CN105866211B - A kind of preparation method and application of ampicillin molecular engram sensor - Google Patents
A kind of preparation method and application of ampicillin molecular engram sensor Download PDFInfo
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- CN105866211B CN105866211B CN201610371665.2A CN201610371665A CN105866211B CN 105866211 B CN105866211 B CN 105866211B CN 201610371665 A CN201610371665 A CN 201610371665A CN 105866211 B CN105866211 B CN 105866211B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
Abstract
The embodiment of the invention discloses a kind of preparation method and application of ampicillin molecular engram sensor, the preparation method of wherein ampicillin molecular engram sensor includes：(1) inert electrode is pre-processed；(2) copolymerization monomer pyrroles, luminol and template molecule ampicillin are dissolved in the buffer solution that pH value is 4~7, obtain polymer fluid, wherein, the ratio of the amount of the substance of copolymerization monomer pyrroles and luminol is (1~8)：1；The ratio of the amount of the substance of pyrroles and ampicillin is 1：(1~6)；(3) pretreated inert electrode is placed in polymer fluid and electropolymerization is carried out using cyclic voltammetry, obtain being combined with pyrroles's luminol co-polymer membrane of ampicillin on the inert electrode surface；(4) ampicillin combined with eluant, eluent removal co-polymer membrane, obtains ampicillin molecular engram sensor.Prepared ampicillin molecular engram sensor has detection sensitivity height, high luminous efficiency and other features.
Description
Technical field
The present invention relates to antibiotic detection field, more particularly to a kind of preparation method of ampicillin molecular engram sensor
And application.
Background technology
Molecularly imprinted polymer (Molecularly imprinted polymer, abbreviation MIP) is to utilize molecular engram skill
Art prepare with the high molecular polymer exactly matched on space structure and binding site with template molecule.The preparation of MIP
It is realized by the following method：Template molecule passes through chemical bond or hydrogen bond knot with function monomer in reaction mixture first
Conjunction forms complex；Then it carries out copolymerization and forms polymer, the functional group on function monomer is made to determine in space arrangement and space
It is fixed up upwards；Again with template molecule is either physically or chemically washed away, obtain hole with a certain size and shape and really
Determine the molecularly imprinted polymer of the functional group of space arrangement.Common preparation method has potentiostatic method, galvanostatic method and cycle
Voltammetry etc..
In recent years, using with specific selectivity, environmental resistance is strong, structure-controllable and the simple molecule of sample treatment
Imprinted polymer realizes that the analysis of target analytes measures as recognition component structure molecular engram sensor becomes one kind very
Promising detection method.Electrochemical luminescence (ECL) is by the current potential controllability of electrochemical techniques and chemiluminescence analysis
High sensitivity is combined, with detection sensitivity is high, the range of linearity is wide, photosignal acquires simultaneously, Instrument structure and operation are simple
The advantages that single, convenient for detection.Structure molecule is combined using the good MIP of the selectivity ECL detection methods high with detection sensitivity
Trace electrochemical luminescence sensor combines front and rear caused ECL signal intensities using template molecule and template molecule is carried out again
Quantitative determination.The different trend of caused electrochemical luminescence signals variation after being identified according to template molecule, and can be by molecular engram
Electrochemical luminescence sensor is divided into Turn-on types (i.e. signal enhancing type) and Turn-off types (i.e. signal attenuated type).Turn-on
Type molecular imprinting electrochemical luminescence sensor have higher detection sensitivity, but it has been reported that molecular imprinting electrochemical hair
Optical sensor is mostly Turn-off types.In addition, ECL sensors can be divided into according to the state of ECL reagents by liquid phase ECL and solid phase again
ECL sensors, liquid phase ECL sensors are directly to be measured using the aqueous solution of ECL reagents, and there is background interference to be difficult to eliminate
And reagent consumption it is big the shortcomings of.ECL reagents are fixed on electrode surface by solid phase ECL, have luminous efficiency height, background interference
It is small, it is reusable the features such as.
At this stage, highly sensitive ECL detection methods are combined to structure molecular imprinting electrochemical hair with the MIP of high specificity
The report of optical sensor is seldom, and the molecular imprinting electrochemical luminescence sensor of structure is mostly measured using liquid phase ECL, and is mostly
Turn-off types.Up to now, ECL reagent tris (bipyridine) rutheniums are fixed on electrode by an only document report using nano material
Surface, and Turn-on type solid phase molecules are constructed as template molecule using the isoniazid that can enhance tris (bipyridine) ruthenium ECL responses
Trace electrochemical luminescence sensor.But the fixation procedure of tris (bipyridine) ruthenium and the polymerization process of MIP are carried out in two steps, and make sensing
The building process of device is more cumbersome to be taken.
Ampicillin (AM) also known as ampicillin are a kind of stronger beta-lactam class antibiotic of anti-infection ability, have
Broad-spectrum antibacterial action.AM is usually used in respiratory tract infection, skin infection, pneumonia, typhoid fever, bronchitis and intestines caused by sensitive bacterial
The treatment of the diseases such as gastritis, is widely used in Animal husbandry production, results in its residual in animal-derived food.People
AM or edible is used for a long time can cause AM to accumulate in vivo containing the remaining meat of AM, egg, dairy produce, cause the resistance to of microorganism in human body
The problems such as pharmacological property and allergic reaction, produces security risk to health.
The method of common detection AM mainly has high performance liquid chromatography, fluorescence method and UV absorption light splitting both at home and abroad at present
Photometry etc..But the shortcomings of high performance liquid chromatography takes there is instrumentation, sample treatment is cumbersome, fluorescence method have selection
Property it is not high the shortcomings that, the detection sensitivity of ultraviolet absorption spectrophotometry and selectivity are not high enough.Therefore further development letter
Just, sensitive, quick AM detection methods have very important significance.
Invention content
The embodiment of the invention discloses a kind of preparation method and applications of ampicillin molecular engram sensor, are used to prepare
Go out the ampicillin molecular engram sensor for being capable of easy, sensitive, quick detection ampicillin.Technical solution is as follows：
Present invention firstly provides a kind of preparation method of ampicillin molecular engram sensor, including：
(1) inert electrode is pre-processed；
(2) copolymerization monomer pyrroles, luminol and template molecule ampicillin are dissolved in the buffer solution that pH value is 4~7
In, polymer fluid is obtained, wherein, the ratio of the amount of the substance of copolymerization monomer pyrroles and luminol is (1~8)：1；Pyrroles and ammonia benzyl
The ratio of the amount of the substance in XiLin is 1：(1~6)；
(3) pretreated inert electrode is placed in polymer fluid and electropolymerization is carried out using cyclic voltammetry, described lazy
Property electrode surface obtains being combined with pyrroles-luminol co-polymer membrane of ampicillin；
(4) ampicillin combined with eluant, eluent removal co-polymer membrane, obtains ampicillin molecular engram sensor.
In the specific embodiment of the present invention, the inert electrode is selected from gold electrode, platinum electrode, glass-carbon electrode or stone
Electrode ink, preferably gold electrode.
The present invention specific embodiment in, the buffer solution be phosphate buffer solution, pH value 6.
In the specific embodiment of the present invention, the polymerizing condition that electropolymerization is carried out using cyclic voltammetry is included：
Scanning current potential is 0~1V；Speed is swept as 30~80mV s-1In any value, the scan round number of turns for 3~10 circle in any value.
In the specific embodiment of the present invention, the eluant, eluent is 9:The methanol-acetic acid solution of 1 (v/v), 0.01M
The HCl solution of NaOH solution or 0.01M.
In the specific embodiment of the present invention, a concentration of (10~30) mM of the pyrroles in buffer solution.
The present invention also provides the ampicillin molecular engram sensors by aforementioned method preparation.
The present invention further additionally provides above-mentioned ampicillin molecular engram sensor in ampicillin molecule is detected
Application.
In the specific embodiment of the present invention, described the step of detecting ampicillin molecule, includes：
(a) different known concentration C are prepared1~CnAmpicillin standard solution and blank sample without ampicillin
Solution；
(b) by ampicillin molecular engram sensor respectively in prepared ampicillin standard solution and blank sample
After impregnating specified time in product solution, it is respectively placed in detection bottom liquid and measures the ampicillin for combining various concentration ampicillin
The ECL intensity of molecular engram sensor；
(c) C is calculated1~CnThe ECL of the corresponding ampicillin molecular engram sensor of concentration ampicillin standard solution is strong
Spend difference DELTA ECL1~Δ ECLn, wherein, Δ ECLi=ECLi–ECL0, 1≤i≤n, ECL0For the corresponding ammonia of blank sample solution
The ECL intensity of benzyl XiLin molecular engram sensor；
(d) with ampicillin concentration C1~CnFor abscissa or ordinate, with strength difference Δ ECLiFor ordinate or horizontal seat
Mark establishes standard curve, obtains ampicillin concentration and strength difference Δ ECLiLinear equation；
(e) ampicillin standard solution is replaced with the ampicillin solution of unknown concentration, performs step (b) and (c)；Really
Determine the ECL strength differences corresponding to the ampicillin solution of unknown concentration；Linear equation in step (d) can obtain not
Know the ampicillin concentration in the ampicillin solution of concentration.
In the specific embodiment of the present invention, 1,1 '-two cyclopentadienyl of electroactive material is contained in the detection bottom liquid in step (b)
The double carboxylic acids of iron.
According to the above-mentioned technical solution, the present invention gathers using pyrroles, luminol as comonomer on the surface of inert electrode
It closes and obtains that there is specific selectivity and pyrroles-luminol co-polymer membrane of chemiluminescent properties to ampicillin, so as to obtain ammonia
Benzyl XiLin molecular engram sensor；The sensor belongs to Turn-on type sensors, has the characteristics that detection sensitivity is high；Simultaneously
Since ECL reagent luminols are fixed on formation solid phase ECL sensors in co-polymer membrane, thus the sensor has the effect that shines again
Rate is high, and background interference is small, it is reusable the features such as.
Using molecular engram sensor in ampicillin provided by the invention, can realize it is easy, sensitive, quickly detect ammonia
Benzyl XiLin can be widely applied to each field, be particularly suitable for the detection of ampicillin in food.
Description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention, for those of ordinary skill in the art, without creative efforts, can be with
Other attached drawings are obtained according to these attached drawings.
Fig. 1 is the cyclic voltammetry curve of electropolymerization in 1 step of the embodiment of the present invention (3), and the curve 1 in figure from top to bottom arrives
5 be respectively curve of the polymerization first lap to the 5th circle；
Fig. 2 is the infrared spectrogram of 4 kinds of substances, and wherein a is the infrared spectrogram of AM, and b is the infrared spectrogram of PPY, and c is
The infrared spectrogram of PL, d are the infrared spectrogram of AM-MIP；
Fig. 3 be naked gold electrode, the gold electrode covered with AM-MIP films, remove AM ampicillin molecular engram sensor and
The ECL curves of the ampicillin molecular engram sensor after 50 μM of AM are recombined, wherein curve a is the ECL of naked gold electrode
Curve, curve b are the ECL curves of the gold electrode covered with AM-MIP films, and curve c is the ampicillin molecular engram biography for removing AM
The ECL curves of sensor, curve d are the ECL curves for being combined with the ampicillin molecular engram sensor after 50 μM of AM again；
The ECL curves that it is the ampicillin molecular engram sensor for being combined with various concentration AM in embodiment 5 that Fig. 4 A, which are, figure
In curve a-h from the bottom to top be respectively a concentration of 0 (i.e. the blank sample solution) for being combined with AM, 0.07,0.5,0.7,3,10,
The ECL curves of 20 and 50 μM of ampicillin molecular engram sensor；
Fig. 4 B are the examination criteria curve for the ampicillin molecular engram sensor that embodiment 5 is established；
Fig. 5 be eluted ampicillin or combine 0.7 μM of ampicillin ampicillin molecular engram sensor exist respectively
Not comprising Fc (COOH)2Or include 2mM Fc (COOH)28.0 phosphate buffers of pH in ECL curves, wherein, curve a is washes
The ampicillin molecular engram sensor of ampicillin has been taken off not comprising Fc (COOH)28.0 phosphate buffers of pH in
ECL curves；Curve b is to have eluted the ampicillin molecular engram sensor of ampicillin comprising 2mM Fc (COOH)2PH
ECL curves in 8.0 phosphate buffers；Curve c is the ampicillin molecular engram sensor with reference to 0.7 μM of ampicillin not
It includes Fc (COOH)28.0 phosphate buffers of pH in ECL curves；Curve d is the ampicillin with reference to 0.7 μM of ampicillin
Molecular engram sensor is including 2mM Fc (COOH)28.0 phosphate buffers of pH in ECL curves；
Fig. 6 is the ECL curves for the ampicillin molecular engram sensor that various concentration AM is combined in embodiment 7, in figure
Curve a-f from the bottom to top is respectively a concentration of 0 (i.e. the blank sample solution) for being combined with AM, 0.7,9,20,500 and 700nM
Ampicillin molecular engram sensor ECL curves.
Specific embodiment
Present invention firstly provides a kind of preparation method of ampicillin molecular engram sensor, including：
(1) inert electrode is pre-processed；
(2) copolymerization monomer pyrroles, luminol and template molecule ampicillin are dissolved in the buffer solution that pH value is 4~7
In, polymer fluid is obtained, wherein, the ratio of the amount of the substance of copolymerization monomer pyrroles and luminol is (1~8)：1；Pyrroles and ammonia benzyl
The ratio of the amount of the substance in XiLin is 1：(1~6)；
(3) pretreated inert electrode is placed in polymer fluid and electropolymerization is carried out using cyclic voltammetry, described lazy
Property electrode surface obtains being combined with pyrroles-luminol co-polymer membrane of ampicillin；
(4) ampicillin combined with eluant, eluent removal co-polymer membrane, obtains ampicillin molecular engram sensor.
In the prior art, it generally is required to first pre-process inert electrode when preparing molecular engram sensor,
To obtain the electrode surface of more smooth pieces.
In the specific embodiment of the present invention, step (1) may be used following scheme and realize：First by inert electrode with not
The polishing powder of one-size is processed by shot blasting, is then cleaned again with ethyl alcohol and distilled water.Preferably, it can successively use
1.0th, 0.5,0.05 μm of alumina powder is processed by shot blasting inert electrode, then uses ethyl alcohol and distilled water sonic oscillation respectively
5 minutes, finally with dry after distilled water flushing.
In the specific embodiment of the present invention, inert electrode is selected from gold electrode, platinum electrode, glass-carbon electrode or graphite electricity
One kind in extremely, preferably gold electrode.
In step (2), for the monomer luminol of polymerization, also referred to as 3- aminobenzenes diformylhydrazine, English name is
luminol。
In the specific embodiment of the present invention, the buffer solution in step (2) is 0.1M phosphate buffers (pH 6.0).
In the specific embodiment of the present invention, the ratio of the amount of the substance of copolymerization monomer pyrroles and luminol for (2~
6)：1；The ratio of the amount of the substance of pyrroles and ampicillin is 1：(2~4), a concentration of (10~30) of the pyrroles in buffer solution
mM。
After step (2) obtains polymer fluid, you can carry out step (3), the present invention carries out electropolymerization using cyclic voltammetry；
In the prior art, cyclic voltammetry is a kind of common electropolymerization method, operating method and polyplant used, example
If constructed three-electrode system is (such as using Ag/AgCl electrodes as reference electrode, using platinum electrode as to electrode, with this hair
Bright inert electrode used is as working electrode) and electrolytic cell, electrochemical workstation for polymerization etc. be the prior art,
Herein without limiting, the polymerizing condition of electropolymerization can include the present invention：Scanning current potential is 0~1V, sweeps speed as 30~80mV
s-1In any value, the scan round number of turns for 3~10 circle in any value.
In the specific embodiment of the present invention, following proposal implementation specifically may be used in step (4)：
Inert electrode after polymerization in step (3) is placed in the eluant, eluent prepared in advance, after stirring 10-30 minutes, just
It removes in the pyrroles-luminol co-polymer membrane that can obtain template molecule ampicillin from step (3), is then rinsed with water
Afterwards, room temperature is dried.Wherein, eluant, eluent used can be 9:Methanol-acetic acid solution (i.e. methanol, the acetic acid body of 1 (v/v)
Product is than being 9:1), the HCl solution of the NaOH solution of 0.01M or 0.01M.
After by step (4) by template molecule elution, you can obtain to ampicillin with specific selectivity and with
The ampicillin molecular engram sensor of chemiluminescent properties.
It, can easy, sensitive, quick detection ammonia benzyl using the ampicillin molecular engram sensor prepared by the present invention
XiLin molecule.
The present invention specific embodiment in, detect ampicillin molecule the step of include：
(a) different known concentration C are prepared1~CnAmpicillin standard solution and blank sample without ampicillin
Solution；Method commonly used in the art may be used in the method for preparing solution, and the present invention is herein without limiting.
(b) by ampicillin molecular engram sensor respectively in prepared ampicillin standard solution and blank sample
After impregnating specified time in product solution, the ampicillin molecular engram sensor for combining various concentration ampicillin is measured respectively
ECL intensity；
In the specific embodiment of the present invention, the soaking time in step (b) can be in 10-30 minutes；
In the specific embodiment of the present invention, step (b) measures the ampicillin for combining various concentration ampicillin
The ECL intensity of molecular engram sensor may be used following scheme and implement：
To impregnate the ampicillin molecular engram sensor of ampicillin standard solution and blank sample solution as
Working electrode forms three-electrode system with reference electrode, to electrode respectively, and three-electrode system is placed in detection bottom liquid, is used
Electrochemical workstation and Electrochemial luminescence detecting instrument detection various concentration ampicillin standard solution and blank sample solution pair
The ECL intensity of ampicillin molecular engram sensor answered obtains testing result and is worked by being equipped with electrochemical analyser
The computer output of software；It should be noted that in a specific embodiment, the ampicillin solution of a concentration is often detected
Afterwards, it needs to carry out removing processing to the ampicillin combined in the molecular engram sensor of ampicillin, preparation side may be used
The scheme of step (4) in method realizes that the present invention is not being repeated herein.After ampicillin is stripped of, then by ammonia benzyl
XiLin molecular engram sensor is soaked in the standard solution of other concentration, is then detected again.
(c) C is calculated1~CnThe ECL of the corresponding ampicillin molecular engram sensor of concentration ampicillin standard solution is strong
Spend difference DELTA ECL1~Δ ECLn, wherein, Δ ECLi=ECLi–ECL0, 1≤i≤n, ECL0For the corresponding ammonia of blank sample solution
The ECL intensity of benzyl XiLin molecular engram sensor；
(d) with ampicillin concentration C1~CnFor abscissa or ordinate, with strength difference Δ ECLiFor ordinate or horizontal seat
Mark establishes examination criteria curve, obtains ampicillin concentration and strength difference Δ ECLiLinear equation；
(e) the ampicillin standard solution in step (a) is replaced with the ampicillin solution of unknown concentration, performs step
(b) and (c)；Determine the ECL strength differences corresponding to the ampicillin solution of unknown concentration；Linear side in step (d)
Journey is the ampicillin concentration in the ampicillin solution for can obtain unknown concentration.
In addition, inventor it has been investigated that, during ampicillin is detected, electric active matter is added in bottom liquid is detected
Matter 1, the double carboxylic acids of 1 '-ferrocene can significantly increase the ECL intensity of molecular engram sensor in ampicillin provided by the invention.
In a kind of specific embodiment of the present invention, 1, a concentration of 1~4mM of the double carboxylic acids of 1 '-ferrocene in bottom liquid is detected.
In the present invention, described 1, the double carboxylic acids of 1 '-ferrocene, structural formula is as follows：
In the present invention, mentioned concentration unit M represents mol/L；
In the present invention, described " ampicillin molecular engram sensor " and " it is stripped of the ampicillin of ampicillin
Molecular engram sensor " has identical meaning, each means the molecular engram sensor for being not bound with ampicillin.
Below in conjunction with the attached drawing in the embodiment of the present invention, the technical solution in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other without creative efforts
Embodiment shall fall within the protection scope of the present invention.
In following each embodiments and full text, AM represents ampicillin；PY represents pyrroles；Luminol represents luminol.
Instrument and material
Electrochemical workstation CHI 660A CH Instruments, Shanghai Chen Hua instrument company；
Fourier Transform Infrared Spectrometer IRAffinity-1 types, Japanese Shimadzu Corporation；
Electrochemial luminescence detecting instrument MPI-E types, Xi'an Analytical Instruments Co., Ltd.；
Required reagent is all commercially available.
Embodiment 1
Prepare ampicillin molecular engram sensor
Step (1) is successively processed by shot blasting gold electrode using 1.0,0.5 and 0.05 μm of alumina powder, after processing
Gold electrode respectively with ethyl alcohol and distilled water sonic oscillation 5 minutes, finally with dry after distilled water flushing；
Pyrroles, luminol and ampicillin are dissolved in 0.1M phosphate buffers (pH 6.0) by step (2), are contained
The polymer fluid of 20mM pyrroles, 5mM luminols and 60mM ampicillins；
Using Ag/AgCl electrodes as reference electrode, platinum electrode is used as to electrode step (3), gold electrode as working electrode,
Cyclic voltammetry is carried out using CHI 660A types electrochemical workstation (CH Instruments, Shanghai Chen Hua instrument company) to sweep
It retouches, voltage range is 0~1V, sweeps speed as 50mV s-1, the scanning number of turns is 5 circles, is obtained in gold electrode surfaces and is combined with ammonia benzyl west
The pyrroles of woods-luminol co-polymer membrane, referred to as AM-MIP films, and be dried at room temperature for handling, the cycle during electropolymerization
Volt-ampere curve figure is as shown in Figure 1；
From figure 1 it appears that during electropolymerization first lap, luminol monomer and pyrrole are respectively obtained in 0.55 and 0.9V or so
Cough up the oxidation peak of monomer.As the polymerization number of turns gradually increases, the oxidation peak current of luminol monomer and pyrrole monomer continuously decreases.
As a result illustrate：The AM-MIP films for being combined with template molecule are successfully aggregated in gold electrode surfaces, and hinder luminol monomers
And further to electrode surface redox reaction occurs for PY monomers.
Further to prove that step (3) polymerization has obtained AM-MIP films, using fourier transform infrared spectroscopy to AM, gather
Pyrroles (abbreviation PPY), poly- luminol (abbreviation PL, by electrochemical polymerization in the 0.1M pH containing 5mM luminol
5 circle of CV scannings is carried out in 6.0 phosphate buffers to be prepared, and is scanned 0~1V of current potential, is swept fast 50mV s-1) and the progress of AM-MIP films
Characterization, as shown in Figure 2.It can be found that AM is in 1773cm in the infrared spectrum of AM-MIP films-1The carboxyl stretching vibration at place
Peak (ν-COOH) and 1384~1398cm-1Neighbouring bimodal (the σ of methyl C-H bending vibrationsC-H), illustrate the template during electropolymerization
Molecule AM is successfully combined in AM-MIP films.It can be found that PPY is in 1529cm in the infrared spectrum of AM-MIP films-1
The C-N asymmetry ring stretching vibrations (ν at placeasC-N) characteristic peak, during illustrating electropolymerization, polypyrrole is formd in AM-MIP films
Structure.In addition, it can not be confirmed successfully to synthesize PL in MIP films with FTIR.But the experiment of subsequent ECL (electrochemical luminescence) can be into
One step confirms PL also in successful polymerization to MIP films.
Gold electrode after polymerization in step (3) is placed in the methanol-acetic acid solution (first that 20mL prepares in advance by step (4)
Alcohol, acetic acid volume ratio are 9:1) in, after twenty minutes, AM is removed for magnetic agitation, and after being then rinsed with water, room temperature is dried.
Embodiment 2
Prepare ampicillin molecular engram sensor
Compared with Example 1, step (1) is identical with (4) for embodiment 2；Step (2), step (3) and embodiment 1
Difference lies in：
Pyrroles, luminol and template molecule ampicillin are dissolved in 0.1M phosphate buffers (pH 6.0) by step (2),
Obtain the polymer fluid containing 30mM pyrroles, 4mM luminols and 30mM ampicillins；
Step (3), voltage range are 0~1V, sweep speed as 80mV s-1, the scanning number of turns is 3 circles.
Embodiment 3
Prepare ampicillin molecular engram sensor
Compared with Example 1, step (1) is identical with (4) for embodiment 3；Step (2), step (3) and embodiment 1
Difference lies in：
Pyrroles, luminol and template molecule ampicillin are dissolved in 0.1M phosphate buffers (pH 6.0) by step (2),
Obtain the polymer fluid containing 10mM pyrroles, 10mM luminols and 80mM ampicillins；
Step (3), voltage range are 0~1V, sweep speed as 30mV s-1, the scanning number of turns is 10 circles.
Embodiment 4
Ampicillin molecular engram sensor prepared by embodiment 1 is detected to act on the specific recognition of AM
By in 1 preparation process of embodiment, the gold electrode (referred to as naked gold electrode) in step (1) before electropolymerization, step (3)
In the obtained gold electrode covered with AM-MIP films, the ampicillin molecular engram sensor of removing AM and again in step (4)
The ampicillin molecular engram sensor after 50 μM of AM is newly combined with respectively as working electrode, and using Ag/AgCl as reference electricity
Pole, Pt are to electrode, form three-electrode system, carry out ECL detections, using the phosphate buffer of pH 8.0 as detection bottom liquid, institute
It obtains testing result and is exported by being equipped with the computer of electrochemical analyser working software, the results are shown in Figure 3；
(the curve a) in Fig. 3, in gold electrode surfaces electropolymerization AM-MIP films from figure 3, it can be seen that compared with naked gold electrode
Afterwards, (curve b) in Fig. 3 is remarkably reinforced in the ECL intensity of electrode.This is because the polypyrrole knot in pyrroles-luminol co-polymer membrane
Structure promotes the electron transmission of Polyluminol structure in film.Meanwhile after electropolymerization obtains AM-MIP films, the template molecule AM in film
Ultra-oxygen anion free radical can be generated with the Effect of Dissolved Oxygen in detection bottom liquid.Ultra-oxygen anion free radical and the poly- Shandong in film
The oxidation intermediate state reaction of minot structure generates a large amount of excitation state intermediates, so as to enhance ECL intensity.When template molecule AM quilts
After washing away, the ECL intensity that removes the ampicillin molecular engram sensor of AM is substantially reduced (curve c) in Fig. 3.It recombines
(curve d) in Fig. 3 is remarkably reinforced in the ECL intensity for having the ampicillin molecular engram sensor after 50 μM of AM again.These experiment knots
Fruit not only shows that the ampicillin molecular engram sensor of removing AM in step (4) has template molecule AM recognition reaction, and
And the AM combined in AM-MIP films can enhance the ECL intensity of Polyluminol structure, therefore ampicillin provided by the invention
Molecular engram sensor is Turn-on type solid phase molecules trace electrochemical sensors, and can utilize and combine the ammonia benzyl after AM again
The ECL intensity enhancings value of XiLin molecular engram sensor quantitative determines the AM contents in detected solution.
Embodiment 5
In the ampicillin solution of ampicillin molecular engram sensor detection unknown concentration prepared by Application Example 1
Ampicillin content, step are as follows：
(a) ammonia of a concentration of 0.07,0.5,0.7,3,10,20,50 μM of ampicillin is prepared respectively using distilled water as solvent
Benzyl XiLin standard solution；And prepare the blank sample solution without ampicillin；
(b) it will be marked respectively in prepared ampicillin by the ampicillin molecular engram sensor of elution ampicillin
It impregnates 15 minutes, is subsequently placed in detection bottom liquid (phosphate buffer solution of pH 8.0) in quasi- product solution and in blank sample solution
Carry out ECL measure；The ECL curves for the ampicillin molecular engram sensor for combining each concentration ampicillin are obtained, such as Fig. 4 A
It is shown；The peak of each curve is attached most importance to reference to the ECL of the ampicillin molecular engram sensor of each concentration ampicillin in Fig. 4 A
Intensity, the ECL intensity with reference to 0,0.07,0.5,0.7,3,10,20,50 μM of ampicillin is respectively 106 (i.e. blank samples again
The corresponding ECL intensity of solution for 106), 131,146,159,201,269,360,651；Wherein, to impregnate ampicillin standard
The ampicillin molecular engram sensor of product solution or blank sample solution is as working electrode, using Ag/AgCl as reference electrode,
Pt is to electrode, forms three-electrode system；
(c) according to formula Δ ECLi=ECLi–ECL0, 1≤i≤n, the ammonia of 0.07,0.5,0.7,3,10,20,50 μM of calculating
The ECL strength differences Δ ECL of benzyl XiLin standard solution, respectively 25,40,53,95,163,254,545；
(d) it using the ECL strength difference Δ ECL of each concentration standards solution obtained as ordinate, is marked with ampicillin
Quasi- product solution concentration establishes the detection linear equation of ampicillin molecular engram sensor for abscissa；Δ ECL=46.23+
10.1C (μM) (r=0.995) (the corresponding standard curve of the equation is as shown in Figure 4 B), the range of linearity are 0.07~50 μM, detection
Sensitivity is 336.67 μM-1cm-2, minimum detection limit (LOD) is 18nM.
(e) a certain unknown concentration ampicillin solution is prepared, chloramphenicol molecular imprinting electrochemistry made from embodiment 1 is sent out
Optical sensor is impregnated in this ampicillin solution, and soaking time is 15 minutes.Step (b) and (c) is performed, obtains unknown concentration ammonia
The corresponding strength difference 126 in benzyl XiLin, the linear equation in step (d) are the substance that can obtain obtaining ampicillin solution
A concentration of 7.9 μM of amount.
Embodiment 6
Determine the amplification that the double carboxylic acids of 1,1 '-ferrocene detect ampicillin
The ampicillin molecular engram sensor of ampicillin will have been eluted respectively not comprising Fc (COOH)2PH 8.0
In phosphate buffer and including 2mM Fc (COOH)28.0 phosphate buffers of pH in carry out ECL detections, will recombine
There is the ampicillin molecular engram sensor of 0.7 μM of ampicillin respectively not comprising Fc (COOH)28.0 phosphoric acid buffers of pH
In liquid and including 2mM Fc (COOH)28.0 phosphate buffers of pH in carry out ECL detections；Testing result is as shown in Figure 5；
From figure 5 it can be seen that 2mM Fc (COOH) are added in detection bottom liquid (8.0 phosphate buffers of pH)2Afterwards, it washes
Nearly 3 times of the ECL intensity enhancings of ampicillin molecular engram sensor after de-, this is because Fc (COOH)2Electrochemistry oxygen
Electrochemical oxidation process of the Polyluminol structure in gold electrode surfaces can be catalyzed by changing product, so as to increase Polyluminol structure oxygen
Change the content of intermediate, effectively enhance its ECL responses.Fc(COOH)2The ECL signal amplifications of Polyluminol structure can be used
In the raising sensor to the detection sensitivity of AM.For example, by again with reference to the ampicillin molecular engram sensing after 0.7 μM of AM
Device is placed in containing 2mM Fc (COOH)28.0 phosphate buffers of pH in carry out ECL measure (curve d), obtained ECL in Fig. 3
Intensity is not add Fc (COOH)2ECL intensity (nearly 3 times of curve c) measurement results in Fig. 3.It follows that utilize Fc
(COOH)2AM can be amplified at double to the electrocatalysis of Polyluminol structure and combine front and rear ampicillin molecular engram sensing again
The ECL Strength Changes of device are expected to improve the detection sensitivity of sensor.
Embodiment 7
It determines to contain Fc (COOH) in detection bottom liquid2Examination criteria curve as electroactive probe
Using the examination criteria curve that ampicillin molecular engram sensor is determined with implementing 5 identical steps, with implementing 5
Difference lies in the ampicillin standard solution concentration that the present embodiment is prepared is respectively 0.7,9,20,500 and 700nM；And
Detection bottom liquid is includes 2mM Fc (COOH)28.0 phosphate buffers of pH；What is obtained combines various concentration ampicillin
Ampicillin molecular engram sensor ECL intensity curves Fig. 6 shown in；With a concentration of abscissa of ampicillin standard solution
Establish the detection linear equation of ampicillin molecular engram sensor；Δ ECL=30.93+0.31C (nM) (r=0.996),
The range of linearity is 0.7~700nM, detection sensitivity 10.33mM-1cm-2, LOD 0.58nM.As it can be seen that at Fc (COOH)2Electricity
Under catalytic amplification, the detection sensitivity of the sensor is enlarged into no Fc (COOH)2When 31 times of (embodiment 5), LOD ratios without
Fc(COOH)2When (embodiment 5) low two orders of magnitude of LOD.
The preparation method and application of provided by the present invention kind of ampicillin molecular engram sensor has been carried out in detail above
It is thin to introduce.Specific embodiment used herein is expounded the principle of the present invention and embodiment, above example
Illustrate to be merely used to help understand method and its central idea of the invention.It should be pointed out that the ordinary skill people for this field
Member for, without departing from the principle of the present invention, can also to the present invention some improvement and modification can also be carried out, these improve and
Modification also falls into the protection of the claims in the present invention.
Claims (10)
1. a kind of preparation method of ampicillin molecular engram sensor, which is characterized in that including：
(1) inert electrode is pre-processed；
(2) copolymerization monomer pyrroles, luminol and template molecule ampicillin are dissolved in the buffer solution that pH value is 4~7, obtained
To polymer fluid, wherein, the ratio of the amount of the substance of copolymerization monomer pyrroles and luminol is (1~8)：1；Pyrroles and ampicillin
The ratio of the amount of substance is 1：(1~6)；
(3) pretreated inert electrode is placed in polymer fluid and electropolymerization is carried out using cyclic voltammetry, in inertia electricity
Pole surface obtains being combined with pyrroles-luminol co-polymer membrane of ampicillin；
(4) ampicillin combined with eluant, eluent removal co-polymer membrane, obtains ampicillin molecular engram sensor.
2. the method as described in claim 1, which is characterized in that the inert electrode is selected from gold electrode, platinum electrode, glass carbon electricity
Pole or graphite electrode.
3. the method as described in claim 1, which is characterized in that the buffer solution be phosphate buffer solution, pH value 6.
4. the method as described in claim 1, which is characterized in that the polymerizing condition that electropolymerization is carried out using cyclic voltammetry
Including：Scanning current potential is 0~1V；Speed is swept as 30~80mV s-1In any value, the scan round number of turns for 3~10 circle in appoint
One value.
5. the method as described in claim 1, which is characterized in that the eluant, eluent is 9:The methanol-acetic acid solution of 1 (v/v),
The NaOH solution of 0.01M or the HCl solution of 0.01M.
6. the method as described in claim 1, which is characterized in that a concentration of (10~30) mM of the pyrroles in buffer solution.
7. the ampicillin molecular engram sensor prepared by method according to any one of claims 1 to 6.
8. application of the ampicillin molecular engram sensor in ampicillin molecule is detected described in claim 7.
9. application as claimed in claim 8, which is characterized in that described the step of detecting ampicillin molecule includes：
(a) different known concentration C are prepared1~CnAmpicillin standard solution and blank sample solution without ampicillin；
(b) by ampicillin molecular engram sensor respectively in prepared ampicillin standard solution and blank sample is molten
After impregnating specified time in liquid, it is respectively placed in detection bottom liquid and measures the ampicillin molecule for combining various concentration ampicillin
The ECL intensity of trace sensor；
(c) C is calculated1~CnThe ECL intensity differences of the corresponding ampicillin molecular engram sensor of concentration ampicillin standard solution
It is worth Δ ECL1~Δ ECLn, wherein, Δ ECLi=ECLi–ECL0, 1≤i≤n, ECL0For the corresponding ammonia benzyl west of blank sample solution
The ECL intensity of woods molecular engram sensor；
(d) with ampicillin concentration C1~CnFor abscissa or ordinate, with strength difference Δ ECLiIt is built for ordinate or abscissa
Day-mark directrix curve obtains ampicillin concentration and strength difference Δ ECLiLinear equation；
(e) ampicillin standard solution is replaced with the ampicillin solution of unknown concentration, performs step (b) and (c)；It determines not
Know the ECL strength differences corresponding to the ampicillin solution of concentration；Linear equation in step (d) can obtain unknown dense
Ampicillin concentration in the ampicillin solution of degree.
10. application as claimed in claim 9, which is characterized in that contain electroactive material 1 in the detection bottom liquid in step (b),
The double carboxylic acids of 1 '-ferrocene.
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