concentration system corresponding to its, reality is obtained
Border sample recovery rate is as shown in table 1, and it is 98.45%-104.5% that detection, which obtains rate of recovery range, may be implemented to actual sample
Trace Detecting Pesticide, and operation is simple for actual sample pretreatment mode described in step (5), convenience and high-efficiency.
The practical tealeaves sample rate of recovery of 1. embodiment of table summarizes
A:5 parallel laboratory test average value
The standard deviation of b:5 parallel laboratory test
In the description of this specification, reference term " one embodiment ", " some embodiments ", " illustrative examples ",
The description of " example ", " specific example " or " some examples " etc. means specific features described in conjunction with this embodiment or example, knot
Structure, material or feature are included at least one embodiment or example of the invention.In the present specification, to above-mentioned term
Schematic representation may not refer to the same embodiment or example.Moreover, specific features, structure, material or the spy of description
Point can be combined in any suitable manner in any one or more of the embodiments or examples.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this
The range of invention is defined by the claims and their equivalents.
SEQUENCE LISTING
<110>Jiangsu University
<120>a kind of trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer
<130>a kind of trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 49
<212> DNA
<213>artificial sequence
<400> 1
tgtaatttgt ctgcagcggt tcttgatcgc tgacaccata ttatgaaga 49
Claims (4)
1. a kind of trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer, which is characterized in that should
Method is using the nano material of label substance markers as enhancing substrate, through pesticide in conjunction with the specific recognition of aptamers, shadow
Ring nano material in detection architecture distribution of charges balance, so as to cause label substance markers nano material in different detection agricultures
Aggregation difference under concentration；The enhancing substrate of different aggregation extents can carry out different degrees of amplification, foundation to Raman signal
The marker raman characteristic peak map of different raman scattering intensities realizes the purpose that sensitive indirect quantification detection is carried out to trace pesticide；
Detection method comprises the following specific steps that:
1) enhance the preparation of substrate: the preparation of gold nano grain is carried out using trisodium citrate heating reduction method, in preparation process
In, keeping concentration is that 0.02% (M/V) chlorauric acid solution is mixed with the citric acid three sodium solution of 1% (M/V) of 2.3mL to prepare
The suitable gold nano grain of particle size, about 30nm；And by 0.8 μM of 20 μ L of Raman active Crystal Violet solution and 100
μ L prepares resulting gold nano grain mixing, so that Raman active Crystal Violet is adsorbed in gold nano in the form of Electrostatic Absorption
The surface of particle；
2) characterization of detection method: here, the specific binding of pesticide and aptamer is characterized using circular dichroism spectra: with containing
The aptamer that the 10mM Tris buffer preparation of 10mM sodium chloride and 5mM magnesium chloride configures equivalent in centrifuge tube is molten
Liquid；Then, 5 μM of Acetamiprid pesticides are added in a wherein pipe, the ultrapure water of equivalent is added in another pipe, finally in 230nm-
The detection of circular dichroism spectrogram is carried out under 300nm wave-length coverage；The enhancing substrate of different coherent conditions is shot using images of transmissive electron microscope:
The mixed solution for drawing a certain amount of different detection architectures, is added dropwise on copper mesh, carries out transmission image shooting；
3) detection of Raman spectrum: firstly, the Acetamiprid solution of 20 μ L various concentrations is mixed in 4 μ L aptamers of same matrix
Close reaction 30 minutes；Enhancing substrate liquid is then added, and it is this mixed system whirlpool is uniform, it is placed in that the reaction was continued at room temperature 8 points
Clock；In this stage, its nucleic acid structure changes with the aptamer of Acetamiprid specific recognition, not specific with Acetamiprid
The aptamer of identification is adsorbed in the surface of gold nano grain in the form of Electrostatic Absorption；Finally, being added 20 μ L 350mM's
Sodium chloride solution and 20 μ L ultrapure waters make the 200 μ L of total volume of its detection architecture；After the detection architecture is sufficiently mixed, it is placed in
Raman spectrum detection is carried out in microcolorimetric ware, according to the Raman spectrum characteristic peak of the Acetamiprid of various concentration system corresponding to its
The peak value of position establishes standard curve；Here, the optical maser wavelength of Raman spectrum is set as 785nm；
4) detection of actual sample: the detection of practical Tea Samples trace pesticide residue uses standard addition method；Firstly, accurate claim
It takes 5g through the baking oven Tea Samples that drying to constant weight, and it is mixed into ultrasonic extraction 45min with 150mL methylene chloride；Then, right
Mixed liquor is filtered, and leaves and takes filtrate, and prepare certain density Acetamiprid mixed liquor with clear extracting solution；Then, one is taken
Half above-mentioned Acetamiprid mixed liquor carries out rotary evaporation concentration, and by gained concentrate with the ultrapure water containing a certain amount of methanol to its into
Row dilution；Finally, carrying out Raman spectrum detection according to step 3), trace pesticide residue in actual sample is quantified to realize
Detection.
2. the trace pesticide residue Surface enhanced Raman spectroscopy detection side according to claim 1 based on aptamer
Method, which is characterized in that enhancing substrate is the gold nano grain of Raman active Crystal Violet absorption.
3. the trace pesticide residue Surface enhanced Raman spectroscopy detection side according to claim 1 based on aptamer
Method, which is characterized in that using circular dichroism spectra characterization pesticide in conjunction with the specific recognition of aptamer.
4. the trace pesticide residue Surface enhanced Raman spectroscopy detection side according to claim 1 based on aptamer
Method, which is characterized in that trace pesticide is Acetamiprid, nucleic acid aptamer sequence are as follows: 5 '-TGT AAT TTG TCT GCA GCG
GTT CTT GAT CGC TGA CAC CAT ATT ATG AAG A-3’。
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| CN106706910B (en) * | 2017-01-05 | 2018-10-09 | 江苏大学 | A kind of food-borne pathogens detection method for modifying gold nanorods based on p-Mercaptoaniline |
| CN106841340B (en) * | 2017-03-14 | 2019-06-14 | 浙江省农业科学院 | The electrochemical sensor and its method system of isocarbophos and Acetamiprid are detected simultaneously |
| CN107389576A (en) * | 2017-08-29 | 2017-11-24 | 贵州大学 | A kind of quick colorimetric detection method of Acetamiprid based on nanometer enzymatic |
| CN107655958B (en) * | 2017-09-22 | 2019-09-24 | 山西大学 | It is the Acetamiprid detection method for indicating probe based on ferronickel cyanide complex nano particle |
| CN108760715B (en) * | 2018-05-07 | 2021-11-09 | 同济大学 | Surface-enhanced Raman scattering aptamer sensor for detecting polychlorinated biphenyl and application thereof |
| CN108645840B (en) * | 2018-07-20 | 2021-02-26 | 江苏大学 | Surface-enhanced Raman pesticide detection method based on gold-magnetic nano sandwich type |
| CN109540866A (en) * | 2018-11-30 | 2019-03-29 | 泉州师范学院 | A kind of compound gold nano-material and preparation method thereof for Detecting Pesticide |
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Inventor after: Chen Quansheng Inventor after: Hu Weiwei Inventor after: Li Huanhuan Inventor after: Zhang Bin Inventor after: Ou Yangqin Inventor after: Zhao Jiewen Inventor before: Hu Weiwei Inventor before: Chen Quansheng Inventor before: Li Huanhuan Inventor before: Zhang Bin Inventor before: Ou Yangqin Inventor before: Zhao Jiewen |
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Application publication date: 20161116 Assignee: Nanjing Rongdian Food Technology Co.,Ltd. Assignor: Jiangsu University Contract record no.: 2019320000096 Denomination of invention: Trace pesticide residue surface enhanced Raman spectrum detection method based on a nucleic acid aptamer Granted publication date: 20190104 License type: Common License Record date: 20190404 |