CN104833716A - Preparation method of estradiol magnetic molecularly imprinted nanoparticle electrochemical sensing membrane - Google Patents

Preparation method of estradiol magnetic molecularly imprinted nanoparticle electrochemical sensing membrane Download PDF

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CN104833716A
CN104833716A CN201510185802.9A CN201510185802A CN104833716A CN 104833716 A CN104833716 A CN 104833716A CN 201510185802 A CN201510185802 A CN 201510185802A CN 104833716 A CN104833716 A CN 104833716A
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nanoparticle
solution
magnetic
preparation
estradiol
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CN104833716B (en
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周学敏
姜慧君
沈心
韩青
徐磊
段白云
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Nanjing University
Nanjing Medical University
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Nanjing Medical University
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Abstract

The invention discloses a preparation method of an estradiol magnetic molecularly imprinted nanoparticle electrochemical sensing membrane. According to the preparation method, an estradiol magnetic molecularly imprinted polymer is obtained via preparation of a molecularly imprinted self-assembled solution, polymerization, and template molecule elution. The estradiol magnetic molecularly imprinted nanoparticle electrochemical sensing membrane is high in selectivity, response speed, sensitivity, stability, tolerability, and universality, and is capable of realizing high efficiency, sensitive, real-time detection of estradiol in composite matrix.

Description

A kind of preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film
Technical field
The invention belongs to technical field of analysis and detection, be specifically related to a kind of preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film (MMIES).
Background technology
17-β estradiol (E2), as one of incretion interferent, there are some researches show children's sexual precocity, prostate cancer, and the rising of women breast cancer and the cancer of the uterus incidence of disease all remains relevant with the estrogen in food.In addition, it also can affect the physiological law of women, even can cause infertile.Other estradiol of trace level just can cause the imbalance of humoral immunity and cellular immunity, causes immune, the cardiovascular and neural pathology of reproduction.But due to reasons such as the residual and illegal interpolations in estrogen production run, often cause the problem such as environmental pollution and food security to occur.In order to ensure that consumers in general's is healthy, specification food products market, setting up a reliable estrogen detection method is fast very necessary and urgent problems.
Electrochemical sensor is a kind of important electron device, because of its simplicity of design, highly sensitive, cheap, human body can be realized detect in real time, the advantages such as clinical diagnosis, environmental analysis, food analysis and Product checking needs can be met, more and more receive the concern of people.Molecular imprinting is the technology that preparation has specific selectivity or single-minded selective polymerisation thing.Its most significant three large feature is: structure effect precordainment, specific recognition and broad applicability.In current magnetic molecule marking nano sensing film and Electrochemical Detection complex system, estradiol correlative study has no report.
Food, health products are due to the complicacy of self component, and the problem such as the diversity of additive types and non-intellectual, detect have higher requirement to its analysis containing estradiol.The detection method of existing bibliographical information has: HPLC, LC-MS, GC-MS etc.Although these traditional methods also can realize the mensuration to estradiol, often need through loaded down with trivial details sample pre-treatments because sample substrate is complicated, this have impact on accuracy and the agility of object detection greatly.Recently popular research field is become based on the biology sensor of nano material and nanometer technology and electrochemical sensor because having quick, sensitive, stable feature.Particularly molecular imprinting electrochemical sensor (MIES) is because having high sensitivity and selectivity becomes a kind of galvanochemistry biomimetic sensor having prospect, has been widely used in identification and the Sensitive Detection of complex system microscratch amount object.MIES often needs complicated electrode modification and electrochemical polymerization sensing membrane forming process, and preparation and operative technique require high, are unfavorable for promotion and application.Although MIPs electropolymerization sensing membrane can repeatedly use, polishing electrode regeneration treatment time is long, and electrode utilization factor is low, and electrode abrasion cause serviceable life limited.In addition, MIPs structure has a significant effect to electrochemical response, and the MIPs building good electric conduction and catalytic activity plays an important role to raising MIES detection sensitivity.Therefore design and prepare electroactive structure MIPs, research has the strong high sensitivity of universality and selectivity MIES by significance.
Summary of the invention
The object of this invention is to provide a kind of preparation method of the magnetic molecule marking nano sensing film for estrogen Electrochemical Detection, it is the magnetic molecule marking nano electrochemical sensing membrane of representative that the advantage of this method combined with electrochemical sensing technology, magnetic absorption technology, molecular imprinting nanoparticle three is first constructed with estradiol, achieve and microscratch amount estradiol in complex matrices is detected fast, this method measures highly sensitive, strong to object selectivity, simple to operate, there is good universality.
Object of the present invention can be reached by following measures:
A kind of estradiol magnetic molecule marking nanoparticle electrochemical sensing membrane preparation method, is characterized in that the method comprises the following steps:
A) preparation of molecular imprinting self assembly solution: at Fe 3o 4add E2-ethanolic solution, stirring reaction in@Au-GSH nanoparticle, solution adds aniline-HCl solution after leading to nitrogen deoxygenation, lucifuge stirs;
B) polyreaction: add initiator ammonium persulfate (APS) in the solution that step a) obtains, lucifuge stirring reaction;
C) eluted template molecule: by step b) solution that obtains, magnetic resolution removes supernatant, and washing, adds the process of HCl-ethanolic solution, slough template molecule, obtain Fe 3o 4the magnetic molecularly imprinted nanoparticle of@Au-GSH-PANI (MMIPs);
D) enrichment template molecule: obtained MMIPs is placed in E2-PBS solution, stirring and adsorbing, obtains E2-MMIPs;
E) Electrochemical Detection: the solution containing E2-MMIPs is placed in reaction tank, with magnetic glassy carbon electrode (MGCE) magnetic absorption 5min ~ 1h, makes electrode surface film forming.
Step a) in, the Fe of every 5mg ~ 20mg 3o 4@Au-GSH nanoparticle, add the E2-ethanolic solution that 5mL ~ 50mL concentration range is 1mmol/L ~ 20mmol/L, the ethanol used is absolute ethyl alcohol, reaction time 1 ~ 20h.Before adding aniline-HCl solution, the time of logical nitrogen is 1 ~ 50min.
In aniline-HCl solution, the volume range of aniline and HCl is 1:10 ~ 20, and the concentration range of HCl is 0.05mol/L ~ 5mol/L.The acidity of solution increases, and electron transfer rate increases, and protonation is strengthened, and be conducive to the synthesis of polyaniline, but acidity is excessive, magnetic resolution difficulty can be made to strengthen, the MMIPs electric conductivity finally obtained weakens.In this experiment, the optimum molar concentration ratio of aniline and HCl is 1:2.5.
The temperature of reaction of step whole process a) is 5 ~ 50 DEG C.
In step b) in, ammonium persulfate adds with the form of ammonium persulfate solution, often containing the Fe of 5mg ~ 20mg 3o 4in the pre-assembled solution of molecular imprinting of@Au-GSH nanoparticle, add ammonium persulfate solution 0.1mL ~ 5mL that concentration range is 10mmol/L ~ 500mmol/L, polymerization reaction time is 2 ~ 20h, and temperature of reaction is 0 ~ 50 DEG C.Polymerization time affects the effect of the marking, and polymerization time is too short, and the response of Ip is very weak, and polymerization reaction time is long, and E2 embedded deeply, and the response of Ip also can reduce.Best polymerization reaction time in this experiment is 6h.
In step c) in, the concentration range of HCl-ethanolic solution is 0.05mol/L ~ 2.0mol/L, and mixing time is 5min ~ 2h, can process under 5 ~ 50 DEG C of temperature conditions.
In steps d) in, the addition mass range of MMIPs is 5mg ~ 50mg, E2-PBS concentration is 0.01 ~ 2.0mol/L, and mixing time is 5min ~ 5h.
Fe 3o 4the preparation of@Au-GSH nanoparticle comprises preparation Fe 3o 4magnetic nano particle, Fe 3o 4surface meets NH 2, preparation Fe 3o 4@Au nanoparticle, linkage function group sulfydryl step.
Preparation Fe 3o 4magnetic nano particle step is specific as follows:
Under nitrogen protection and magnetic agitation, by FeCl 36H 2o and FeCl 24H 2the mixed aqueous solution of O, after being heated to 20-80 DEG C, dropwise adds NH 3h 2o reacts 20 ~ 60min, and magnetic resolution removes supernatant, collects the washing of reaction precipitation thing, obtains Fe 3o 4magnetic nano particle.Wherein, FeCl 36H 2o and FeCl 24H 2the mol ratio of O is 3:1 ~ 1:1, FeCl 36H 2o is 0.05 ~ 0.1mmol/mL in the volumetric concentration of mixed aqueous solution, and every 100-300ml mixed aqueous solution adds NH 3h 2the volume range of O is 5mL ~ 20mL.
At Fe 3o 4surface meets NH 2step is specific as follows: get Fe 3o 4magnetic nano particle, adds APTES-ethanolic solution, and temperature is 0 ~ 80 DEG C of stirring reaction 1-48h, and magnetic resolution removes supernatant, adds ethanol purge and dissolves, and obtains APTES and wraps up Fe 3o 4nanoparticle.Wherein, the volume concentration range of APTES-ethanol is 0.1% ~ 5%, every 5-20mgFe 3o 4the volume range that magnetic nano particle adds is 5mL ~ 20mL.
Preparation Fe 3o 4@Au nanoparticle step is specific as follows: get 1-100mg APTES and wrap up Fe 3o 4nanoparticle, adds HAuCl4 aqueous solution and 1.4-140mL redistilled water that 0.1-10mL volumetric concentration is 1%-5%, and after ultrasonic disperse, continuing the ultrasonic concentration range that simultaneously adds is 2mmol/L ~ 20mmol/L NaBH 4until solution colour becomes purple potato look from tawny obtain Fe 3o 4@Au nanoparticle solution, temperature of reaction is 10 ~ 100 DEG C.At preparation Fe 3o 4in@Au nanoparticle step, NaBH 4make reductive agent in the reaction, gold ion is reduced into Jenner's grain of rice and is wrapped in Fe 3o 4surface, the NaBH of preparation 4solution concentration is unsuitable too high, and rate of addition should slow ultrasonic disperse simultaneously, otherwise reduction reaction can be made to carry out rapidly, and Jenner's grain of rice of generation can not be wrapped in Fe equably 3o 4surface.
At above-mentioned Fe 3o 4in@Au nanoparticle solution, add 0.5mL ~ 30mL 5mmol/L ~ 100mmol/L GSH, stirring reaction 1-48h, temperature of reaction is 5 ~ 50 DEG C.Magnetic resolution removes supernatant, and washing, obtains Fe 3o 4@Au/GSH nanoparticle.The GSH added also can be that other contain the amino acid of sulfydryl (-SH), as halfcystine.
The preparation method of above-mentioned estradiol magnetic molecule marking nanoparticle electrochemical sensing film, specifically can comprise the following steps:
1, the preparation of magnetic molecule marking nanoparticle electrochemical sensing film
A) Fe is prepared 3o 4nanoparticle: under nitrogen protection, heating FeCl 36H 2o and FeCl 24H 2the mixed aqueous solution of O, drips NH under the condition of vigorous stirring 3h 2o reacts, and reaction terminates rear magnetic resolution and removes supernatant, and water washing and precipitating thing obtains Fe 3o 4magnetic nano particle.
B) Fe 3o 4surface meets-NH 2: get Fe 3o 4nanoparticle, add APTES-ethanolic solution, stirring reaction, magnetic resolution removes supernatant, adds ethanol purge and dissolves, and obtains APTES and wraps up Fe 3o 4nanoparticle.
C) Fe is prepared 3o 4@Au nanoparticle: get step b) in the APTES that obtains wrap up Fe 3o 4nanoparticle, adds HAuCl 4with appropriate redistilled water, ultrasonic disperse, continue ultrasonicly to add NaBH4 until solution colour becomes purple potato look from tawny simultaneously;
D) linkage function group-sulfydryl: in step c) in the solution that obtains, then add GSH stirring reaction, magnetic resolution removes supernatant, washing; Obtain Fe 3o 4@Au-GSH nanoparticle.
E) molecular imprinting self assembly solution: by the Fe obtained 3o 4add E2-ethanolic solution, stirring reaction in@Au-GSH nanoparticle, solution adds aniline-HCl solution after leading to nitrogen deoxygenation, lucifuge stirs;
F) polyreaction: in step e) add initiator ammonium persulfate (APS), lucifuge stirring reaction in the solution that obtains;
G) eluted template molecule: by step f) solution that obtains, magnetic resolution removes supernatant, and washing, adds the process of HCl-ethanolic solution, slough template molecule, obtain Fe 3o 4@Au-GSH-PANI magnetic molecule marking nanoparticle (MMIPs);
H) enrichment template molecule: obtained MMIPs is placed in E2-PBS solution, stirring and adsorbing, obtains E2-MMIPs;
I) Electrochemical Detection: by step h) in be placed in reaction tank containing the solution of E2-MMIPs, with magnetic glassy carbon electrode (MGCE) magnetic absorption, carry out electrochemical gaging.
By the following method magnetic control electrochemical sensing film of the present invention is detected:
Electrochemical detection method and condition:
Cyclic voltammetry (CV) method: detection potential range is-0.2 ~ 0.6V, sweep speed is 100mVs -1.Test end liquid is 0.1molL -1kCl and 1mmolL -1k 3fe (CN) 6solution.
Differential pulse method (DPV) method: detection potential range is-0.2 ~ 0.8V, sweep speed is 50mVs -1, current potential increment is 0.005V, and amplitude is 0.05V, and pulse width is 0.1s, and sampling width is 0.02s, and rest time is 2s.Electrode Al before test 2o 3powder polishing, ethanol, distilled water ultrasonic cleaning, nitrogen dry up.Test end liquid is 0.05molL -1pBS (pH 7.0)-0.1molL -1kCl solution.
Sample determination: to measure the estradiol in milk powder, takes certain milk powder powder 1g, and after dissolving 1h with 20mL EtOH Sonicate, the centrifugal 5min of 12000rpm, gets supernatant, vacuum drying.Redissolve with 5mL ethanol after dry, get 0.5mL pH7.0 phosphate buffer and be diluted to 5mL, the MMIPs that embodiment 1 method is obtained is placed in solution stirring and adsorbing 30min.MGCE after the E2-MMIPs solution surface magnetic absorption 10min obtained, adopt above-mentioned in DPV method working sample in the concentration of estradiol.Magnetic molecule marking nanoparticle sensing membrane prepared by this method is for estradiol electrochemical gaging, and its detectability (LOD) can reach 2.76 × 10 -9molL -1(S/N=3), highly sensitive, it also has, and selectivity is high, response fast, stability and the feature such as tolerance is good, the efficient, sensitive of estradiol in complex matrices can be realized and detect in real time.
The preparation process of magnetic non-molecular imprinting nanoparticle electrochemical sensing film (MNIES): the preparation of magnetic non-molecular imprinting nanoparticle electrochemical sensing film is exactly do not add E2-ethanolic solution, and other steps are identical with embodiment.
The sign of MMIPs preparation process
1.UV-VIS
In uv-visible absorption spectra figure, the absorption peak of Au NPs, at 526nm, is close with the 520nm in bibliographical information.And Fe 3o 4at 560nm, compared with the absorption peak of Au NPs, there is obvious red shift in the absorption peak of@Au NPs, shows that Jenner's grain of rice is successfully wrapped in Fe 3o 4surface.
2.FTIR
In infrared spectrogram, a series of Fe can be observed 3o 4@Au/GSH characteristic absorption peak, 543cm -1fe-O peak, 637cm -1c-S peak, 1084cm -1si-O peak, 1300cm -1c-N peak, 1670cm -1be the C=O peak in second amido link, the appearance of these characteristic peaks all shows Fe 3o 4@Au/GSH nanoparticle successfully synthesizes.The characteristic peak of polyaniline is at 1593cm -1and 1502cm -1place, be the C=C peak on quinone and phenyl ring, the appearance of these two characteristic peaks shows that polyaniline is successfully cross-linked at Fe 3o 4the surface of@Au NPs.
3.SEM
Fe 3o 4the morphosis of@Au/GSH and MMIPs can be obtained by scanning electron microscope.From Fig. 2 A, the Fe of preparation 3o 4@Au/GSH NPs mean grain size is within 200nm scope.
4.MMIPs electrochemical Characterization-each step cycle volt-ampere contrast
By above-mentioned CV method, cyclic voltammetric sign is carried out to the MGCE that MMIPs modifies.Comparison diagram 3a curve and b curve, find that the MGCE cyclic voltammetric current-responsive value of modifying through MMIPs raises, CV area under the curve becomes large, is because Fe 3o 4@Au nanoparticle and polyaniline have excellent electric conductivity, accelerate the speed of electro transfer.Relatively c curve and b curve, find that the MGCE cyclic voltammetric current-responsive value that E2-MMIPs modifies declines, CV area under the curve reduces, and may be because template molecule occupies marking hole, hinder probe molecule K 3[Fe (CN) 6] redox reaction, cyclic voltammetric current-responsive value reduce.
The assessment of MMIPs absorption property
1. Static Adsorption test
Fig. 4 A is the Static Adsorption curve of MMIPs and MNIPs to 0.025 μm of ol/L ~ 5mmol/L series estradiol-PBS solution.MMIPs and MNIPs is placed in serial estradiol-PBS solution stirring and adsorbing 30min respectively, measures Ip value by above-mentioned DPV method.In MMIPs adsorption isothermal curve, along with the continuous increase of template molecule E2 concentration, Ip value also increases gradually, when concentration is 2mmolL -1time, absorption reaches balance, and Ip value no longer changes, and thinks that now marking hole is almost all occupied by template molecule.Show that MMIPs marking hole and active binding site have high affinity and special recognition capability to estradiol.And in MNIPs sorption isotherm, owing to not forming marking hole in MNIPs, Ip value is almost without significant change.
2. dynamic adsorption test
Fig. 4 B is the Dynamic Adsorption curve of MMIPs to 0.1 μm of ol/L and 1 μm ol/L estradiol.MMIPs is placed in the estradiol-PBS solution stirring and adsorbing of 0.1 μm of ol/L and 1 μm ol/L, two kinds of concentration, respectively at 5,10,20,30,40min time take out, measure Ip value by above-mentioned DPV method.Curve is very fast in starting stage increase ratio, is tending towards saturated about 30min.Even if the initial concentration of estradiol is different, the state adsorbance of reaching capacity is substantially identical, and in visible film, effective marking number of cavities is certain, further demonstrates in MMIPs the specificity hole defining estradiol.
3. selective adsorption test
Selective adsorption test is the important indicator investigating molecularly imprinted polymer marking effect and performance quality, is also the prerequisite that can sensor be applied to actual sample detection.Whether the experiment of design interference is for investigating molecular imprinting effect, and whether the marking hole namely studying MMIPs has specific selectivity to template molecule, not by the interference of other competitive analogs.
Oestrone, ethinylestradiol, estriol and estradiol is adopted to carry out contrast experiment in experiment.After MMIPs and MNIPs being placed in A, B, C, solution D stirring and adsorbing 30min respectively, at 0.05molL -1pBS (pH 7.0)-0.1molL -1the DPV method is above adopted to carry out DPV scanning analysis in KCl solution.Result shows, and the oxidation current response of MNIPs-ES to A, B, C, D is all very low, and the oxidation current response of MMIPs-ES to A, B, C, D is all higher and close.Show under 10 times of concentration chaff interference existent condition, MMIPs is substantially constant to the adsorbance of estradiol, namely the MMIPs prepared is not subject to the interference of oestrone, estriol and ethinylestradiol to the absorption of estradiol, thus the MMIPs showing preparation in this experiment has stable specific adsorption ability to estradiol.
Estradiol-the PBS solution of the estradiol of the estradiol of A:0.1 μm of ol/L and 1 μm of ol/L oestrone-PBS solution B:0.1 μm ol/L and the estradiol of 1 μm of ol/L ethinylestradiol-PBS solution C:0.1 μm ol/L and 1 μm of ol/L estriol-PBS solution D:0.1 μm ol/L.
Sample determination methodology
1. typical curve and detectability
Accurate compound concentration is 0.025 μm of ol L -1~ 10 μm of ol L -1estradiol series standard solution.After estradiol standard solution MMIPs obtained under optimum experiment condition being placed in respectively series concentration adsorbs 30min, carry out DPV mensuration by method described above, response peak electric current and its concentration are 2.5 × 10 -8~ 5.0 × 10 -7molL -1with 5.0 × 10 -7~ 1.0 × 10 -5molL -1scope in good linear relationship, equation of linear regression is respectively Ip 1=0.08+1.08C (r=0.902), Ip 2=0.58+1.05C (r=0.980).Detection is limited to 2.76nmol/L, S/N=3.
The stability of 2.MMIPs and reappearance
With embodiment 1 legal system for 5 parts of MMIPs, measure it at 1 μm of olL -1ip value after estradiol-PBS solution absorption, RSD is 3.9%; Same electrode replication Ip value 5 times, RSD is 2.5%.Show that MMIPs preparation method stablizes, favorable reproducibility, and it is excellent to measure repeatability.
After separately MMIPs being preserved 10 days as 4 DEG C, refrigerator, when its current-responsive is reduced to initial 91%, and the profile at oxidation potential and peak does not significantly change.
3. actual sample analysis
The MMIPs obtained with the present embodiment 1 measures the estradiol in milk powder, takes certain milk powder powder 1g, and after dissolving 1h with 10mL EtOH Sonicate, the centrifugal 5min of 12000rpm, gets supernatant, vacuum drying.Redissolve with 1mL ethanol after dry, be diluted to 5mL with pH7.0 phosphate buffer, under getting optimum experiment condition, obtained MMIPs 10mg is placed in solution stirring and adsorbing 30min.MGCE after the E2-MMIPs solution surface magnetic absorption 10min obtained, adopt above-mentioned in DPV method working sample in the concentration of estradiol, the estradiol content in result display milk powder is 14.71ng/g.In milk powder, add the estradiol standard solution of basic, normal, high 3 concentration respectively, carry out average recovery test, concrete outcome is in table 1.
The assay of estradiol in table 1 milk powder
Beneficial effect of the present invention compared with the prior art:
1. by an assembling magnetic pole, the soft modification sensing membrane of the magnetic molecule marking is formed to E2-MMIPs induced by magnetic field, through E2, detection is completed to electrode interface electrochemical response, when taking out the magnetic core in electrode, sensing membrane disappears, make loaded down with trivial details electrode reprocessing become very simple, there is good automatically cleaning effect.
2. utilize the controlled structure sensing membrane in external magnetic field, the preparation of MMIES and service condition gentleness, at room temperature can complete.
3. soft modification sensing membrane of the present invention, little on electrode surface impact, the sanding and polishing of electrode and cleaning course are greatly simplified, extend the serviceable life of electrode.
4. based on Fe 3o 4the E2-MMIPs that@Au-GSH@PANI builds, has good molecule distinguishability and superior chemical property.
5. the MMIPs built can be prepared and repetitive cycling utilization in batches, and cost is low.
6. MMIPs of the present invention preparation and all electrode is not relied on to the Selective Separation of sample analytes and enrichment and the regeneration (eluted template) after measuring, greatly improve electrode detection efficiency.
7. electrode of the present invention does not need complicated modification, and soft modification film forming and detect and can complete successively, simple to operate and quick, general analysis personnel easily grasp, and have good universality.
8. the magnetic molecule marking nano electrochemical sensing membrane that prepared by the present invention have selectivity high, respond the features such as quick, highly sensitive, stability and better tolerance, universality be strong, the efficient, sensitive of estradiol in complex matrices can be realized and detect in real time.
Accompanying drawing explanation
Fig. 1 is MMIPs preparation process medium ultraviolet-visible absorption spectra figure (UV-VIS) and FTIR spectrum figure (FT-IR)
In figure, A is Au NPs and Fe 3o 4the UV-VIS of@Au/GSH
B is Fe 3o 4the FT-IR of@Au/GSH and MMIPs
Fig. 2 is the electron-microscope scanning figure (SEM) in MMIPs preparation process
In figure, A is Fe 3o 4@Au/GSH
B is MMIPs
Fig. 3 is each step cycle volt-ampere comparison diagram in MMIPs preparation process
In figure, a is the CV curve of naked-MGCE
B is the CV curve after modifying MMIPs
C is the CV curve after modifying E2-MMIPs
Fig. 4 is the absorption property evaluation map of MMIPs
In figure, A is the adsorption isothermal curve figure of MMIPs and MNIPs
B is the Dynamic Adsorption curve map of MMIPs
C is the selective adsorption figure of MMIPs and MNIPs, and wherein (1) to be 0.1 μm of ol/L estradiol and 1 μm of ol/L oestrone-PBS solution (2) be 0.1 μm of ol/L estradiol and 1 μm of ol/L ethinylestradiol-PBS solution (3) are 0.1 μm of ol/L estradiol and 1 μm of ol/L estriol-PBS solution (4) is 0.1 μm of ol/L estradiol-PBS solution.
Embodiment
Medicine and reagent: estradiol (E2), estriol (E3), ethinyloestradiol (EE), estrone (E1) (Zhejiang Xian Ju pharmaceutical Co. Ltd), gold chloride (HAuCl 4), glutathione (GSH) (West Asia reagent company limited), aniline, 3-aminopropyl triethoxysilane (APTES) (Shanghai Aladdin reagent company limited), experimental water is redistilled water, 0.05molL -1kH 2pO 4and 0.05molL -1k 2hPO 4the phosphate buffered solution (phosphate buffered solution, PBS) of solution pH 7.0.
Embodiment 1
The preparation of 1.1 magnetic molecule marking nanoparticles electrochemical sensing film (MMIES)
(1) Fe is prepared 3o 4@Au nanoparticle
Under nitrogen protection and magnetic agitation, in 250mL flask, add 180mL containing 11.2mmol FeCl 36H 2o and 5.6mmol FeCl 24H 2the aqueous solution of O, after being heated to 50 DEG C, dropwise adds 12.5mL NH 3h 2o reacts 30min, and magnetic resolution removes supernatant, collects reaction precipitation thing and washes three times, obtain Fe 3o 4magnetic nano particle.
Get 20mgFe 3o 4magnetic nano particle, adds 10mL 1% (v/v) APTES-ethanolic solution, stirring at room temperature reaction 12h, and magnetic resolution removes supernatant, adds ethanol purge and dissolves, and obtains APTES and wraps up Fe 3o 4nanoparticle.
Get obtained 12.5mg APTES and wrap up Fe 3o 4nanoparticle, adds 2.5mL 1%HAuCl4 and 35mL redistilled water, after ultrasonic disperse, continues ultrasonicly to add 10mL 10mmol/L NaBH simultaneously 4purple potato look is become to solution colour from tawny.
(2) linkage function group-sulfydryl
At Fe 3o 4in@Au nanoparticle solution, add 2.2mL 100mmol/L GSH, stirring at room temperature reaction 24h, magnetic resolution removes supernatant, and washing, obtains Fe 3o 4@Au/GSH nanoparticle.
(3) the pre-assembled solution of molecular imprinting
Get 20mg Fe 3o 4@Au/GSH nanoparticle, adds the E2-ethanolic solution that 22mL concentration is 10mmol/L, stirring at room temperature 4h.Logical nitrogen 30min, adds 150 μ L aniline and 2mL 2mol/L HCl, and room temperature lucifuge stirs 30min.
(4) polyreaction
In the pre-assembled solution of molecular imprinting, add 1.0mL 200mmol/L ammonium persulfate APS, lucifuge stirring reaction 6h under room temperature.
(5) eluted template molecule
By the solution magnetic resolution after polyreaction, remove supernatant, alcohol wash, washing, add 20mL 0.5mol/L HCl-ethanolic solution and stir 20min, slough template molecule, obtain Fe 3o 4@Au/GSH-PANI magnetic molecule marking nanoparticle (MMIPs).
(6) enrichment template molecule
Get obtained MMIPs 10mg and be placed in E2-PBS (0.05mol/L pH7.0) solution, stirring and adsorbing 30min, obtains E2-MMIPs.
(7) Electrochemical Detection
Solution containing E2-MMIPs is placed in reaction tank, and magnetic glassy carbon electrode (MGCE) uses Al 2o 3powder polishing, ethanol, distilled water ultrasonic cleaning, nitrogen dries up, and MGCE, at E2-MMIPs solution surface magnetic absorption 30min, carries out electrochemical gaging.
The preparation of 1.2 magnetic non-molecular imprinting nanoparticle electrochemical sensing film (MNIES)
Do not add E2-ethanolic solution, other steps are identical with 1.1
Embodiment 2
The preparation of 1.1 magnetic molecule marking nanoparticles electrochemical sensing film (MMIES)
(1) Fe is prepared 3o 4@Au nanoparticle
Under nitrogen protection and magnetic agitation, in 250mL flask, add 200mL containing 13.1mmol FeCl 36H 2o and 6.5mmol FeCl 24H 2the aqueous solution of O, after being heated to 50 DEG C, dropwise adds 12.5mL NH 3h 2o reacts 30min, and magnetic resolution removes supernatant, collects reaction precipitation thing and washes three times, obtain Fe 3o 4magnetic nano particle.Get 25mgFe 3o 4magnetic nano particle, adds 15mL 1% (v/v) APTES-ethanolic solution, stirring at room temperature reaction 15h, and magnetic resolution removes supernatant, adds ethanol purge and dissolves, and obtains APTES and wraps up Fe 3o 4nanoparticle.Get obtained 15mg APTES and wrap up Fe 3o 4nanoparticle, adds 4mL 1%HAuCl4 and 45mL redistilled water, ultrasonic disperse, continues ultrasonicly to add 15mL10mmol/L NaBH simultaneously 4until solution colour becomes purple potato look from tawny.
(2) linkage function group-sulfydryl
At Fe 3o 4in@Au nanoparticle solution, add 3mL 100mmol/L GSH, stirring at room temperature reaction 30h, magnetic resolution removes supernatant, and washing, obtains Fe 3o 4@Au/GSH.
(3) molecular imprinting self assembly solution
Get 20mg Fe 3o 4@Au-GSH nanoparticle, adds the E2-ethanolic solution that 30mL concentration is 10mmol/L, stirring at room temperature 6h.Logical nitrogen 45min, adds 180 μ L aniline and 3mL 2mol/L HCl, and room temperature lucifuge stirs 45min.
(4) polyreaction
In molecular imprinting self assembly solution, add 2.0mL 200mmol/L ammonium persulfate APS, lucifuge stirring reaction 10h under room temperature.
(5) eluted template molecule
By the solution magnetic resolution after polyreaction, remove supernatant, washing, adds 30mL 0.5mol/L HCl-ethanolic solution process 30min, sloughs template molecule, obtain Fe 3o 4the magnetic molecularly imprinted nanoparticle of@Au/GSH-PANI (MMIPs).
(6) enrichment template molecule:
Get obtained MMIPs 20mg and be placed in E2-PBS (0.05mol/L pH7.0) solution, stirring and adsorbing 45min, obtains E2-MMIPs.
(7) Electrochemical Detection
Solution containing E2-MMIPs is placed in reaction tank, and magnetic glassy carbon electrode (MGCE) uses Al 2o 3powder polishing, ethanol, distilled water ultrasonic cleaning, nitrogen dries up, and MGCE, at solution surface magnetic absorption 45min, carries out electrochemical gaging.
The preparation of 1.2 magnetic non-molecular imprinting nanoparticle electrochemical sensing film (MNIES)
Do not add E2-ethanolic solution, other steps are identical with 1.1
Embodiment 3
The preparation of 1.1 magnetic molecule marking nanoparticles electrochemical sensing film (MMIES)
(1) Fe is prepared 3o 4@Au nanoparticle
Under nitrogen protection and magnetic agitation, in 250mL flask, add 150mL containing 10.2mmol FeCl 36H 2o and 4.9mmol FeCl 24H 2the aqueous solution of O, after being heated to 50 DEG C, dropwise adds 10mL NH 3h 2o reacts 30min, and magnetic resolution removes supernatant, collects reaction precipitation thing and washes three times, obtain Fe 3o 4magnetic nano particle.Get 20mgFe 3o 4magnetic nano particle, adds 8mL 1% (v/v) APTES-ethanolic solution, stirring at room temperature reaction 10h, and magnetic resolution removes supernatant, adds ethanol purge and dissolves, and obtains APTES and wraps up Fe 3o 4nanoparticle.Get the Fe that obtained 10.5mg APTES wraps up 3o 4nanoparticle, adds 2mL 1%HAuCl4 and 30mL redistilled water, ultrasonic disperse, continues ultrasonicly to add 8mL 10mmol/L NaBH simultaneously 4until solution colour becomes purple potato look from tawny.
(2) linkage function group-sulfydryl
At Fe 3o 4in@Au nanoparticle solution, add 1.8mL 100mmol/L GSH, stirring at room temperature reaction 20h, magnetic resolution removes supernatant, and washing, obtains Fe 3o 4@Au/GSH.
(3) molecular imprinting self assembly solution
Get 10mg Fe 3o 4@Au/GSH nanoparticle, adds the E2-ethanolic solution that 18mL concentration is 10mmol/L, stirring at room temperature 4h.Logical nitrogen 20min, adds 120 μ L aniline and 1.5mL 2mol/L HCl, and room temperature lucifuge stirs 20min.
(4) polyreaction
In molecular imprinting self assembly solution, add 1.0mL 200mmol/L ammonium persulfate APS, lucifuge stirring reaction 8h under room temperature.
(5) eluted template molecule
By the solution magnetic resolution after polyreaction, remove supernatant, washing, adds 20mL 0.5mol/L HCl-ethanolic solution process 20min, sloughs template molecule, obtain Fe 3o 4the magnetic molecularly imprinted nanoparticle of@Au/GSH-PANI (MMIPs).
(6) enrichment template molecule
Get obtained MMIPs 15mg and be placed in E2-PBS (0.05mol/L pH7.0) solution, stirring and adsorbing 20min, obtains E2-MMIPs.
(7) Electrochemical Detection
Solution containing E2-MMIPs is placed in reaction tank, and magnetic glassy carbon electrode (MGCE) uses Al 2o 3powder polishing, ethanol, distilled water ultrasonic cleaning, nitrogen dries up, and MGCE, at solution surface magnetic absorption 20min, carries out electrochemical gaging.
The preparation of 1.2 magnetic non-molecular imprinting nanoparticle electrochemical sensing film (MNIES)
Do not add E2-ethanolic solution, other steps are identical with 1.1
Embodiment 4
The preparation of 1.1 magnetic molecule marking nanoparticles electrochemical sensing film (MMIES)
(1) Fe is prepared 3o 4@Au nanoparticle
Under nitrogen protection and magnetic agitation, in 500mL flask, add 300mL containing 21mmol FeCl 36H 2o and 11mmol FeCl 24H 2the aqueous solution of O, after being heated to 50 DEG C, dropwise adds 20mL NH 3h 2o reacts 45min, and magnetic resolution removes supernatant, collects reaction precipitation thing and washes three times, obtain Fe 3o 4magnetic nano particle.Get 50mgFe 3o 4magnetic nano particle, adds 18mL 1% (v/v) APTES-ethanolic solution, stirring at room temperature reaction 24h, and magnetic resolution removes supernatant, adds ethanol purge and dissolves, and obtains APTES and wraps up Fe 3o 4nanoparticle.Get obtained 40mgAPTES and wrap up Fe 3o 4nanoparticle, adds 5mL 1.5%HAuCl4 and 60mL redistilled water, ultrasonic disperse, continues ultrasonicly to add 15mL12mmol/L NaBH simultaneously 4until solution colour becomes purple potato look from tawny.
(2) linkage function group-sulfydryl
At Fe 3o 4in@Au nanoparticle solution, add 3.5mL 80mmol/L GSH, stirring at room temperature reaction 36h, magnetic resolution removes supernatant, and washing, obtains Fe 3o 4@Au/GSH.
(3) molecular imprinting self assembly solution
Get 15mg Fe 3o 4@Au/GSH nanoparticle, adding 35mL concentration is 8mmol/L E2-ethanol, stirring at room temperature 12h.Logical nitrogen 50min, adds 150 μ L aniline and 2mL 2mol/L HCl, and room temperature lucifuge stirs 50min.
(4) polyreaction
In molecular imprinting self assembly solution, add 2.0mL 200mmol/L ammonium persulfate APS, lucifuge stirring reaction 12h under room temperature.
(5) eluted template molecule
By the solution magnetic resolution after polyreaction, remove supernatant, washing, adds 30mL 1mol/L HCl-ethanolic solution process 30min, sloughs template molecule, obtain Fe 3o 4the magnetic molecularly imprinted nanoparticle of@Au/GSH-PANI (MMIPs).
(6) enrichment template molecule
Get the E2-PBS solution that obtained MMIPs 25mg is placed in 0.1mol/L, stirring and adsorbing 1h, obtains E2-MMIPs.
(7) Electrochemical Detection
Solution containing E2-MMIPs is placed in reaction tank, and magnetic glassy carbon electrode (MGCE) uses Al 2o 3powder polishing, ethanol, distilled water ultrasonic cleaning, nitrogen dries up, and MGCE, at solution surface magnetic absorption 45min, carries out electrochemical gaging.
Above-described embodiment electrochemical detection method and condition:
Cyclic voltammetry (CV) method: detection potential range is-0.2 ~ 0.6V, sweep speed is 100mVs -1.Test end liquid is 0.1molL -1kCl and 1mmolL -1k 3fe (CN) 6solution.
Differential pulse method (DPV) method: detection potential range is-0.2 ~ 0.8V, sweep speed is 50mVs -1, current potential increment is 0.005V, and amplitude is 0.05V, and pulse width is 0.1s, and sampling width is 0.02s, and rest time is 2s.Electrode Al before test 2o 3powder polishing, ethanol, distilled water ultrasonic cleaning, nitrogen dry up.Test end liquid is 0.05molL -1pBS (pH 7.0)-0.1molL -1kCl solution.

Claims (10)

1. a preparation method for estradiol magnetic molecule marking nanoparticle electrochemical sensing film, is characterized in that the method comprises the following steps:
A) preparation of molecular imprinting self assembly solution: at Fe 3o 4add E2-ethanolic solution, stirring reaction in@Au-GSH nanoparticle, solution adds aniline-HCl solution after leading to nitrogen deoxygenation, lucifuge stirs;
B) polyreaction: add initiator ammonium persulfate in the solution that step a) obtains, lucifuge stirring reaction;
C) eluted template molecule: by step b) solution that obtains, magnetic resolution removes supernatant, and washing, adds the process of HCl-ethanolic solution, slough template molecule, obtain Fe 3o 4the magnetic molecularly imprinted nanoparticle of@Au-GSH-PANI;
D) enrichment template molecule: by obtained Fe 3o 4the magnetic molecularly imprinted nanoparticle of@Au-GSH-PANI is placed in E2-PBS solution, stirring and adsorbing, obtains E2-MMIPs;
E) Electrochemical Detection: the solution containing E2-MMIPs is placed in reaction tank, with magnetic glassy carbon electrode magnetic absorption 5min ~ 1h, makes electrode surface film forming.
2. the preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film according to claim 1, it is characterized in that step a) in, the Fe of every 5mg ~ 20mg 3o 4@Au-GSH nanoparticle, adds the E2-ethanolic solution that 5mL ~ 50mL concentration range is 1mmol/L ~ 20mmol/L, reaction time 1 ~ 20h.
3. the preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film according to claim 1, it is characterized in that step a) in, adding the time that aniline-HCl solution leads to nitrogen is 1 ~ 50min.
4. the preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film according to claim 1, it is characterized in that step a) in, the volume range of aniline and HCl is 1:10 ~ 20, and the concentration range of HCl is 0.05mol/L ~ 5mol/L.
5. the preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film according to claim 1, it is characterized in that step a) in, the temperature of reaction of whole process is 5 ~ 50 DEG C.
6. the preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film according to claim 1, is characterized in that in step b) in, often containing the Fe of 5mg ~ 20mg 3o 4in the pre-assembled solution of molecular imprinting of@Au-GSH nanoparticle, add ammonium persulfate solution 0.1mL ~ 5mL that concentration range is 10mmol/L ~ 500mmol/L.
7. the preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film according to claim 1, is characterized in that in step b) in, polymerization reaction time is 2 ~ 20h, and temperature of reaction is 0 ~ 50 DEG C.
8. the preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film according to claim 1, is characterized in that in step c) in, the concentration range of HCl-ethanolic solution is 0.05mol/L ~ 2.0mol/L, and mixing time is 5min ~ 2h.
9. the preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film according to claim 1, is characterized in that in steps d) in, the concentration of E2-PBS is 0.01 ~ 2.0mol/L, and mixing time is 5min ~ 5h.
10. the preparation method of estradiol magnetic molecule marking nanoparticle electrochemical sensing film according to claim 1, is characterized in that Fe 3o 4the preparation of@Au-GSH nanoparticle comprises preparation Fe 3o 4magnetic nano particle, Fe 3o 4surface meets NH 2, preparation Fe 3o 4@Au nanoparticle, linkage function group sulfydryl step.
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CN105891295A (en) * 2016-03-29 2016-08-24 南京医科大学 Insulin electrochemical detection method based on conducting imprinting nanoparticles and magnetic control induction filming
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CN110006977A (en) * 2019-05-15 2019-07-12 北京理工大学 A kind of CuFe2O4The preparation of nanosphere electrochemical sensor and detection method to lysozyme
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刘峻等: ""内分泌干扰物17β-雌二醇荧光分子印迹识别方法"", 《环境科学研究》 *

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CN105891295A (en) * 2016-03-29 2016-08-24 南京医科大学 Insulin electrochemical detection method based on conducting imprinting nanoparticles and magnetic control induction filming
CN105891295B (en) * 2016-03-29 2018-06-05 南京医科大学 Based on electric conductivity trace nanoparticle and magnetic control induction filming to the electrochemical detection method of insulin
CN106560703A (en) * 2016-10-13 2017-04-12 苏州科技大学 Preparation method of molecular imprinting sensor for detecting melamine
CN110006977A (en) * 2019-05-15 2019-07-12 北京理工大学 A kind of CuFe2O4The preparation of nanosphere electrochemical sensor and detection method to lysozyme
CN113567520A (en) * 2021-07-08 2021-10-29 西北农林科技大学 Electrochemical sensing method for accurate detection of trace protein molecules

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