CN106124475A - A kind of trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer - Google Patents
A kind of trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer Download PDFInfo
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Abstract
The present invention relates to a kind of trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer, belong to field of pesticide detection.The present invention, using the gold nano grain of Raman active dye marker as strengthening substrate, utilizes the specific binding of pesticide and aptamers, causes the gold nano grain of the reactive dye labelling gathering difference in saline solution；By Portable Raman spectrometer, obtain the SERS spectrogram of corresponding reactive dye, it is achieved the detection by quantitative to trace pesticide, and in Folium Camelliae sinensis actual sample, carry out method feasibility checking.The detection method that the present invention provides, raman characteristic peak using detection object in tradition Raman technology can be changed into, as the conventional thinking of rational judgment, the quantitative criterion detected using the raman characteristic peak of reactive dye label as object, not only increase detection sensitivity, simplify complicated strengthen substrate preparation flow, more trace pesticide residues food and the research of industrial circle, detect, supervision etc. provides conveniently.
Description
Technical field
The present invention relates to a kind of Raman spectra detection process for pesticide residues, refer in particular to trace based on aptamer
Pesticide residues surface enhanced raman spectroscopy (SERS) detection method, belongs to field of pesticide detection.
Background technology
Pesticide, with its outstanding contributions in terms of crop with the prevention and control of plant diseases, pest control so that its agricultural life at home and abroad
Produce and be widely used in cultivating.But, when pesticide ensure vegetable increase income, while Food Production, pesticide residues phenomenon also with
?.Extensive and serious Pesticide Residue, the worry being not only in terms of ecological environment brought, the most residual
Pesticide is stayed to take in by passive or proceed to food safety hidden danger that is internal and that cause from nature by the way of enrichment.At present, I
State's Pesticides Testing method is mainly traditional detection mode, including: the methods such as gas phase, liquid phase and mass spectrometry thereof, they are permissible
Realize the accurate residue detection of pesticide, but need instrument and equipment and the professional operator of complex and expensive, sample pre-treatments in addition
Loaded down with trivial details time-consuming, testing cost is high, it is difficult to meet on-the-spot, the needs of low cost detection.And immunoassay side of based on antigen-antibody
Although method specificity is high, there is false positive, the shortcoming that price is high.
Raman spectroscopy is one and has high sensitivity, ease for operation, the spectrum skill of the multiple advantages such as low cost detection
Art, and it is widely used in multiple fields such as medical science, biology, environment, archaeology.Wherein, surface enhanced raman spectroscopy (SERS) technology
Discovery, promoted Raman spectroscopy in individual molecule detection, the application of trace materials context of detection and development especially.Mesh
Before, single nanometer gold/silver colloid is the most quite varied as the application strengthening substrate, and the Raman with label strengthens substrate
The trace Detecting Pesticide mode of bind nucleic acid aptamers is but rarely reported.The present invention is residual as the trace pesticide of a kind of novelty
Stay Raman detection method, improve surface enhanced Raman technique detection efficiency the most to a certain extent, widened trace simultaneously
The detection thinking of pesticide residues.
Summary of the invention
It is an object of the invention to provide the inspection of a kind of trace pesticide residues surface enhanced raman spectroscopy based on aptamer
Survey method, the synthesis of its base material is simple, detection sensitivity is high, easy to operate, with low cost, high specificity, is widely used in
Food and the research of industrial circle, detect, supervision etc..
To achieve these goals, the present invention uses surface enhanced raman spectroscopy technology, and the method utilizes label labelling
Nano material as strengthen substrate, be combined with the specific recognition of aptamers by pesticide, affect nano material in detection bodies
CHARGE DISTRIBUTION balance in system, thus cause the gathering under difference detection pesticide concentration of the nano material of label labelling poor
Different；The enhancing substrate of different aggregation extents can carry out amplification in various degree to Raman signal, according to the mark of different raman scattering intensities
Note thing raman characteristic peak collection of illustrative plates, it is achieved trace pesticide is carried out the purpose of sensitive indirect quantification detection.Meanwhile, the method is applied to
The detection of Tea Samples, to verify its method reliability in actual applications.
Above-mentioned trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer, described Raman
Strengthen the gold nano grain that substrate is the absorption of Raman active Crystal Violet.
Above-mentioned trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer, described trace
The specific binding sign of pesticide aptamers corresponding to it is to use circular dichroism spectra.
Above-mentioned trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer, described 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’。
Above-mentioned trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer, its concrete steps
As follows:
1) preparation of substrate is strengthened: use trisodium citrate to add thermal reduction and carry out the preparation of gold nano grain.In preparation
During, keep concentration be 0.02% (M/V) chlorauric acid solution mix with the citric acid three sodium solution of 1% (M/V) of 2.3mL with
The gold nano grain of preparation size suitable (about 30nm)；And by the Raman active Crystal Violet solution of 20 μ L 0.8 μM
With the gold nano grain mixing that 100 μ L prepare gained so that Raman active Crystal Violet is adsorbed in the form of Electrostatic Absorption
The surface of gold nano grain.
2) sign of detection method: here, the specific binding employing circular dichroism spectra of pesticide and aptamer characterizes: use
10mM Tris buffer preparation containing 10mM sodium chloride and 5mM magnesium chloride is the core of configuration equivalent in centrifuge tube (1.5mL)
Acid aptamers solution；Subsequently, wherein in a pipe add 5 μMs of Acetamiprid pesticide, another pipe add equivalent ultra-pure water, finally in
Circular dichroism spectrogram detection is carried out under 230nm-300nm wave-length coverage.The enhancing substrate of different coherent conditions uses transmission electron microscope picture
As shooting: draw the mixed solution of a certain amount of different detection system, be added dropwise on copper mesh, carry out transmission image shooting.
3) detection of Raman spectrum: first, the Acetamiprid solution of 20 μ L variable concentrations is adaptive in 4 μ L nucleic acid of same matrix
Body hybrid reaction 30 minutes.Subsequently, add and strengthen substrate liquid, and this mixed system whirlpool is uniform, put and continue instead at room temperature
Answer 8 minutes.In this stage, change, not with Acetamiprid with its nucleic acid structure of aptamer of Acetamiprid specific recognition
The aptamer of specific recognition is adsorbed in the surface of gold nano grain with the form of Electrostatic Absorption.Finally, 20 μ L are added
Sodium chloride (NaCl) solution of 350mM and 20 μ L ultra-pure waters make the cumulative volume of its detection system be 200 μ L.This detection system is filled
After dividing mixing, it is placed in microcolorimetric ware and carries out Raman spectrum detection.Here, the optical maser wavelength of Raman spectrum is set as 785nm.
4) detection of actual sample: the detection of actual Tea Samples trace pesticide residues uses standard addition method.First, accurate
Really weigh the 5g Tea Samples through oven for drying to constant weight, and by itself and 150mL dichloromethane (CH2Cl2) mixing supersound extraction
45min；Subsequently, mixed liquor is filtered, leave and take filtrate, and prepare the mixing of certain density Acetamiprid with the extracting solution of clarification
Liquid；Then, take half above-mentioned Acetamiprid mixed liquor and carry out rotary evaporation concentration, and by gained concentrate with containing a certain amount of methanol
(CH3It is diluted by ultra-pure water OH)；Finally, according to step 3) carry out Raman spectrum detection, thus realize actual sample
The detection by quantitative of trace pesticide residues in product.
Beneficial effects of the present invention:
The gold nano grain substrate of the Raman active Crystal Violet absorption that the present invention provides, on the one hand can utilize nanometer
The reinforcement of intergranular phasmon coupled electromagnetic field adjusts surface enhanced raman spectroscopy signal；On the other hand drawing of absorption is utilized
Graceful reactive dye crystal violet, can by tradition Raman technology using the raman characteristic peak of detection object as the routine of rational judgment
Thinking is changed into the quantitative criterion detected using the raman characteristic peak of label as object, and this utilizes marker peak to replace material special
Levy opening one's minds of peak and not only increase detection sensitivity, simplify the step of substrate preparation and chemical modification, and avoid
The fluorescence interference of background material under complex detection environment.
The present invention uses circular dichroism spectra to characterize the specific binding of pesticide and aptamer, utilizes the acquisition spectrum can be straight
Obtain the specific binding and nucleic acid conformation change information taking pesticide with aptamer；Use transmission electron microscope shooting difference inspection
The image of survey system, utilizes acquisition image can intuitively obtain the enhancing substrate information of different coherent condition.These two kinds of characterizing methods
Feasibility analysis offer for experiment 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, synthesize low cost, stable chemical nature,
And it is suitable for the detection extending to other objects.
The trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer of present invention design, operation
Flow process is simple, and detection confidence is strong, can food and the research of industrial circle, detect, the direction such as supervision is extensively applied.
Accompanying drawing explanation
Fig. 1 is aptamer and the specific binding sign of Acetamiprid of embodiment；
Fig. 2 is the Acetamiprid Raman standard curve of embodiment；
Fig. 3 be the surface-enhanced Raman curve chart of embodiment with corresponding transmission electron microscope Cleaning Principle phenogram: (A) strengthens base
End a and the surface-enhanced Raman curve chart corresponding transmission electron microscope Cleaning Principle strengthening substrate-sodium chloride b solution system characterize
Figure；(B) aptamer-enhancing substrate-sodium chloride c and Acetamiprid-aptamer-enhancing substrate-sodium chloride d solution system
Surface-enhanced Raman curve chart corresponding transmission electron microscope Cleaning Principle phenogram.
Detailed description of the invention
The present invention has versatility to trace pesticide residues, but owing to pesticide variety is more, the present invention has only selected main flow
In pesticide classification, the most widely used Acetamiprid is referred to this embodiment as embodiment, the detection of other pesticide
Method.
Example enforcement step combines accompanying drawing and is described in detail.
(1) preparation of gold nano grain
Use trisodium citrate to add thermal reduction and carry out the preparation of gold nano grain.First, with the chloroazotic acid of new preparation (1:
3HNO3/ HCl) soak the required glass drying oven used in preparation, and be rinsed with distilled water before use, dry in air
Use after dry.Secondly, take 0.0200g gold chloride and add 100ml ultra-pure water, be configured to 0.02% (M/V) solution and be placed in three necks burnings
In Ping, and utilize electric heating to be placed under 550rpm rotating speed to carry 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 solution colour is changed into claret-red by faint yellow
Color；It is further continued for being heated at reflux 30min.Finally, obtain the gold nano grain solution of 30nm size, this solution is placed in room temperature
Lower natural cooling, and it is stored at 4 DEG C standby.
(2) aptamer and the sign detecting object specific recognition
Two 1.5mL centrifuge tubes of the 10mM Tris buffer solution containing 10mM sodium chloride and 5mM magnesium chloride will be added
The aptamer of equivalent (5ml, 5 μMs)；The most respectively toward centrifuge tube adds Acetamiprid and the ultra-pure water of equivalent (5ml, 5 μMs),
After reacting 30 minutes, carry out circular dichroism spectra mensuration.Result shows, at feature peak position (279nm, 251nm) place it can clearly be seen that
Aptamer and the peak value difference of Acetamiprid-aptamer.This phenomenon explanation Acetamiprid and aptamer can be special
Property combine, and change the space structure of aptamer, the gold nano grain of crystal violet labelling is at salt to cause it to be unable to maintain that
Dispersity under solution；The Acetamiprid of variable concentrations causes the gathering of gold nano grain in various degree.
(3) detection of Raman spectrum
The Acetamiprid solution of 20 μ L variable concentrations adds the 4 μ L aptamers of 20 μ L, and hybrid reaction added after 30 minutes
The Raman active Crystal Violet of 20 μ L0.8 μM and 100 μ L prepare the gold nano grain of gained, continue hybrid reaction after 8 minutes,
Add sodium chloride solution and the 20 μ L ultra-pure waters of 20 μ L 350mM so that it is the cumulative volume of detection system is 200 μ L, finally by this inspection
After survey system is sufficiently mixed, it is placed in microcolorimetric ware and carries out Raman spectrum detection.The testing conditions of Raman spectrum is: laser wave
A length of 785nm, laser power is 0.3W-0.5W, and the time of integration is 5-10s, and times of collection is 2-5 time, and detection range is 500-
1700cm-1。
The peak value of the Raman spectrum 1175nm feature peak position of the system corresponding to it of the Acetamiprid according to variable concentrations sets up mark
Directrix curve is analyzed calculating.Result such as Fig. 2 shows, the linear relationship between Acetamiprid concentration and 1175nm peak are strong is good, pyridine
The detection of worm amidine is limited to 1.76 × 10-8M, the range of linearity is 3 × 10-8M-4×10-6M。
(4) collection of images of transmissive electron microscope
From above-mentioned detection system, draw 30 μ L mixed solutions, be added dropwise on copper mesh, carry out images of transmissive electron microscope and adopt
Collection.Fig. 3 shows the Raman spectrum under different testing conditions and the transmission electron microscope picture of correspondence thereof.Fig. 3 (A) a display explanation preparation
Gained monodisperse gold granule, favorable dispersibility, substantially there is no Raman signal；Fig. 3 (A) b display explanation is when there being salt ion
Add fashionable, the monodisperse gold granule Severe aggregation of crystal violet labelling, and produce powerful Raman signal；Fig. 3 (B) c shows
Illustrating, in the system of the gold nano grain-sodium chloride of aptamer-crystal violet labelling, aptamer can protect knot
The gold nano grain of crystalviolet labelling produces gathering, and interdependent ratio Fig. 3 (A) a under saline solution, it was observed that significantly crystal violet Raman
Characteristic peak, illustrates to utilize label can expand Raman signal；Fig. 3 (B) d shows explanation, in the presence of Acetamiprid pesticide, and nucleic acid
Aptamers and object produce specific binding and make aptamer conformation change, the gold nano of crystal violet labelling accordingly
Granule produces under saline solution and again produces gathering, and produces stronger Raman signal.Therefore, from the images of transmissive electron microscope gathered
In it can be seen that the number of Acetamiprid persticide residue directly determines the gold nano grain of crystal violet labelling under NaCl solution
Coherent condition, and the characteristic peak being proportional to label crystal violet is strong and weak.
(5) detection of actual Tea Samples
Utilize standard addition method that Folium Camelliae sinensis actual sample trace pesticide residues are detected.Accurately weigh 5g through oven for drying
Tea Samples to constant weight is ultrasonic with 150mL dichloromethane solution mixes 45min；Subsequently, filter tea grounds, retain supernatant；Its
Secondary, with the Acetamiprid pesticide of the extracting solution preparation variable concentrations clarified, after uniformly mixing, take half mixed liquor carry out rotating steaming
Send out and concentrate；Then, with the ultra-pure water containing a certain amount of methanol, mixed liquor is diluted；Finally, Raman light is carried out according to step (3)
Spectrum detection.The peak value of the Raman spectrum 1175nm feature peak position of the system corresponding to it of the Acetamiprid according to variable concentrations, obtains reality
Border sample recovery rate is as shown in table 1, and it is 98.45%-104.5% that detection is recycled rate scope, it is possible to achieve to actual sample
Trace Detecting Pesticide, and actual sample pretreatment mode described in step (5) operation is simple, convenience and high-efficiency.
The actual Folium Camelliae sinensis sample response rate of table 1. embodiment collects
A:5 parallel laboratory test meansigma methods
The standard deviation of b:5 parallel laboratory test
In the description of this specification, reference term " embodiment ", " some embodiments ", " illustrative examples ",
The description of " example ", " concrete example " or " some examples " etc. means to combine this embodiment or the specific features of example description, knot
Structure, material or feature are contained at least one embodiment or the example of the present invention.In this manual, to above-mentioned term
Schematic representation is not necessarily referring to identical embodiment or example.And, the specific features of description, structure, material or spy
Point can combine in any one or more embodiments or example in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
These embodiments can be carried out multiple change in the case of departing from the principle of the present invention and objective, revise, replace and modification, this
The scope of invention is limited by claim and equivalent thereof.
SEQUENCE LISTING
<110>Jiangsu University
<120>a kind of trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer
<130>a kind of trace pesticide residues 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 (5)
1. a trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer, it is characterised in that should
Method utilizes the nano material of label labelling as strengthening substrate, is combined with the specific recognition of aptamers by pesticide, shadow
Ring nano material CHARGE DISTRIBUTION balance in detection system, thus cause the nano material of label labelling to detect agriculture in difference
Gathering difference under concentration；The enhancing substrate of different aggregation extents can carry out amplification in various degree, foundation to Raman signal
The label raman characteristic peak collection of illustrative plates of different raman scattering intensities, it is achieved trace pesticide is carried out the purpose of sensitive indirect quantification detection.
Trace pesticide residues surface enhanced raman spectroscopy detection side based on aptamer the most according to claim 1
Method, it is characterised in that strengthen the gold nano grain that substrate is the absorption of Raman active Crystal Violet.
Trace pesticide residues surface enhanced raman spectroscopy detection side based on aptamer the most according to claim 1
Method, it is characterised in that use circular dichroism spectra to characterize pesticide and be combined with the specific recognition of aptamer.
Trace pesticide residues surface enhanced raman spectroscopy detection side based on aptamer the most according to claim 1
Method, it is characterised in that trace pesticide is Acetamiprid, nucleic acid aptamer sequence is: 5 '-TGT AAT TTG TCT GCA GCG
GTT CTT GAT CGC TGA CAC CAT ATT ATG AAG A-3’。
Trace pesticide residues surface enhanced raman spectroscopy detection side based on aptamer the most according to claim 1
Method, it is characterised in that detection method comprises the following specific steps that:
1) preparation of substrate is strengthened: use trisodium citrate to add thermal reduction and carry out the preparation of gold nano grain.In preparation process
In, keeping concentration is that 0.02% (M/V) chlorauric acid solution mixes with the citric acid three sodium solution of 1% (M/V) of 2.3mL with preparation
The suitable gold nano grain of size, about 30nm；And by the Raman active Crystal Violet solution and 100 of 20 μ L 0.8 μM
μ L prepares the gold nano grain mixing of gained so that Raman active Crystal Violet is adsorbed in gold nano with the form of Electrostatic Absorption
The surface of granule；
2) sign of detection method: here, the specific binding employing circular dichroism spectra of pesticide and aptamer characterizes: 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；Subsequently, adding 5 μMs of Acetamiprid pesticide wherein in a pipe, another pipe adds the ultra-pure water of equivalent, finally in 230nm-
Circular dichroism spectrogram detection is carried out under 300nm wave-length coverage；The enhancing substrate of different coherent conditions uses images of transmissive electron microscope to shoot:
Draw the mixed solution of a certain amount of different detection system, be added dropwise on copper mesh, carry out transmission image shooting；
3) detection of Raman spectrum: first, the Acetamiprid solution of 20 μ L variable concentrations mixes in 4 μ L aptamers of same matrix
Close reaction 30 minutes；It is subsequently added enhancing substrate liquid, and this mixed system whirlpool is uniform, put and continue reaction 8 points at room temperature
Clock；In this stage, change with its nucleic acid structure of aptamer of Acetamiprid specific recognition, not with Acetamiprid specificity
The aptamer identified is adsorbed in the surface of gold nano grain with the form of Electrostatic Absorption；Finally, the chlorine of 20 μ L350mM is added
Change sodium solution and 20 μ L ultra-pure waters make the cumulative volume of its detection system be 200 μ L；After this detection system is sufficiently mixed, it is placed in micro-
Amount cuvette carries out Raman spectrum detection, here, the optical maser wavelength of Raman spectrum is set as 785nm；
4) detection of actual sample: the detection of actual Tea Samples trace pesticide residues uses standard addition method；First, accurately claim
Take the 5g Tea Samples through oven for drying to constant weight, and it is mixed supersound extraction 45min with 150mL dichloromethane；Subsequently, right
Mixed liquor filters, and leaves and takes filtrate, and prepares certain density Acetamiprid mixed liquor with the extracting solution of clarification；Then, one is taken
Half above-mentioned Acetamiprid mixed liquor carries out rotary evaporation concentration, and the gained concentrate ultra-pure water containing a certain amount of methanol is entered it
Row dilution；Finally, according to step 3) carry out Raman spectrum detection, thus realize trace pesticide residues quantitative in actual sample
Detection.
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| CN106770173A (en) * | 2017-01-05 | 2017-05-31 | 江苏大学 | A kind of MCNCs/PMAA magnetic control Raman aptamer sensor preparation methods for antibiotics leftover detection |
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| CN106841340A (en) * | 2017-03-14 | 2017-06-13 | 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 |
| CN107655958A (en) * | 2017-09-22 | 2018-02-02 | 山西大学 | Acetamiprid detection method based on ferronickel cyanide complex nano particle for instruction probe |
| 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 |
| CN108760715A (en) * | 2018-05-07 | 2018-11-06 | 同济大学 | Detect Polychlorinated biphenyls Surface enhanced Raman scattering aptamer Sensors & Application |
| CN108645840A (en) * | 2018-07-20 | 2018-10-12 | 江苏大学 | One kind being based on the sandwich surface-enhanced Raman Pesticides Testing method of gold-magnetic nanometer |
| 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 |
| CN110044862A (en) * | 2019-02-15 | 2019-07-23 | 东莞出入境检验检疫局检验检疫综合技术中心(东莞国际旅行卫生保健中心) | Method based on ferroso-ferric oxide polypyrrole and fluorescent material detection Acetamiprid |
| CN110044862B (en) * | 2019-02-15 | 2021-06-25 | 东莞海关综合技术中心 | Method for detecting acetamiprid based on ferroferric oxide polypyrrole and fluorescent material |
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