CN106053790A - Method for detecting ochratoxin A based on near-infrared up-conversion luminescence marking and magnetic separation - Google Patents
Method for detecting ochratoxin A based on near-infrared up-conversion luminescence marking and magnetic separation Download PDFInfo
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- CN106053790A CN106053790A CN201610353399.0A CN201610353399A CN106053790A CN 106053790 A CN106053790 A CN 106053790A CN 201610353399 A CN201610353399 A CN 201610353399A CN 106053790 A CN106053790 A CN 106053790A
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
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Abstract
A method for detecting ochratoxin A based on near-infrared up-conversion luminescence marking and magnetic separation is used for detecting ochratoxin A (OTA) content in wheat and its products and the like. By connecting near-infrared up-conversion luminescent material with NaYF4:Yb 0.2 and Tm 0.02 with ochratoxin A aptamer to form a signal probe, the signal probe and aptamer complementary oligonucleotide single strand modified Fe3O4 magnetic nanomaterial form a nano composite, and at the moment up-conversion luminescence signal is maximum; when OTA is present in a detection system, OTA specifically binds with OTA aptamer so that double strands are unlinked, and by monitoring up-conversion luminescence signal strength 804 nm from a near-infrared zone, it is possible to quantitatively detect OTA, with a linear range of 0.01-100 ng/mL and a detection limit of 0.005 ng/mL. The method for OTA detection has the advantages of high sensitivity, high speed and good simplicity and is applied to detecting beer samples, with accurate and reliable results.
Description
Technical field
A kind of based on near-infrared up-conversion luminescence labelling and the Determination Methods for Ochratoxin A of Magneto separate, relate to nanometer material
Material and technical field of analytical chemistry, for detecting ochratoxin A in food.
Background technology
Ochratoxin A (Ochratoxins A, OTA) mainly by pure green cyan mould (Penicillium Verrucosum),
Aspergillus ochraceus (Aspergillus achraceus) and three kinds of funguses of carbon black aspergillosis (A.carbonarius) produce, and much other produce
Toxic bacterial strain is the most constantly in the news.OTA main Pollution Plant product, such as corn, beans, Semen arachidis hypogaeae, dried Fructus Vitis viniferae, coffee, beer
Wine and red wine, much report display, there is also OTA in Some Animals product, this is because food chain cumulative action, animal is taken the photograph
Food part is caused OTA to be enriched with in animal body by the plant product that OTA pollutes.OTA has stronger liver, kidney and nerve
Toxicity and teratogenesis, carcinogenic, mutagenic action, to human body is healthy and food safety causes the biggest harm.Therefore, standard is set up
Really, OTA detection technique sensitive, quick is significant for food safety.
The main method of detection OTA has thin layer chromatography (TLC), high performance liquid chromatography (HPLC), liquid chromatograph at present
Matter combination method (LC-MS/MS), enzyme linked immunosorbent assay (ELISA) and highly-pathogenic avian influenza method (GICA) etc..ELISA
It is based primarily upon antigen-antibody compatible reaction, is usually used in OTA field quick detection and batch samples screening.But antibody is easily by the external world
Condition affects, especially temperature, and the preparation of antibody needs through animal or cell experiment, loaded down with trivial details time-consuming, relatively costly；
Analysis method based on instrument then needs substantial amounts of manpower and financial resources to put into, and is usually served only for lab analysis.So exploitation
A kind of quick and convenient, good stability, highly sensitive, high specificity, novel detection method with low cost are necessary.
Aptamer (Aptamer) is by index concentration Fas lignand system evolution (systematic evolution of
Ligands by exponential enrichment, SELEX) technology obtains from in-vitro screening, and can be single-minded with respective ligand
The class single strand oligonucleotide acid sequence that property is combined closely.It is the most right that the higher structure that this oligonucleotide sequence is formed is capable of identify that
The target substances such as any kind of albumen answered and low molecule, and with target substance, there is high affinity.Compared with antibody, aptamers
Target molecule scope wide, in-vitro screening, easy synthetic and modification, molecule is less, good stability, to temperature-insensitive, easily
Preserve, and aptamers is as identifying that molecule is at clinical diagnosis, clinical treatment, medicament transport, proteome research and food
Product safety is used widely.
In recent years, novel nano-material fast development in analyzing detection is by the green grass or young crops of increasing researcher
Look at, and emerged the nano material much with excellent characteristic.Wherein up-conversion luminescence nanomaterial causes the optics of uniqueness
Characteristic is known.
The luminous mechanism of up-conversion luminescence nanomaterial (Upconversion Nanoparticles, UCNPs) be based on
Two-photon or multiphoton processes, be a kind of effective way that infrared light is transformed into visible ray.Organic glimmering relative to traditional
Photoinitiator dye and other fluorescent nano materials, up-conversion luminescence nanomaterial has the biggest as completely inert phosphor
Advantage, spectrochemical property is stable, and monochromaticity is strong, has longer luminescent lifetime, can select different host materials and mix
Heteroion regulates up-conversion luminescence, and the real list excitation multi that realizes is penetrated, and beneficially in living things system, multicomponent labelling is examined simultaneously
Survey.In view of above-mentioned advantage, up-conversion luminescence nanomaterial has become a kind of outstanding biological labled material and has been widely used in life
Thing monitoring analysis field.
First the present invention has synthesized the near-infrared up-conversion luminescence nanomaterial having strong emission peak at 804nm, secondly uses
One-step synthesis is prepared for amination Fe3O4Magnetic Nano material, and modify in near-infrared up-conversion luminescence nanomaterial respectively
OTA aptamers and on Fe3O4 magnetic Nano material modification of complementarity oligonucleotide chain, separately constitute signal probe and magnetic and visit
Pin, based on both base pair complementarity principles so that two kinds of probes form near-infrared up-conversion luminescence nanomaterial-magnetic Nano
Material nano complex, luminous intensity now reaches maximum.When adding different amounts of OTA, OTA carries out special with aptamers
Property combine so that duplex structure dissociates, and part near-infrared up-conversion luminescence nanomaterial comes off from magnetic Nano material surface, closely
Infrared up conversion luminous intensity weakens, and detects signal using 980nm laser as excitation source record near-infrared up-conversion luminescence, logical
Cross the detection to variable concentrations ochratoxin A standard substance, Criterion curve, reach to carry out containing ochratoxin A sample
The purpose of detection by quantitative.This invention may be used for ochratoxin A in the samples such as medicated beer, Semen Maydis, corn, feedstuff and goods thereof and contains
The detection of amount.
Summary of the invention:
A kind of based on near-infrared up-conversion luminescence labelling and the Determination Methods for Ochratoxin A of Magneto separate: by NaYF4:
Yb0.2,Tm0.02Near-infrared up-conversion luminescence nanomaterial and Fe3O4Magnetic Nano material is respectively by EDC/NHS reaction and penta 2
Aldehyde cross-linking method coupling Avidin, utilizes the high-affinity between Avidin and biotin, respectively couple biotin OTA aptamers
And complementary strand, make bi-material form nano-complex based on hybridization, now nano-complex has the luminous letter of maximum
Number.After adding object OTA in this complex systems, owing to OTA aptamers is combined with the specific recognition of OTA, OTA fits
Part preferentially combines target OTA, causes dissociating of OTA aptamers and complementary strand, causes near-infrared up-conversion luminescence nanomaterial
With the separation of magnetic Nano material, by externally-applied magnetic field effect, collect near-infrared up-conversion luminescence in the nano-complex obtained
Nano material reduces, and luminous signal now will weaken accordingly, according to adding target concentration and luminous signal Strength Changes
Relation between amount, it is possible to achieve the detection by quantitative of OTA；Step is:
(1) by high temperature pyrolysis law technology, NaYF is prepared4:Yb0.2,Tm0.02Near-infrared up-conversion luminescence nanomaterial, and
It is done surface modify.Weigh YCl3·6H2O, YbCl3·6H2O, TmCl3·6H2O (Ln:78mol%Y3+, 20mol%Yb3+,
2mol%Tm3+；1mmol altogether) join in 100mL there-necked flask, add 6mL oleic acid and 15mL 1-octadecylene.In stirring
Under, it is gradually heating to 160 DEG C, after homogeneous solution to be formed, naturally cools to room temperature.Accurately weigh 2.5mmol NaOH and
4mmol NH4F is dissolved in 10mL methanol solution.Above-mentioned solution is slowly added in there-necked flask, magnetic agitation 30min.Again
Solution is slowly heated under removal methanol, argon shield and is heated to 300 DEG C, and continue 1h.After reaction terminates, naturally cool to
Room temperature, is centrifuged resulting materials with water and ethanol and cleans three times, store for future use.
(2) utilize ligand exchange method that up-conversion luminescence nano-particle is carried out surface carboxyl groups modification.Weigh 30mg oleic acid
Modify NaYF4:Yb0.2,Tm0.02UCNPs is scattered in 4mL chloroformic solution.In round-bottomed flask, add 200mg polyacrylic acid (phase
To molecular weight 2000) and 8mL ultra-pure water, stir and fully dissolve.UCNPs chloroformic solution is slowly added into round-bottomed flask
In, after magnetic agitation 24h, centrifugal collection material, cleans for several times with ultra-pure water, obtains polyacrylic acid and modify NaYF4:Yb0.2,
Tm0.02Near-infrared up-conversion luminescence nanomaterial, stores for future use.
(3) a step solvent method is used to prepare amination Fe3O4Magnetic Nano material.Weigh 2.0g anhydrous sodium acetate, 1.0g
FeCl3·6H2O and 6.5g 1,6-hexamethylene diamine adds in 30mL ethylene glycol, is heated to 50 DEG C under magnetic agitation, to be formed bright all
After the colloid solution of one, transfer in the reactor of band liner, under 198 DEG C of parts, react 6h.After the cooling of question response still, by still
Liquid is transferred in beaker, and Magnet separates and collects black solid, supernatant discarded, repeatedly cleans several times.Gained black solid is 60
Under the conditions of DEG C, dried overnight is standby.
(4) utilize EDC/NHS method by carboxylated up-conversion luminescence nanomaterial and Avidin (Avidin) coupling.Weigh
The NaYF that 10mg polyacrylic acid is modified4:Yb0.2,Tm0.02Near-infrared up-conversion luminescence nanomaterial is scattered in 5mL 10mmol/L
In MES (pH 5.6) buffer, abundant ultrasonic disperse, adds 0.6mL 2mg/mL EDC solution and 0.2mL 2mg/mL NHS is molten
Liquid, activates 2h in 37 DEG C of shaking tables.Centrifugal collection material, cleans three times with phosphate buffer.It is then dispersed in phosphate buffer,
Add 1mL 0.5mg/mL Avidin solution, 37 DEG C of shaking tables react 12h.Centrifugal collection material and supernatant, measure Avidin
Uv absorption before and after reaction.
(5) utilize glutaraldehyde method by amination Fe3O4Magnetic Nano material and Avidin coupling.Accurately weigh 10mg amino
Change Fe3O4Magnetic Nano material is scattered in 5mL 10mmol/L phosphate buffer, and abundant ultrasonic disperse is subsequently adding 1.25mL
25% glutaraldehyde solution, hatches 2h for 37 DEG C, after Magneto separate, cleans for several times with phosphoric acid, after Magneto separate is collected, is scattered in phosphoric acid buffer
In liquid, adding 1mL 0.5mg/mL Avidin solution, after reacting 12h in 37 DEG C of shaking tables, Magneto separate is collected.
(6) utilize and by specific binding between Avidin (Avidin) and biotin (Biotin), surface is modified close
The Fe modified with near-infrared up-conversion luminescence nano-particle and the Avidin of element3O4Magnetic Nano material respectively with biotin
(Biotin) the ochratoxin A aptamers DNA strand modified and complementary strand connect.Method particularly includes: Avidin is modified
NaYF4:Yb0.2,Tm0.02Near-infrared up-conversion luminescence nanomaterial is scattered in phosphate buffer, adds a certain amount of biotin
Change OTA aptamers, in 37 DEG C of shaking tables, hatch 12h, centrifugal collection material and supernatant, clean three times with phosphate buffer, will be
After near-infrared up-conversion luminescence signal probe (apt-UCNPs) that obtains be scattered in BB buffer (10mM Tris, pH8.5,
120mM NaCl, 5mM KCl and 20mM CaCl2In).
The Fe that Avidin is modified3O4Magnetic Nano material is scattered in 5mL phosphate buffer, adds a certain amount of biology
Elementization OTA aptamers complementary strand, hatches 12h, Magneto separate collection material in 37 DEG C of shaking tables, cleans for several times with phosphate buffer,
By the complementary strand finally obtained modify magnetic Nano probe (cDNA-MNPs) be scattered in BB buffer (10mmol/L Tris,
PH 8.5,120mmol/L NaCl, 5mmol/L KCl and 20mmol/L CaCl2In), 4 DEG C of preservations.
(7) ochratoxin A standard substance are detected, Criterion curve.By ochratoxin A aptamers DNA
It is complementary to the hybridization of DNA strand, takes 75 μ L signal probes and 80 μ L magnetic probes 37 DEG C of reaction 2h in BB buffer, obtain
Near-infrared up-conversion luminescence nanomaterial-magnetic Nano material complex, Magneto separate collects complex, with BB buffer solution for cleaning number
Secondary, it is ensured that free near-infrared up-conversion luminescence nanomaterial completely removes, and is resuspended in BB buffer, swash at 980nm laser
Give the up-conversion luminescence signal obtained at 804nm, the now signal (I of complex0) maximum.The OTA mark of preparation variable concentrations
Quasi-product join in nanocomposite system, and after 37 DEG C of reaction 2h, external magnetic field separates lower cleaning for several times, it is ensured that split away off
UCNPs wash away completely.In fluorescence spectrophotometer, under 980nm laser excitation, measure luminous intensity, along with ochratoxin A
The increase luminous signal (I) of concentration progressively reduces.According to luminous difference (△ I=I-I0) with corresponding ochratoxin A standard
Product concentration Criterion curve, experimental result obtains good linear relation in 0.01-100ng/mL interval.
(8) ochratoxin A sample is detected: sample is done simple process, is added directly into above-mentioned receiving subsequently
Rice complex systems in 37 DEG C hatch 2h after, under 980nm laser, directly excite the up-conversion luminescence signal obtained at 804nm,
The concentration of the ochratoxin A of correspondence is tried to achieve from standard curve.
The invention have the advantage that
(1) utilize aptamers that tested substance realizes specificity capture, be effectively increased the stability of detection with accurate
Property.
(2) utilize aptamers compared with antibody, have can synthetic, be independent of animal and cell, the cycle is short, cost
Between low, batch, difference is little, it is simple to chemical modification, stability might as well, can preserve for a long time.
(3) utilize induced with laser up-conversion luminescence, detect the low sensitivity substantially increasing detection of background.
(4) a step solvent method is utilized to prepare Fe3O4Magnetic Nano material uses it for analyte detection process, can simplify point
From process, improve detection efficiency.
Accompanying drawing explanation
Fig. 1: the experiment of the Determination Methods for Ochratoxin A being based on near-infrared up-conversion luminescence labelling and Magneto separate is former
Reason figure
Fig. 2: NaYF4:Yb0.2,Tm0.02Electronic Speculum figure (a) of near-infrared up-conversion luminescence nanomaterial；Polyacrylic acid is modified
NaYF4:Yb0.2,Tm0.02Electronic Speculum figure (b) of near-infrared up-conversion luminescence nanomaterial
Fig. 3: amination Fe3O4The Electronic Speculum figure of magnetic Nano material
Fig. 4: near-infrared NaYF4:Yb0.2,Tm0.02The XRD figure of up-conversion luminescence nanomaterial
Fig. 5: amination Fe3O4The XRD figure of magnetic Nano material
Fig. 6: NaYF4:Yb0.2,Tm0.02The luminescent spectrum of near-infrared up-conversion luminescence nanomaterial
Fig. 7: near-infrared Up-conversion Intensity changes stacking chart (a) with ochratoxin A；Ochratoxin A detection mark
Directrix curve figure (b), concentration range is at 0.01-100ng/mL.
Detailed description of the invention
The present invention includes but not limited to above example, every any equivalent carried out under the spirit and principles in the present invention
Replace or local improvement, all will be regarded as within protection scope of the present invention.
Embodiment 1: in medicated beer actual sample, ochratoxin A examination criteria curve is set up and detection sample pretreatment: beer
Wine sample is placed in 4 DEG C of refrigerator cold-storage 30min, ultrasonic degassing.Take beer sample 20g after degassing, be placed in 25mL volumetric flask, add
2% sodium bicarbonate and 15% sodium chloride mixed extract, to scale, mixing, are filtered until clear by glass fiber filter paper, collect filter
Liquid is standby.
Buy medicated beer different classes of 5 from local supermarket, utilize the inventive method and enzyme-linked immunoassay method to measure respectively
Wherein the content of ochratoxin A, the results are shown in Table one.Two kinds of method testing results are consistent, no significant difference.
Table one: medicated beer actual sample detects, the inventive method contrasts with ELISA method
Note: ND is not for detect
Embodiment 2: in medicated beer actual sample, detection and the recovery of standard addition laboratory sample pretreatment of ochratoxin A are same real
Execute example 1.
The 5 groups of ochratoxin A concentration datas obtained with embodiment 1, as background values, are added thereto to five kinds of differences respectively
The OTA standard substance of concentration, again detect the content of wherein OTA, obtain detected value also with the inventive method.Response rate %=
(detected value-background values)/addition × 100%.From table two data it can be seen that the response rate is 90.0%～105.0%, explanation
The present invention is stable, sensitive, accurately, it is adaptable to the detection of OTA in medicated beer actual sample.
Table two: the detection of ochratoxin A and recovery of standard addition in medicated beer actual sample
Claims (1)
1. one kind based on near-infrared up-conversion luminescence labelling and the Determination Methods for Ochratoxin A of Magneto separate, it is characterised in that: will
Ochratoxin A (OTA) aptamers and near-infrared NaYF4:Yb0.2,Tm0.02Up-conversion luminescence nanomaterial coupling forms signal and visits
Pin, simultaneously biotinylation OTA aptamers complementary oligonucleotide strand and Fe3O4Magnetic Nano material connects formation magnetic probe, logical
Cross double-strand hybridization and form NaYF4:Yb0.2,Tm0.02Near-infrared up-conversion luminescence nanomaterial Fe3O4Magnetic Nano material is combined
Thing；In detection system, add OTA, OTA can preferentially be combined with aptamers, cause complementary strand to dissociate with OTA aptamers, so that
A part of NaYF4:Yb0.2,Tm0.02Near-infrared up-conversion luminescence nanomaterial Fe3O4Magnetic Nano material complex decomposes, and fills
After dividing Magneto separate to remove supernatant, utilize 980nm to excite, collect up-conversion luminescence luminous signal at 804nm；At 0.01-
In 100ng/mL concentration range, at the concentration of OTA and 804nm, up-conversion luminescence signal intensity is proportionate, Criterion curve,
Reach the detection by quantitative to OTA, can be used for the detection of OTA in medicated beer.
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| CN107607722A (en) * | 2017-07-21 | 2018-01-19 | 南京医科大学 | A kind of method for quantitatively detecting beta lactoglobulin in milk powder |
| CN108132235A (en) * | 2018-02-02 | 2018-06-08 | 首都师范大学 | A kind of method of fluorinion concentration in fluoroscopic examination solution |
| CN108918863A (en) * | 2018-05-23 | 2018-11-30 | 江南大学 | A kind of preparation method of the upper conversion aptamers test strips quickly detected for ochratoxin A |
| CN112129732A (en) * | 2020-08-13 | 2020-12-25 | 江苏大学 | Method for rapidly detecting bacillus cereus based on up-conversion magnetic separation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107607722A (en) * | 2017-07-21 | 2018-01-19 | 南京医科大学 | A kind of method for quantitatively detecting beta lactoglobulin in milk powder |
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| CN108918863A (en) * | 2018-05-23 | 2018-11-30 | 江南大学 | A kind of preparation method of the upper conversion aptamers test strips quickly detected for ochratoxin A |
| CN112129732A (en) * | 2020-08-13 | 2020-12-25 | 江苏大学 | Method for rapidly detecting bacillus cereus based on up-conversion magnetic separation |
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Application publication date: 20161026 |