CN106053439B - A kind of flower-like nanometer gold based on ABEI modification detects terramycin, the method for tetracycline and kanamycins simultaneously - Google Patents
A kind of flower-like nanometer gold based on ABEI modification detects terramycin, the method for tetracycline and kanamycins simultaneously Download PDFInfo
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Abstract
A kind of flower-like nanometer gold based on ABEI modification detects terramycin, the method for tetracycline and kanamycins simultaneously.It is characterized by: based on N- constructed by microwell plate (4- ammonia butyl)-N- ethyl different luminol (ABEI)-H2O2Iodophenol (PIP) steadystate chemical luminescent system can effectively be extended the chemiluminescence time.The flower-like nanometer gold of sulfhydrylation complementation chain link ABEI modification is used for signal probe.The aptamers being fixed on microwell plate are used as molecular recognition elements, and the aptamers of three kinds of antibiotic are separately fixed at three regions of microwell plate to detect the resolution provided spatially while target.Detection while to terramycin, tetracycline and kanamycins may be implemented in constructed aptamer sensor, and in certain concentration range, chemiluminescence intensity is related in negative logarithm to the amount of target.In addition, the sensor is also used for actual sample detection, it was demonstrated that the excellent performance of sensor.
Description
Technical field
Flower-like nanometer gold and microwell plate of the one kind based on the modification of N- (4- ammonia butyl)-N- ethyl different luminol (ABEI) are simultaneously
Terramycin is detected, the method for three kinds of antibiotic of tetracycline and kanamycins is related to nano material and technical field of analytical chemistry, uses
The terramycin in food, tetracycline and kanamycins are detected.
Background technique
Terramycin and tetracycline belong to tetracycline antibiotics, and kanamycins belongs to aminoglycoside antibiotics, they are all
Belong to spectrum antibiotic, and is widely applied in animal husbandry.But the abuse of these antibiotic can not only cause animal itself
Damage, can also have a adverse impact to the mankind, because antibiotic can be enriched in food chain transmittance process.These side effect packets
Damage hearing is included, kidney is damaged, causes allergic reaction and induce the animal of the mankind to develop drug resistance, furthermore abuse of antibiotics
Undesirable effect can be generated to environment.Therefore European Union has formulated antibiotic stringent limit standard, and standard regulation soil is mould
Element, the maximum residue limit of tetracycline and kanamycins in milk are respectively 100 μ g/kg, 100 μ g/kg and 150 μ g/kg.I.e.
Make in this way, there are still the exceeded situations of antibiotic residue in food, and often multiple antibiotic residues and deposit.Therefore,
Establishing a kind of is quickly vital with high-throughput detection method.
According to it has been reported that food in antibiotic residue high-flux detection method.So far, it can be realized to more
There are mainly two types of the modes that component detects simultaneously, and one is multi-color marking methods, and one is spatial resolutions.For multi-color marking mould
For formula, one of part and parcel is exactly the nano material of synthesis such as multicolor fluorescence label, color quantum point, time point
Distinguish nano material, up-conversion nano material etc..And the nano material for synthesizing multi-color marking is not a simple job, and
The difficulty of detection method has been significantly greatly increased.In addition, increasing marker or label during synthesizing the nano material of multi-color marking
The quantity of signal be it is relatively difficult to achieve, this largely constrains the type of target in multi-analyte immunoassay.However, based on sky
Between the detection pattern differentiated a kind of general marker can be used and realize multi-component detection by the segmentation in space,
There may be wider application in multi-analyte immunoassay.
Aptamers are one section of single stranded DNA or RNA with three-D space structure, and specificity knot can occurs in it with target
It closes.Therefore, in biosensor, aptamers are used as a kind of excellent molecular recognition elements.Aptamers are in many sides
The good characteristic of method can compare favourably with antibody.Also, aptamers possess more more excellent than antibody in terms of bio-sensing
Characteristic.Firstly, aptamers are to design and screen in vitro, therefore, in principle, any target can have its corresponding adaptation
Body, secondly, aptamers have more excellent characteristic in terms of signal transduction and chemical modification.In recent years, chemiluminescence method
It is had a wide range of applications in terms of food safety detection.It is well known that chemiluminescent realize depends on chemical illuminating reagent, this
A little chemical illuminating reagents usually require additional markers and are just able to achieve chemiluminescent detection into detection architecture.However, with receiving
The continuous development of rice material, the nano material of chemical illuminating reagent label are thin to more and more concerns.Our seminars it
In a preceding report, we have successfully synthesized the flower-like nanometer gold of ABEI modification, and successfully construct ABEI-H2O2-
PIP steadystate chemical luminescence system.
The present invention is based on the spatial discrimination modes of microwell plate, construct a kind of novel multicomponent chemiluminescence detection system,
And successfully realize detection while to three kinds of antibiotic.Building and ABEI based on steadystate chemical luminescence system are modified flower-shaped
The synthesis of nanogold, to provide possibility using luminol type chemiluminescence on microwell plate.The constructed aptamers of invention
Sensor can using the easy modified for the spatial discrimination performance and nano material that microwell plate carries, be more suitable for it is high-throughput and
Multi-analyte immunoassay.
Summary of the invention:
Flower-like nanometer gold and microwell plate of the one kind based on the modification of N- (4- ammonia butyl)-N- ethyl different luminol (ABEI) are simultaneously
Detect terramycin, the method for three kinds of antibiotic of tetracycline and kanamycins.It is characterized by: based on constructed by microwell plate
ABEI-H2O21500s. sulfydryl can effectively be extended to by the chemiluminescence time to iodophenol (PIP) steadystate chemical luminescent system
The flower-like nanometer gold (ABEI-AuNFs) for changing complementary strand (cDNA) connection ABEI modification is used for signal probe.It is fixed on microwell plate
On aptamers be used as molecular recognition elements, be by three regions that the aptamers of three kinds of antibiotic are separately fixed at microwell plate
Detection provides resolution spatially while target.Aptamers first with signal probe by base pair complementarity in conjunction with,
In the presence of target, aptamers can be tended in conjunction with target lead to chemistry so as to cause signal probe partial exfoliation
The variation of luminous signal intensity.Therefore constructed aptamer sensor may be implemented to terramycin, tetracycline and kanamycins
While detect, in certain concentration range, chemiluminescence intensity and the amount of target are related in negative logarithm.In addition, also will
The sensor is detected for actual sample, and testing result is similar with the commercialization obtained result of ELISA detection method, it was demonstrated that
The excellent performance of the sensor.Constructed detection method has many advantages, such as that easy to operate, selectivity and specificity are high, in food
There is biggish application potential in terms of safety testing field especially multi-analyte immunoassay.Detecting step are as follows:
(1) preparation of ABEI-AuNFs nano material.Firstly, 0.075g chitosan is dissolved in 25mL glacial acetic acid solution
(v/v), then 1.5mL ABEI stock solution and 42mL water are added in above-mentioned solution, are heated to boiling with vigorous stirring.
8mL gold chloride stock solution is added dropwise, fluidized state 2h is kept.Later by material natural cooling at room temperature, it is stored in 4 DEG C
It is spare.
(2) preparation of ABEI-AuNFs signal probe.The material for taking 5mL to prepare is centrifuged 10min under 10000rpm, and
1.5mL is resuspended in, in 30mM Tris-HCl ().CDNA and three (2- carboxyethyl) phosphines (TCEP) (final concentration 100nM) is preparatory
In 25 DEG C of incubation 30min.This mixed liquor and BSA (final concentration 1%) are jointly added in above-mentioned ABEI-AuNFs solution, room temperature
Lower incubation 20h.Above-mentioned mixed liquor is centrifuged to 10min under 8000rpm and obtains the ABEI-AuNFs signal probe spy of cDNA modification
Needle.
(3) preparation of the coated microwell plate of aptamers.As follows by terramycin, tetracycline, card receive mycin adaptation
Body is coated on the different zones of microwell plate.Firstly, 100 μ L 0.02mg/mL streptavidins is taken to be added in microwell plate, and
It is dried for standby at 37 DEG C.Binding site extra on microwell plate is closed with 0.05%BSA.Microwell plate is divided into 3 areas
Domain.The 100 biotinylated aptamers of μ L are added in corresponding 3 regions, are incubated for 1h at 37 DEG C, with PB-T board-washing 3 times,
And drying for standby in air.
(4) based on the ABEI-H of microwell plate2O2Building to iodophenol (PIP) steadystate chemical luminescent system.It is fixed on micro-
Aptamers DNA on orifice plate is complementary the single-stranded hybridization of DNA, signal probe is fixed on micropore plate surface, then to micropore
150 μ L of steadystate chemical luminescence buffer is added in plate, buffer group becomes, 0.2mol/L H2O2,0.05mol/L PIP,
0.00095mol/L Na2HPO4·2H2O,0.00405mol/L NaH2PO4·12H2O and 0.1mol/L NaOH。
(5) mycin is received to terramycin, tetracycline, card to detect, establish standard curve.To the coated microwell plate of aptamers
The corresponding ABEI-AuNFs signal probe of 100 μ L of middle addition, is incubated for 40min at 37 DEG C, will be different after PB-T board-washing 3 times
Concentration terramycin, tetracycline and Ka Na mycin are added in corresponding microwell plate, are incubated for 50min at 37 DEG C, with PB-T board-washing 3
The detection buffer of 150mL is added after secondary.Luminous intensity is measured after 30min.It is cross with the negative logarithm of the antibiotic of various concentration
Coordinate establishes standard working curve using chemiluminescence intensity as ordinate.With the increase of target concentration, chemiluminescence intensity
It reduces, the range of linearity of OTC working curve is 0.05-5ng/mL (Y=-123.688+30.308*X)；The property range of TET is
0.05-5ng/mL (Y=-21.271+22.957*X)；The property range of kanamycins is 0.005-1ng/mL (Y=-74.064+
27.089*X), their minimum detection limit is respectively as follows: 0.02/0.02/0.002ng/mL.
(6) terramycin, tetracycline and kanamycins sample are detected: by various concentration terramycin, tetracycline, Ka Na
Mycin is added in milk sample, dilutes 40 times.Chemiluminescence intensity then is obtained according to operating procedure in (5), from standard song
The concentration of corresponding target is acquired in line.
The invention has the advantages that
(1) specificity capture is realized to tested substance using aptamers, effectively increases the stability of detection and accurate
Property.
(2) using aptamers compared with antibody, having can be artificial synthesized, does not depend on animal and cell, and the period is short, cost
Difference is small between low, batch, is convenient for chemical modification, stability can long-term preservation.
(3) the steadystate chemical luminescence system based on microwell plate is constructed using the flower-like nanometer gold of ABEI modification.Stable state system
Building the chemiluminescence detection time can effectively be extended to 1500s, avoid instantaneous light emission measurement generate error.
(4) using the ABEI-AuNFs of complementary chain link as a universal tag, the spatial discrimination based on microwell plate is special
Point constructs the high-flux detection method of a kind of pair of terramycin, tetracycline and kanamycins while detection.
Detailed description of the invention
Fig. 1: flower-like nanometer gold and microwell plate based on the modification of N- (4- ammonia butyl)-N- ethyl different luminol (ABEI) are simultaneously
Detect terramycin, the detection principle diagram of tetracycline and kanamycins.
The flower-like nanometer gold electron microscope (A) and power spectrum phenogram (B) of Fig. 2: ABEI modification
Fig. 3: ABEI-AuNFs-H2O2-PIP steady generation figure
Fig. 4: target detection canonical plotting (a- terramycin, b- tetracycline, c- kanamycins)
Specific embodiment
The present invention includes but is not limited to above embodiments, it is all carried out under the spirit and principles in the present invention it is any equivalent
Replacement or local improvement, all will be regarded as within protection scope of the present invention.
Embodiment 1: terramycin in milk actual sample, tetracycline and kanamycins detection.Test sample pretreatment:: it is first
Various concentration terramycin, tetracycline, card are first received mycin to be added in milk sample, and dilute 40 times with ELISA treatment fluid.From
Local supermarket buys the milk of 3 kinds of different brands, measures wherein Aspergillus ochraceus respectively using the method for the present invention and enzyme-linked immunoassay method
The content of toxin A, the results are shown in Table one.Two methods testing result is consistent, no significant difference.
Table one: the detection of milk actual sample, the method for the present invention and ELISA method compare
Note: ND is to be not detected
Embodiment 2: the detection of terramycin in milk actual sample, tetracycline and kanamycins and recovery of standard addition test sample
Product are pre-processed with embodiment 1.The 3 groups of terramycin obtained with embodiment 1, tetracycline and kanamycins concentration data are background values,
The antibiotic standard items of three kinds of various concentrations are added thereto respectively, detects also with the method for the present invention and is wherein added again
Antibiotic content, obtain detected value.Rate of recovery %=(detected value-background values)/additive amount X100%.It can be with from two data of table
See that the rate of recovery 96.04%~102.66%, illustrates stabilization of the present invention, it is sensitive, it is accurately, native suitable for milk actual sample
The detection of mycin, tetracycline and kanamycins.
Table two: terramycin, the detection of tetracycline and kanamycins and recovery of standard addition in milk actual sample
Note: ND is to be not detected
Claims (3)
1. a kind of flower-like nanometer gold based on ABEI modification detects terramycin simultaneously, the method for tetracycline and kanamycins is special
Sign is: the different zones being first respectively separately fixed at the aptamers of terramycin, tetracycline and kanamycins on microwell plate；
Again by ABEI modify flower-like nanometer gold chemiluminescence probe be fixed on microwell plate by base pair complementarity, building be based on pair
Iodophenol (PIP)-hydrogen peroxide (H2O2) steadystate chemical luminescent system；The corresponding addition of certain density three kinds of antibiotic is micro-
Three regions of orifice plate measure chemiluminescence signal after board-washing, it can be achieved that chemiluminescence detection while to three kinds of antibiotic.
2. a kind of flower-like nanometer gold based on ABEI modification as described in claim 1 detects terramycin, tetracycline and card simultaneously
The method of that mycin, it is characterised in that: the flower-like nanometer gold of ABEI modification synthesizes by the following method to be obtained, and 0.075g shell is gathered
Sugar is dissolved in 25mL glacial acetic acid solution (2%, v/v), and then 1.5mLABEI stock solution and 2mL water are added in above-mentioned solution,
It is heated to boiling with vigorous stirring, 8mL gold chloride stock solution is added dropwise, keep fluidized state 2h, material exists later
Natural cooling at room temperature, be stored in 4 DEG C it is spare；The flower-like nanometer Au probe of ABEI modification is by by BSA and TCEP and sulfhydrylation
Complementary strand is added in the flower-like nanometer gold of ABEI modification simultaneously and is prepared, and wherein final concentration of 1%, the TCEP of BSA is final concentration of
100nM is incubated for 20h at room temperature, obtains the flower-like nanometer gold signal probe of ABEI modification.
3. a kind of flower-like nanometer gold based on ABEI modification as described in claim 1 detects terramycin, tetracycline and card simultaneously
The method of that mycin, it is characterised in that: by specific buffer (0.00095M Na2HPO4·2H2O,0.00405M
NaH2PO4·12H2O and 0.1M NaOH) in be added 0.05M PIP and 0.2M H2O2It, can structure as steadystate chemical luminescence buffer
The steadystate chemical luminescent system for building the flower-like nanometer gold signal probe based on ABEI modification is multicomponent chemical luminescent detection system
Building provide premise and basis.
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| CN107290526A (en) * | 2017-08-24 | 2017-10-24 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of tetracycline and preparation method thereof |
| CN114397294B (en) * | 2022-01-13 | 2023-12-29 | 东南大学 | Preparation method of tetracycline sensor based on chitosan nanofiber membrane |
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