CN101787125B - Preparation method of pyrrole/N-(2-carboxyethyl) pyrrole composite nanoparticles and application thereof - Google Patents
Preparation method of pyrrole/N-(2-carboxyethyl) pyrrole composite nanoparticles and application thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of pyrrole/N-(2-carboxyethyl) pyrrole composite nanoparticles, comprising the following steps of: pretreating pyrrole monomer, chemically reacting, separating and drying. The preparation method has the advantages of simple and convenient operation as well as nano-grade and epigranular prepared product. The prepared pyrrole/N-(2-carboxyethyl) pyrrole composite nanoparticles have higher specific surface area and more carboxyl functional groups, increase the load capacity of amino ruthenium bipyridine derivatives and ensure that electrochemical luminescence signals are enhanced. A thrombin electrochemical luminescence probe prepared from the pyrrole/N-(2-carboxyethyl) pyrrole composite nanoparticles can be used for detecting thrombin, has favorable selectivity, sensitivity and detection limit and can be used on a medical trace detection instrument.
Description
Technical field
The invention belongs to heterogeneous ring compound, contain two or more heterocycles, as only ring hetero atom technical field, be specifically related to the direct-connected pyrroles/N-of a kind of key (2-propyloic) pyrrole composite nanoparticles by annular atoms-annular atoms with nitrogen-atoms.
Background technology
Zymoplasm is a kind of important physical proteolytic enzyme, in physiology such as blood coagulation, inflammation and wound healing and pathologic process, plays a significant role.Because malignant tumor patient often has bleeding tendency in various degree; Tumor tissues is to the invasion and attack of surrounding tissue, the formation of transfer and the variation of tumor tissues and tumour patient blood itself; All can cause the change of patient's clotting mechanism; And the concentration of zymoplasm and active in the important indicator of weighing clotting mechanism to disclosing the mechanism of tumour, or is judged as early diagnosis, molecule parting, curative effect and prognosis and to be significant.Therefore it is significant medically to detect zymoplasm.Tradition is directly measured the zymoplasm method and is mainly contained fluorescent method and chromophoric substrate method.Fluorescent method is with fluorogenic substrate mark zymoplasm, and through measuring the content that fluorescence intensity draws zymoplasm, wherein markers step is more loaded down with trivial details.The chromophoric substrate method utilizes zymoplasm to cut off itself and the point of application of color development peptide, makes p-Nitroaniline dissociate out, and solution is apparent light yellow, in 405～410nm wavelength light is had maximum absorption, can carry out quantitatively determined in view of the above, and this method is simple.But it is exactly that sensitivity is not high that two kinds of methods all have a drawback, should not carry out trace analysis, can't adapt to the trace detection of modern medicine requirement.
That the electrochemiluminescence analysis method has is highly sensitive, linearity range is wide, equipment is simple, easy to operate, quick, easily be automated, controllability is strong, highly active luminous reaction material can be provided and save characteristics such as reagent, has been applied to fields such as environmental science, life science and Materials science.In the numerous electrochemiluminescence reagent of electrochemiluminescence analysis, tris (bipyridine) ruthenium Ru (bpy)
3 2+Owing to have good water solubility, stable chemical performance, redox reversible, characteristics such as luminous efficiency is high, pH a wider range of application and being widely used in the research of electrochemiluminescence analysis.But the liquid phase electrochemiluminescence analysis at current system detects in the application, because expensive Ru (bpy)
3 2+Or related derivatives as flowing reactive reagent by mass consumption, cause higher analysis cost, thereby limited Ru (bpy)
3 2+Application.And present permanent load method mainly contains physically trapping, electrostatic adhesion, covalent bonding etc.; Physically trapping wherein, the problem that ubiquities such as electrostatic adhesion leak, covalent bonding fixing comparatively firm; But if be fixed on the general block materials, fixed amount is little, if be fixed on the inorganic nano material; On silicon dioxide microsphere,, the electrochemiluminescence analysis target compound is produced certain influence because its bad electroconductibility can influence the conduction of electronics.
The polypyrrole base polymer has plurality of advantages such as good environmental stability, simple and easy synthetic, high conductivity and excellent biological compatibility; These character are given its specific performance that is different from other conductive polymerss, become one of preferential material of selecting of chemistry or biosensor.Especially the polypyrrole base polymer of nanostructure has special mechanics, magnetics, calorifics, optics and electrochemical activity; And polypyrrole base polymer nanometer is simple, and chemical oxidising polymerisation and electrooxidation polymerization all can be implemented, and makes it show wide application prospect in fields such as biomedical engineering, clinical diagnosis, environmental monitoring, food sanitation and science.Especially nanotechnology is introduced biosensor, improved detection sensitivity, shorten the detection time of response, increased the service life, can realize high-throughout real-time detection.Polypyrrole base polymer nano material is fixed biomolecules can improve supported quantity; Basal electrode modified to improve electro catalytic activity, thereby prepare the responsiveness height, susceptibility is high, selectivity is high and stable high novel nano biosensor.The research that at present N-(2-propyloic) pyrrole monomer (English name is 1H-Pyrrole-1-propanoic acid) is prepared pyrroles/N-(2-propyloic) pyrrole composite nanoparticles for the method for functionalized reagent and pyrrole monomer employing chemical oxidising polymerisation and be applied to prepare zymoplasm electrochemiluminescence probe does not appear in the newspapers as yet.
Summary of the invention
It is the preparation method of nano level and even-grained pyrroles/N-(2-propyloic) pyrrole composite nanoparticles that a technical problem to be solved by this invention is to provide a kind of easy and simple to handle, prepared product.
Another technical problem to be solved by this invention is for pyrroles/N-(2-propyloic) pyrrole composite nanoparticles a kind of purposes to be provided.
Solving the problems of the technologies described above the technical scheme that is adopted is that it is made up of following step:
1, pre-treatment pyrrole monomer
Commercially available pyrroles in the 0.02MPa underpressure distillation, is collected 70～80 ℃ of cuts, obtain pyrrole monomer, the inflated with nitrogen protection is kept in Dark Place in 4 ℃ of refrigerators, and is subsequent use.
2, chemical reaction
With pyrrole monomer and N-(2-propyloic) pyrrole monomer is 1: 0.125～1 to join in the Erlenmeyer flask that fills Pentyl alcohol in molar ratio; The concussion dissolving; Add the water phase surfactant mixture that mass concentration is 86.8g/L again; Fully stirred 30 minutes, slowly add oxygenant, pyrrole monomer and Pentyl alcohol, mass concentration are that the water phase surfactant mixture of 86.8g/L, the volume ratio of oxygenant are 1: 1.5～2.7: 80～160: 12～21.1; Stirring at room was reacted 12 hours, obtained containing the mixed solution of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
Above-mentioned pyrroles and N-(2-propyloic) pyrrole monomer is purchased the company in Sigma; Tensio-active agent is sodium lauryl sulphate or Supragil GN or X 2073, purchases in Aladdin company; Oxygenant is FeCl
36H
2O or (NH
4)
2S
2O
8, produce by Xi'an chemical reagent factory.
3, separate
In the mixed solution that contains pyrroles/N-(2-propyloic) pyrrole composite nanoparticles, add the methyl alcohol breakdown of emulsion; Containing pyrroles/N-(2-propyloic) mixed solution of pyrrole composite nanoparticles and the volume ratio of methyl alcohol is 1: 2; 8000 rev/mins of spinnings 10 minutes; Throw out is with methanol wash 2～3 times, again with zero(ppm) water be washed till zero(ppm) water colourless till.
4, drying
Throw out after the washing 60 ℃ of dryings 3～5 hours in vacuum tightness is the electric vacuum drying oven of 0.01MPa are prepared into pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
In chemical reaction step 2 of the present invention; Is to join in the Erlenmeyer flask that fill Pentyl alcohol at 1: 0.5 pyrrole monomer and N-(2-propyloic) pyrrole monomer by optimum mole ratio; The concussion dissolving adds the water phase surfactant mixture that mass concentration is 86.8g/L again, fully stirs 30 minutes; Slowly add oxygenant; Pyrrole monomer and Pentyl alcohol, mass concentration are that the water phase surfactant mixture of 86.8g/L, the optimum volume ratio of oxygenant are 1: 2: 120: 16, and stirring at room was reacted 12 hours, obtained containing the mixed solution of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
The purposes of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles in preparation zymoplasm electrochemiluminescence probe.
The method of use of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles in preparation zymoplasm electrochemiluminescence probe is following:
It is in 7.40 the phosphate buffer soln that the amino succinic diamide of N-and 1-ethyl-3-(3-dimethylamino) carbodiimide hydrochloride are dissolved in pH; The amount of substance concentration of the amino succinic diamide of N-and 1-ethyl-3-(3-dimethylamino) carbodiimide hydrochloride is 0.1mol/L in phosphate buffer soln; Add pyrroles/N-(2-propyloic) pyrrole composite nanoparticles; Room temperature activation 1 hour; Adding amount of substance concentration again is the fit II of zymoplasm of 10 μ mol/L and the amino ruthenium dipyridyl derivatives that amount of substance concentration is 6.3 μ mol/L; The mass ratio of amino ruthenium dipyridyl derivatives and the fit II of zymoplasm, the amino succinic diamide of N-, 1-ethyl-3-(3-dimethylamino) carbodiimide hydrochloride, pyrroles/N-(2-propyloic) pyrrole composite nanoparticles is 1: 11: 4020: 6678: 700; Stirred overnight at room temperature, 12000 rev/mins of spinnings 10 minutes, throw out uses amount of substance concentration to be the NaCl solution washing of 0.5mol/L 3 times; Gained deposition concussion is scattered in pH and in 7.40 the phosphate buffer soln, is prepared into zymoplasm electrochemiluminescence probe.
Graphite Electrodes is inserted in the aqueous solution contain the Repone K that Sulphanilic Acid that amount of substance concentration is 0.005mol/L and amount of substance concentration is 0.10mol/L; Sweep four circles with the speed of sweeping of 10mV/s from 0.5～1.40V with cyclic voltammetry; Take out electrode; With distilled water flushing 1 minute, be the PCl of 0.04mol/L in amount of substance concentration
5Acetone soln in soaked 30 minutes; Take out electrode; Using pH is 7.40 phosphate buffer soln flushing electrode surface 3 minutes, and dripping amount of substance concentration to electrode surface is the fit I of zymoplasm of 10 μ mol/L, and room temperature was cultivated 6 hours; Using pH is 7.40 phosphate buffer soln cleaning electrode surface 3 minutes, is prepared into the fit transmitter of zymoplasm.On the fit transmitter of zymoplasm, dripping amount of substance concentration is 1.0 * 10
-15～1.0 * 10
-12The zymoplasm of mol/L; Room temperature was cultivated 30 minutes; Using pH is 7.40 phosphate buffer soln cleaning electrode surface 3 minutes, drips zymoplasm electrochemiluminescence probe again, and room temperature was cultivated 20 minutes; Using pH is 7.40 phosphate buffer soln cleaning electrode surface 3 minutes, and room temperature is placed to the electrode surface drying.The gained electrode is used to detect zymoplasm.
The amino succinic diamide of above-mentioned N-and 1-ethyl-3-(3-dimethylamino) carbodiimide hydrochloride are all available from Sigma company; (english name is that (2,2 '-bipyridine) (2,2 '-bipyridine-4,4 '-dicarboxylic acid)-ethylenediamine) are that this laboratory is according to document (Terpetschnig, E. to Ruthenium bis to amino ruthenium dipyridyl derivatives; Szmacinski, H.; Malak, H.; Lakowicz, J.R.Biophys.J.1995,68,342-350.) synthetic; The fit I of zymoplasm is 5 '-NH
2-(CH
2)
6-GGT TGG TGT GGTTGG-3 ' (15 bases), the fit II of zymoplasm be 5 '-NH
2-(CH
2)
6-AGT CCG TGG TAG GGC AGG TTG GGGTGA CT-3 ' (29 bases) all purchases the biotechnology ltd in Shanghai; Zymoplasm is purchased in magnificent biotech firm; Sulphanilic Acid is purchased in Chemical Reagent Co., Ltd., Sinopharm Group.
Pyrroles/N-(2-propyloic) pyrrole composite nanoparticles of the present invention's preparation has higher specific surface area and more carboxyl functional group, has increased the charge capacity of amino ruthenium dipyridyl derivatives, and the electrochemiluminescence signal is strengthened.Adopt pyrroles/N-(2-propyloic) pyrrole composite nanoparticles to prepare zymoplasm electrochemiluminescence probe, can be used for detecting zymoplasm, have good selectivity, sensitivity and detection limit, can on medical science trace detection instrument, use.
Description of drawings
Fig. 1 is the transmission electron microscope picture of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles of embodiment 1 preparation.
Fig. 2 is the sem photograph of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles of embodiment 1 preparation.
Fig. 3 is the infrared spectrogram of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles of embodiment 1 preparation.
Fig. 4 is the X ray electron diffraction spectrogram of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles of embodiment 1 preparation.
Fig. 5 is the infrared spectrogram of prepared pyrroles/N-(2-propyloic) pyrrole composite nanoparticles of pyrrole monomer and N-(2-propyloic) pyrroles's different mol ratio.
Embodiment
To further explain of the present invention, but the invention is not restricted to these embodiment below in conjunction with accompanying drawing and embodiment.
1, pre-treatment pyrrole monomer
Commercially available pyrroles in the 0.02MPa underpressure distillation, is collected 70～80 ℃ of cuts, obtain pyrrole monomer, the inflated with nitrogen protection is kept in Dark Place in 4 ℃ of refrigerators, and is subsequent use.
2, chemical reaction
Get N-(2-propyloic) pyrrole monomer 0.5026g, pyrrole monomer 0.5mL; Join in the Erlenmeyer flask that fills the 1mL Pentyl alcohol; The mol ratio of pyrrole monomer and N-(2-propyloic) pyrrole monomer is 1: 0.5, the concussion dissolving, and adding the 60mL mass concentration is the lauryl sodium sulfate aqueous solution of 86.8g/L; Fully stirred 30 minutes, slowly adding 8mL amount of substance concentration is the FeCl of 1.35mol/L
36H
2The O aqueous solution, pyrrole monomer and Pentyl alcohol, mass concentration are that lauryl sodium sulfate aqueous solution, the amount of substance concentration of 86.8g/L is the FeCl of 1.35mol/L
36H
2The volume ratio of the O aqueous solution is 1: 2: 120: 16, and stirring at room was reacted 12 hours, obtained containing the mixed solution of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
3, separate
In the mixed solution that contains pyrroles/N-(2-propyloic) pyrrole composite nanoparticles, add the methyl alcohol breakdown of emulsion; Containing pyrroles/N-(2-propyloic) mixed solution of pyrrole composite nanoparticles and the volume ratio of methyl alcohol is 1: 2; 8000 rev/mins of spinnings 10 minutes; Throw out is with methanol wash 2～3 times, again with zero(ppm) water be washed till zero(ppm) water colourless till.
4, drying
Throw out after the washing 60 ℃ of dryings 3～5 hours in vacuum tightness is the electric vacuum drying oven of 0.01MPa are prepared into pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
Prepared product characterizes with transmission electron microscope, sem, IR, x-ray diffractometer, and the transmission electron microscope observed result is seen Fig. 1, and electron scanning micrograph is seen Fig. 2, and infrared spectrogram is seen Fig. 3, and the X-ray diffraction spectrogram is seen Fig. 4.
Visible by Fig. 1, Fig. 2, product type of being sphere is disperseed comparatively evenly, and particle diameter is 40～50nm.
Visible by Fig. 3,3440,1545cm
-1For on the pyrrole ring-charateristic avsorption band of NH, 1308cm
-1And 1043cm
-1Be respectively the formation vibration absorption peak of C-N stretching vibration absorption peak and c h bond on the pyrrole ring, 1702cm
-1The charateristic avsorption band at place has then confirmed the existence of carboxyl on the polymkeric substance.Through the ir data analysis, prepared product is pyrroles and two kinds of monomeric mixtures of N-(2-propyloic) pyrroles, and the surface has the functionalization carboxyl functional group.
Visible by Fig. 4; The unformed diffraction peak of polypyrrole has appearred in 2 θ between 15 ° and 30 °; Some spikes also appear in addition; The better crystallinity degree that prepared pyrroles/N-(2-propyloic) pyrrole composite nanoparticles is described is in polypyrrole, and polypyrrole does not insolublely melt for improving, unmanageable character has certain meaning.At present, calculating the most frequently used method of percent crystallinity through X-ray diffracting spectrum is the swarming method.The swarming method is at first carried out swarming to diffracting spectrum and is handled, and calculates the area at each peak, then with total peak area divided by total diffraction area, calculate percent crystallinity.Through the computer software simulation, the percent crystallinity of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles is 7.9%.
Embodiment 2
In the chemical reaction step 2 of present embodiment; Get N-(2-propyloic) pyrrole monomer 0.1256g, pyrrole monomer 0.5mL, join in the Erlenmeyer flask that fills the 0.75mL Pentyl alcohol, the mol ratio of pyrrole monomer and N-(2-propyloic) pyrrole monomer is 1: 0.125; The concussion dissolving; Adding the 40mL mass concentration is the lauryl sodium sulfate aqueous solution of 86.8g/L, fully stirs 30 minutes, and slowly adding 6mL amount of substance concentration is the FeCl of 1.35mol/L
36H
2The O aqueous solution, pyrrole monomer and Pentyl alcohol, mass concentration are that lauryl sodium sulfate aqueous solution, the amount of substance concentration of 86.8g/L is the FeCl of 1.35mol/L
36H
2The volume ratio of the O aqueous solution is 1: 1.5: 80: 12, and stirring at room was reacted 12 hours, obtained containing the mixed solution of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.Other steps are identical with embodiment 1.Be prepared into pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
Embodiment 3
In the chemical reaction step 2 of present embodiment; Get N-(2-propyloic) pyrrole monomer 1.0052g, pyrrole monomer 0.5mL, join in the Erlenmeyer flask that fills the 1.35mL Pentyl alcohol, the mol ratio of pyrrole monomer and N-(2-propyloic) pyrrole monomer is 1: 1; The concussion dissolving; Adding the 80mL mass concentration is the lauryl sodium sulfate aqueous solution of 86.8g/L, fully stirs 30 minutes, and slowly adding 10.55mL amount of substance concentration is the FeCl of 1.35mol/L
36H
2The O aqueous solution, pyrrole monomer and Pentyl alcohol, mass concentration are that lauryl sodium sulfate aqueous solution, the amount of substance concentration of 86.8g/L is the FeCl of 1.35mol/L
36H
2The volume ratio of the O aqueous solution is 1: 2.7: 160: 21.1, and stirring at room was reacted 12 hours, obtained containing the mixed solution of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.Other steps are identical with embodiment 1.Be prepared into pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
Embodiment 4
In the chemical reaction step 2 of the foregoing description 1～3, used tensio-active agent sodium lauryl sulphate such as uses at the Supragil GN replacement of quality, oxygenant FeCl
36H
2O is with equimolar (NH
4)
2S
2O
8Replacement, other steps of this step are identical with respective embodiments.Other steps are identical with embodiment 1.Be prepared into pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
Embodiment 5
In the chemical reaction step 2 of the foregoing description 1～3, used tensio-active agent sodium lauryl sulphate such as uses at the X 2073 replacement of quality, and other steps of this step are identical with respective embodiments.Other steps are identical with embodiment 1.Be prepared into pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
Embodiment 6
Adopt the application of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles in preparation zymoplasm electrochemiluminescence probe of the embodiment of the invention 1 preparation following:
It is in 7.40 the phosphate buffer soln that the amino succinic diamide of N-and 1-ethyl-3-(3-dimethylamino) carbodiimide hydrochloride are dissolved in pH; The amount of substance concentration of the amino succinic diamide of N-and 1-ethyl-3-(3-dimethylamino) carbodiimide hydrochloride is 0.1mol/L in phosphate buffer soln; Add 2mg pyrroles/N-(2-propyloic) pyrrole composite nanoparticles; Room temperature activation 1 hour; Adding fit II of zymoplasm and the 500 μ L amount of substance concentration that 350 μ L amount of substance concentration are 10 μ mol/L again is the amino ruthenium dipyridyl derivatives of 6.3 μ mol/L; The mass ratio of amino ruthenium dipyridyl derivatives and the fit II of zymoplasm, the amino succinic diamide of N-, 1-ethyl-3-(3-dimethylamino) carbodiimide hydrochloride, pyrroles/N-(2-propyloic) pyrrole composite nanoparticles is 1: 11: 4020: 6678: 700; Stirred overnight at room temperature, 12000 rev/mins of spinnings 10 minutes, throw out uses amount of substance concentration to be the NaCl solution washing of 0.5mol/L 3 times; Gained deposition concussion is scattered in 1mL pH and in 7.40 the phosphate buffer soln, is prepared into zymoplasm electrochemiluminescence probe.
Graphite Electrodes is inserted in the aqueous solution that 2mL contains the Repone K that Sulphanilic Acid that amount of substance concentration is 0.005mol/L and amount of substance concentration is 0.10mol/L; Sweep four circles with the speed of sweeping of 10mV/s from 0.5～1.40V with cyclic voltammetry; Take out electrode; With distilled water flushing 1 minute, be the PCl of 0.04mol/L in 2mL amount of substance concentration
5Soaked 30 minutes in the acetone soln; Take out electrode; Using pH is 7.40 phosphate buffer soln flushing electrode surface 3 minutes, is the fit I of zymoplasm of 10 μ mol/L to electrode surface Dropwise 5 μ L amount of substance concentration, and room temperature was cultivated 6 hours; Using pH is 7.40 phosphate buffer soln cleaning electrode surface 3 minutes, is prepared into the fit transmitter of zymoplasm.Dropwise 5 μ L amount of substance concentration is 1.0 * 10 on the fit transmitter of zymoplasm
-15～1.0 * 10
-12The zymoplasm of mol/L; Room temperature was cultivated 30 minutes; Using pH is 7.40 phosphate buffer soln cleaning electrode surface 3 minutes, drips 3 μ L zymoplasm electrochemiluminescence probes again, and room temperature was cultivated 20 minutes; Using pH is 7.40 phosphate buffer soln cleaning electrode surface 3 minutes, and room temperature is placed to the electrode surface drying.The gained electrode is used to detect zymoplasm.
The method that pyrroles/N-(2-propyloic) pyrrole composite nanoparticles of other embodiment preparation is used to prepare zymoplasm electrochemiluminescence probe is identical with aforesaid method.
In order to confirm optimised process step of the present invention, the contriver has carried out a large amount of laboratory study tests, and various test situation are following:
Laboratory apparatus: high resolution transmission electron microscopy, model are JEM-2100, are produced by company of NEC; Buddhist nun's high-tensile strength FTIR Spectrometer FTIR, model are AVTAR360, are produced by Buddhist nun's high-tensile strength instrument company.
1, N-(2-propyloic) pyrrole monomer consumption is to the influence of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles particle diameter
Get totally 6 parts of N-(2-propyloic) pyrrole monomer 0.1256g, 0.1884g, 0.2512g, 0.3768g, 0.5026g, 1.0052g; Join respectively in the Erlenmeyer flask that fills the 1mL Pentyl alcohol; Add the 0.5mL pyrrole monomer more respectively, the mol ratio of pyrrole monomer and N-(2-propyloic) pyrrole monomer was respectively 1: 0.125,1: 0.167,1: 0.25,1: 0.333,1: 0.5,1: 1.Other steps are identical with embodiment 1, are prepared into pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
Prepared product is observed with transmission electron microscope, and observed result is seen table 1; Adopt FTIR Spectrometer FTIR to characterize, infrared spectrogram is seen Fig. 5.
Table 1 N-(2-propyloic) pyrrole monomer consumption is to the influence of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles particle diameter
| Pyrrole monomer and N-(2-propyloic) pyrrole monomer mol |
1∶0.125 | 1∶0.167 | 1∶0.25 | 1∶0.333 | 1∶0.5 | 1∶1 |
| Pyrroles/N-(2-propyloic) pyrrole composite nanoparticles particle diameter (nm) | 200 | 100 | 50 | 40 | 40 | 80 |
Can be known that by table 1 mol ratio of pyrrole monomer and N-(2-propyloic) pyrrole monomer is bigger to the grain diameter influence of product, the mol ratio of pyrrole monomer and N-(2-propyloic) pyrrole monomer is 1: 0.125 o'clock; The particle diameter of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles is 200nm, and mol ratio is 1: 0.167 o'clock, and pyrroles/N-(2-propyloic) pyrrole composite nanoparticles particle diameter is 100nm; Increasing along with N-(2-propyloic) pyrrole monomer amount; Particle diameter reduces gradually, and mol ratio is 1: 0.5 o'clock, and the particle diameter of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles is 40nm; Type of being is spherical; And disperse comparatively evenly, mol ratio is 1: 1 o'clock, and particle diameter increases again and is 80nm.
In Fig. 5, curve a, b, c, d, e, f are respectively that the mol ratio of pyrrole monomer and N-(2-propyloic) pyrrole monomer is 1: 0.125,1: 0.167,1: 0.25,1: 0.333,1: 0.5,1: 1 an ir spectra curve.Visible by Fig. 5, carboxyl peak, last 1702 place of curve a and curve b is not too obvious, along with the increasing of N-(2-propyloic) pyrrole monomer amount, 1702cm on the ir spectra
-1The intensity at peak is grow gradually, and the mol ratio of pyrrole monomer and N-(2-propyloic) pyrrole monomer is 1: 0.5,1: 1 o'clock, and the carboxyl peak is comparatively obvious, has confirmed the existence of carboxyl on the nanoparticle, is applicable to preparation zymoplasm electrochemiluminescence probe.
Can be known that by above experimental data the mol ratio of pyrrole monomer and N-(2-propyloic) pyrrole monomer is at 1: 0.125～1 o'clock, the particle diameter of prepared pyrroles/N-(2-propyloic) pyrrole composite nanoparticles is all less.It is 1: 0.125～1 that the present invention selects the mol ratio of pyrrole monomer and N-(2-propyloic) pyrrole monomer, optimal selection 1: 0.5.
In order to verify beneficial effect of the present invention, the contriver utilizes the electrochemiluminescence analysis system that the effect of the electrode detection zymoplasm of embodiment 6 preparations is tested, and various test situation are following:
Laboratory apparatus: MPI-E type Electrochemiluminescprocess process system, limited instrument company produces by the auspicious analysis advanced in years in Xi'an.
Experimental technique: with the electrode of the embodiment of the invention 6 preparation as working electrode; Being inserted into 2mL and containing in the electrochemiluminescence pond of phosphate buffer soln of positive tripropyl amine that amount of substance concentration is 0.1mol/L, is to the utmost point with platinum electrode, is reference electrode with Ag/AgCl (saturated KCl); On MPI-E Electrochemiluminescprocess process appearance; Adopt chronoamperometry, when current potential is 1.35V, the zymoplasm of different concns is carried out the electrochemiluminescence strength test.
Experimental result shows that the concentration of zymoplasm is 1.0 * 10
-15～1.0 * 10
-12During mol/L, the logarithm of concentration of thrombin and electrochemiluminescence intensity are linear dependence, and its equation of linear regression is:
S＝722.01×lgC+10956
Wherein S is an electrochemiluminescence intensity, and C is the concentration of zymoplasm, coefficient R
2=0.9958, detect and be limited to 5.0 * 10
-16Mol/L (S/N=3) is lower than reported values 6 * 10
-11Mol/L.
Simultaneously in order to verify that the fit I of zymoplasm, the fit II of zymoplasm only have the specific recognition effect to zymoplasm, respectively to 1.0 * 10
-10Mol/L bovine serum albumin, 1.0 * 10
-10Mol/L oxyphorase, 1.0 * 10
-10Mol/L immunoglobulin G, 1.0 * 10
-10Mol/L N,O-Diacetylmuramidase, 1.0 * 10
-12The zymoplasm of mol/L and 1.0 * 10
-12The zymoplasm of mol/L and above-mentioned four kinds of composite mixing solutionss of the albumen with isoconcentration detect; Not add zymoplasm is blank; Measure electrochemiluminescence intensity under the same conditions, horizontal survey three times, calculate electrochemiluminescence intensity enhancing value according to following formula:
Test and calculation result are seen table 2.
The different proteic electrochemiluminescence intensity of the electrode detection of table 2 embodiment 6 preparations
| N,O-Diacetylmuramidase | Oxyphorase | Bovine serum albumin | Immunoglobulin G | Blank | Zymoplasm | Mixing solutions | |
| Luminous intensity (a.u.) | 126±29 | 136±35 | 125±22 | ?139±21 | 110±23 | 2342±137 | ?2333±240 |
| Luminous intensity enhancing value (%) | 0.72 | 1.2 | 0.67 | ?1.3 | —— | —— | ?99.6 |
Visible by table 2; Use bovine serum albumin, oxyphorase, immunoglobulin G, when N,O-Diacetylmuramidase experimentizes; Can only detect the electrochemiluminescence signal close with blank value; Compare with the electrochemiluminescence intensity enhancing value of zymoplasm, increase 0.67%, 1.2%, 1.3%, 0.72% respectively.When this explanation has only zymoplasm to exist, could make up sandwich structure, detect very strong electrochemiluminescence signal.The electrochemiluminescence intensity and 1.0 * 10 that mixing solutions records
-12The electrochemiluminescence intensity of mol/L zymoplasm gained does not have significant difference, explains that the zymoplasm electrochemiluminescence probe for preparing is used to detect zymoplasm and has good selectivity.
Claims (3)
1. the preparation method of a pyrroles/N-(2-propyloic) pyrrole composite nanoparticles is characterized in that it is made up of following step:
(1) pre-treatment pyrrole monomer
Commercially available pyrroles in the 0.02MPa underpressure distillation, is collected 70～80 ℃ of cuts, obtain pyrrole monomer, the inflated with nitrogen protection is kept in Dark Place in 4 ℃ of refrigerators, and is subsequent use;
(2) chemical reaction
With pyrrole monomer and N-(2-propyloic) pyrrole monomer is 1: 0.125～1 to join in the Erlenmeyer flask that fills Pentyl alcohol in molar ratio; The concussion dissolving; Add the water phase surfactant mixture that mass concentration is 86.8g/L again; Fully stirred 30 minutes, slowly add oxygenant, pyrrole monomer and Pentyl alcohol, mass concentration are that the water phase surfactant mixture of 86.8g/L, the volume ratio of oxygenant are 1: 1.5～2.7: 80～160: 12～21.1; Stirring at room was reacted 12 hours, obtained containing the mixed solution of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles;
Above-mentioned tensio-active agent is sodium lauryl sulphate or Supragil GN or X 2073; Oxygenant is that amount of substance concentration is the FeCl of 1.35mol/L
36H
2The O aqueous solution;
(3) separate
In the mixed solution that contains pyrroles/N-(2-propyloic) pyrrole composite nanoparticles, add the methyl alcohol breakdown of emulsion; Containing pyrroles/N-(2-propyloic) mixed solution of pyrrole composite nanoparticles and the volume ratio of methyl alcohol is 1: 2; 8000 rev/mins of spinnings 10 minutes; Throw out is with methanol wash 2～3 times, and it is colourless using zero(ppm) water to be washed till zero(ppm) water again;
(4) drying
Throw out after the washing 60 ℃ of dryings 3～5 hours in vacuum tightness is the electric vacuum drying oven of 0.01MPa are prepared into pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
2. according to the preparation method of the described pyrroles/N-of claim 1 (2-propyloic) pyrrole composite nanoparticles; It is characterized in that: in chemical reaction step (2); With pyrrole monomer and N-(2-propyloic) pyrrole monomer is to join in the Erlenmeyer flask that fill Pentyl alcohol at 1: 0.5 in molar ratio; The concussion dissolving adds the water phase surfactant mixture that mass concentration is 86.8g/L again, fully stirs 30 minutes; Slowly add oxygenant; Pyrrole monomer and Pentyl alcohol, mass concentration are that the water phase surfactant mixture of 86.8g/L, the volume ratio of oxygenant are 1: 2: 120: 16, and stirring at room 12 hours obtains containing the mixed solution of pyrroles/N-(2-propyloic) pyrrole composite nanoparticles.
3. pyrroles/N-(2-propyloic) pyrrole composite nanoparticles of the preparation method of the described pyrroles/N-of claim 1 (2-propyloic) pyrrole composite nanoparticles preparation, the purposes in preparation zymoplasm electrochemiluminescence probe.
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