CN107356571A - A kind of method for determining surface charge - Google Patents
A kind of method for determining surface charge Download PDFInfo
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- CN107356571A CN107356571A CN201710498660.0A CN201710498660A CN107356571A CN 107356571 A CN107356571 A CN 107356571A CN 201710498660 A CN201710498660 A CN 201710498660A CN 107356571 A CN107356571 A CN 107356571A
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- determinand
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a kind of method for determining surface charge, for making visualization quantitative determination to the surface charge distribution state and its charge density of rough surface and irregular particle or membranoid substance, determinand is placed in buffer solution, add the fluorescent nano particles with determinand surface charge contrary sign electric charge, fluorescent nano particles and the electric charge on determinand surface combine because electrostatic attraction acts on, wash the free fluorescent nano particles for being not bonded to determinand surface charge off, by the way that determinand to be placed under fluorescence microscope to the fluorescence distribution state and intensity that carry out fluorescence imaging and observe fluorescent nano particles, the distribution of determinand surface charge is can determine that from its fluorescence distribution state, fluorescence intensity by determining every bit can determine that the charge density of the point.This method is easy to operate, quick, and energy Visual retrieval has the surface charge distributed in three dimensions state that various configurations of surface include considerably complicated coarse and irregular surface particle or membranoid substance, and detection resolution is tens nanometer.
Description
Technical field
The invention belongs to optical technical field, is related to a kind of fluorescence imaging with powered fluorescent nano particles and detects skill
Art, and in particular to a kind of fluorescence by determining the fluorescent nano particles combined with body surface electric charge by electrostatic attraction effect is strong
Spend includes the table of coarse and irregular surface particle or membranoid substance to visualize quantitative determination with various configuration of surface objects
The technical method of Surface charge distribution state and its charge density.
Background technology
The surface of many materials all carries electric charge.The presence of these electric charges is between material characteristic in itself and material
Interaction has material impact.For example many organic and inorganic particle, its surface electriferous state are just carrying out relevantization to it
Course of reaction is learned to play an important role；And for biological cell and molecule, its surface charge state not only influences its interaction, also
Determine its own shape of cell so that life state.So in actual applications, often to determine the electricity of various material surfaces
Lotus, including its powered positive and negative, distribution and charge density.But existing e measurement technology has significant limitation.Mainly
It is because they can only all detect smooth and regular surface.A kind of such as Kelvin probe force microscopy (atomic force of repacking
Microscope), electrostatic force microscope, X-ray energy dispersive spectrometer etc., can only all detect smooth with regular surface, can not examine
Survey biological cell living and albumen etc..And most of organic its surface such as with inorganic particle, film, biological cell be all it is coarse not
Rule, or even form are extremely complex and extraordinary-looking.
Therefore, either nano science, material, Surface Science, polymer, film, semiconductor, biomaterial are cured to biology
Etc. is researched and analysed and all tight demands of the practical application of many industrial or agricultural are a kind of can be to various organic and inorganic particles or film
Shape object so that biological cell etc. have it is coarse with the surface charge distribution state and its charge density of the object of irregular surface
Technical method.
The content of the invention
The invention aims to solve drawbacks described above of the prior art, there is provided one kind measure has arbitrary surfaces shape
The method of the surface charge of state object.
The purpose of the present invention can be reached by adopting the following technical scheme that：
A kind of method for determining surface charge, for including rough surface and irregular to tool arbitrary surfaces form object
Particle or membranoid substance surface charge distribution state and its charge density make visualization quantitative determination, under described method includes
Row step：
Determinand is placed in buffer solution, adds the fluorescent nano particles with determinand surface charge contrary sign electric charge, it is glimmering
Light nano-particle and the electric charge on determinand surface are combined so as to be adhered to determinand surface because electrostatic attraction acts on；
Wash the free fluorescent nano particles for being not bonded to determinand surface charge off；
Determinand is placed under fluorescence microscope and carries out imaging；
Using computer picture reconfiguration technique, the two dimension or three-dimensional fluorescence for visualizing fluorescent nano particles are distributed shape
State, from the distribution of its fluorescence distribution condition adjudgement determinand surface charge；
Using Computer imaging analysis system, the fluorescence intensity of each point on determinand is determined, then according to being obtained ahead of time
Fluorescence intensity and the relation curve of zeta current potentials, determine the zeta potential value ζ of every bit, every bit are drawn by zeta potential values ζ
Charge density σ.
Further, described fluorescent nano particles are the nano-particle that diameter can fluoresce in 2~100nm, pass through table
The processing such as face modification make its surface with the electric charge with determinand surface charge contrary sign, including fluorescence semiconductor microcrystal, rare earth
Up-conversion nanoparticles, metal nanoparticle are adulterated, and directly by organic or inorganic nanoparticle made of fluorescent material, dyestuff
Son.
Further, described rough surface and irregular particle or membranoid substance include organic matter, inorganic matter and life
Thing cell.
Further, described buffer solution is different and different with the property of determinand, and its effect is determinand is kept
Make fluorescent nano particles that there is stable dispersiveness while its nature, when described determinand is organic matter or inorganic
Deionized water, ethanol, n-hexane, chloroform and toluene may be selected in thing, described buffer solution, when described determinand is thin for biology
Born of the same parents, described buffer solution are the mixed liquor of sodium chloride and glucose, and PBS liquid.
Further, the formula of the charge density σ that every bit is drawn by zeta potential values ζ is as follows：
N in formula is Avogadro number, and k is the graceful constant of Bohr thatch, and ε is dielectric constant, and T is absolute temperature, and I is ion
Intensity, e are quantities of charge.
Further, described fluorescence microscope is general fluorescence microscope for two-dimensional detection, for three-dimensional values, then
For fluorescent scanning microscope or confocal microscope.
Further, if the powered property of determinand is unknown in advance, determinand is first determined into its zeta with zeta potentiometers
Current potential, to determine its potential value ζ sizes and powered property.
The present invention is had the following advantages relative to prior art and effect：
(1) can surface of the various configurations of surface of Visual retrieval tool including the coarse and particle of irregular surface or membranoid substance
Charge distribution state；
(2) charge density of determinand surface every bit can be quantitative determined simultaneously；
(3) detection to surface charge distribution and charge density size can be carried out in two dimension or three-dimensional, and detection resolution be
Nanoscale；
(4) method is simple and easy, and operation is easy, it is not necessary to the situation of expensive instrument, such as two dimension measure, only needs in general glimmering
Light microscope can be completed to detect.
Brief description of the drawings
Fig. 1 is that configuration of surface is considerably complicated and the polyolefin particles of very irregular；
Fig. 2 is a kind of flow chart of method for determining surface charge disclosed by the invention.
Embodiment
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is
Part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art
The every other embodiment obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.
Embodiment one
The present embodiment disclose it is a kind of determine that various bands are coarse and the miniature object of irregular surface, it is including organic and inorganic
The technical side of the surface charge property of the thing and particle of organism, membranoid substance and cell etc., distribution and its charge density
Method.And its detection is not still visual or quantitative, has nano level resolution ratio.
First, determinand is placed in buffer solution, adds the fluorescence nano grain with determinand surface charge contrary sign electric charge
All electric charges on son, fluorescent nano particles and determinand surface combine because of the electrostatic attraction effect between heterocharge, so as to viscous
It is attached to determinand surface.Then, the free fluorescent nano particles for being not coupled to determinand surface charge are washed off, determinand is put
Imaging is carried out under fluorescence microscope, can be intuitively depending on seeing the fluorescence distribution state and intensity of fluorescent nano particles, from it
Fluorescence distribution state can determine whether the distribution of determinand surface charge, and the electricity of every bit can be determined from the fluorescence intensity of every bit
Lotus density.
As shown in Fig. 2 the method for the measure surface charge specifically includes following step：
S1, determinand is placed in buffer solution, described buffer solution is different and different with the property of determinand, and its effect is
Determinand is set to make fluorescent nano particles that there is stable dispersiveness while keeping its nature.Optional deionized water,
Ethanol, n-hexane, chloroform and toluene.For biological cell, for the mixing of the sodium chloride of 5ml 0.9% and the glucose of 10ml 5%
Liquid, and 0.1M PBS liquid.
S2, fluorescent nano particles are added in buffer solution, described fluorescent nano particles are that diameter can be sent out in 2~100nm
The nano-particle of fluorescence, its surface is made with the electric charge with determinand surface charge contrary sign by surface modification.
S3,10 minutes are stood after the solution with determinand is gently shaken up, make fluorescent nano particles and determinand surface
Electric charge is acted on by electrostatic attraction to combine.
Three times free fluorescent nano particles for making to be not coupled in determinand surface charge of S4 and then cleaning are disposed；
S5, determinand is placed in the micro- Microscopic observation of fluorescent scanning and carries out three-dimensional imaging；As only needed two-dimensional imaging, then only
It need to be imaged with general fluorescence microscope；
S6, using computer picture reconfiguration technique, visualize the fluorescent three-dimensional distribution of fluorescent nano particles i.e.
The distribution of determinand surface charge；
S7, using Computer imaging analysis system, the fluorescence intensity of each point on determinand is determined, then according to being obtained ahead of time
Fluorescence intensity and zeta current potentials relation curve, the zeta potential values of every bit are determined, finally according to formula (1), by zeta electricity
Position ζ draws the charge density σ of every bit：
N in formula is Avogadro number, and k is the graceful constant of Bohr thatch, and ε is dielectric constant, and T is absolute temperature, and I is ion
Intensity, e are quantities of charge.
Powered property such as determinand is unknown in advance, then determinand first should be determined into its zeta current potential with zeta potentiometers,
To determine its current potential size and powered property (positive or negative).
The present invention action principle be：Fluorescent nano particles are a kind of nano-particles that can be fluoresced, including fluorescence is partly
Conductor microcrystal, rear-earth-doped up-conversion nanoparticles, metal nanoparticle (nanogold, Nano Silver), and directly by fluorescence
Organic or inorganic nano-particle made of material, dyestuff.In 1~100nm, its table typically is handled by surface modification etc. for its diameter
Face positively chargeable or negative electrical charge.When it interacts with the particle or membranoid substance of surface charge to be measured in aqueous, because
Acted on for the electrostatic attraction of heterocharge, fluorescent nano particles can with all electric charges on determinand surface it is in situ with reference to and be adhered to
Determinand surface.After now the fluorescent nano particles not combined with determinand surface charge are removed by way of elution,
The left fluorescent nano particles for being all bonded to determinand surface charge.The irradiation of fluorescence excitation light source can now be passed through
Each fluorescent nano particles is sent fluorescence, so as to by the fluorescence distribution states of fluorescent nano particles by determinand surface charge
Distribution visualization is shown.Due to the fluorescent nano particles number in determinand some local point and the surface of the point
Electric charge is directly proportional, and fluorescent nano particles are directly proportional to the fluorescence intensity that it is launched in the number of the point.So pass through measure
The fluorescent nano particles fluorescence intensity of every bit, so that it may determine the charge density of the point.Because no matter the configuration of surface of determinand
How, the combination of the fluorescent nano particles with heterocharge and determinand surface charge can all be carried out, and institute can survey in this way
Fixed various rough surface and irregular or even extremely complicated particle (see Fig. 1) and membranoid substance.And by using fluorescent scanning
Microscope, the three-dimensional fluorescence distribution of fluorescent nano particles can be shown, so as to reach Three-dimensional Display determinand surface electricity
The purpose of lotus distribution.In quantitative determination, first the surface zeta potential current potential of determinand is measured, to determine its surface
The powered property (positive electricity or negative electricity) of electric charge, and its average charge density.Then with a series of with different charge densities
Composition granule to be measured determines the relation curve of its fluorescent nano particles fluorescence intensity and charge density as calibration, so that it may according to treating
The fluorescence intensity of thing every bit is surveyed, corresponds to the charge density on calibration curve, quantitatively determines the charge density of the point.
Embodiment two
With the surface charge of the PP GRANULES of the technology of the present invention fluorescent nano particles imaging method measure positively charged：Will be to be measured
PP GRANULES is placed in by the sodium chloride solutions of 5ml 0.9%, then adding electronegative fluorescence nano gold particle, and solution is light
10 minutes are stood after jog is even；Then clean three times the fluorescence nano goldc grains being not coupled in PP GRANULES surface charge
Son washes.PP GRANULES is placed under fluorescent scanning microscope, carries out three-dimensional imaging, and imaging is carried out with computer
Three-dimensionalreconstruction, obtain the distributed in three dimensions state of PP GRANULES surface charge.Then according to the fluorescence intensity of every bit, using pre-
The fluorescence intensity that first obtains and the relation curve of zeta current potentials, the zeta potential values of every bit are determined, finally according to formula (1), by
Zeta current potentials draw the charge density of every bit.The verified resolution ratio of measurement is horizontal 65nm, longitudinal 66nm.
In order to verify whether fluorescent nano particles are really all marked the surface charge of PP GRANULES, also to knot
The PP GRANULES for having closed fluorescent nano particles has carried out Zeta potential measure.Acquired results are zero, this explanation fluorescence nano grain
Son with all surfaces charge bonded on PP GRANULES really by making it electrically neutralize, therefore Zeta potential is zero.
Embodiment three
The surface of electronegative live body people burr cell is determined with the technology of the present invention fluorescent nano particles imaging method
Electric charge：By burr cell to be measured be placed in by the sodium chloride solutions of 5ml 0.9% and the glucose groups of 10ml 5% into mixing
In liquid, then by fluorescence semiconductor (CdSe/ZnS) particle and the 0.1M PBS solutions of positively charged that 1 μ L concentration is 4.88 μM/L
Add, 10 minutes are stood after these sample solutions are gently shaken up；Then clean red thin crenation is not coupled to three times
Fluorescence nano semiconductor particle on born of the same parents' Particle surface charge washes.Burr cell is placed in fluorescent scanning microscope
Under, three-dimensional imaging is carried out, and three-dimensionalreconstruction is carried out to imaging with computer, obtain the three of burr cell surface charge
Tie up distribution.Then it is bent using the fluorescence intensity being obtained ahead of time and the relation of zeta current potentials according to the fluorescence intensity of every bit
Line, the zeta potential values of every bit are determined, finally according to formula (1), the charge density of every bit is drawn by zeta current potentials.It is detected
Resolution ratio is identical with embodiment two.
Equally, in order to verify whether fluorescent nano particles really all mark the surface charge of burr cell
Come, Zeta potential measure also has been carried out to the burr cell for combining fluorescent nano particles.Acquired results are similarly zero,
Illustrate fluorescent nano particles really by making it electrically neutralize with all surfaces charge bonded on burr cell,
Therefore Zeta potential is zero.In addition, negative control experiment is also further carried out, to prove fluorescence intensity and surface charge density
It is proportional.Experiment uses a series of red blood cells that part surface electric charge is removed through trypsin treatment, then respectively simultaneously
Its Zeta potential and fluorescence intensity are measured, it is found that the two has good linear relationship.With the reduction of charge density, fluorescence intensity
It is corresponding to reduce, so prove, with the inventive method of the fluorescent nano particles combination determinand surface charge with heterocharge, really
The distributing labels of determinand all surfaces electric charge can be come out in fact, and the density of its fluorescence intensity reflection determinand surface charge.
Obviously, above-described embodiment is only intended to clearly illustrate patent example of the present invention, and is not to this hair
The restriction of the embodiment of bright patent.For those of ordinary skill in the field, may be used also on the basis of the above description
To make other changes in different forms.Herein without being exhaustive to all embodiments.It is all in the present invention
Any change, modification, replacement, combination, the simplification made within the spirit and principle of patent, should be equivalent substitute mode,
It should be included within the protection domain of Patent right requirement of the present invention.
Claims (7)
1. a kind of method for determining surface charge, for including rough surface and irregular to the object with various configurations of surface
Particle or membranoid substance surface charge distribution state and its charge density make visualization quantitative determination, it is characterised in that it is described
Method comprise the following steps：
Determinand is placed in buffer solution, adds the fluorescent nano particles with determinand surface charge contrary sign electric charge, fluorescence is received
Rice corpuscles and the electric charge on determinand surface combine because electrostatic attraction acts on；
Wash the free fluorescent nano particles for being not bonded to determinand surface charge off；
Determinand is placed under fluorescence microscope and carries out imaging；
Using computer picture reconfiguration technique, the two dimension or three-dimensional fluorescence distribution of fluorescent nano particles are visualized, from
The distribution of its fluorescence distribution condition adjudgement determinand surface charge；
Using Computer imaging analysis system, the fluorescence intensity of each point on determinand is determined, then according to the fluorescence being obtained ahead of time
Intensity and the relation curve of zeta current potentials, the zeta potential value ζ of every bit are determined, the electricity of every bit is drawn by zeta potential values ζ
Lotus density σ.
A kind of 2. method for determining surface charge according to claim 1, it is characterised in that described fluorescent nano particles
The nano-particle that can be fluoresced in 2~100nm for diameter, its surface is set to carry and determinand surface by surface modification and processing
The electric charge of electric charge contrary sign, including fluorescence semiconductor microcrystal, rear-earth-doped up-conversion nanoparticles, metal nanoparticle, and
Directly by organic or inorganic nano-particle made of fluorescent material, dyestuff.
A kind of 3. method for determining surface charge according to claim 1, it is characterised in that described rough surface and not
The particle or membranoid substance of rule include organic matter, inorganic matter and biological cell.
4. a kind of method for determining surface charge according to claim 3, it is characterised in that described buffer solution is with to be measured
The property of thing is different and different, and it is to enable determinand fluorescent nano particles is had surely while keeping its nature that it, which is acted on,
Fixed dispersiveness, when described determinand is organic matter or inorganic matter, deionized water, ethanol, just may be selected in described buffer solution
Hexane, chloroform and toluene, when described determinand is biological cell, described buffer solution is the mixing of sodium chloride and glucose
Liquid, and PBS liquid.
5. a kind of method for determining surface charge according to claim 1, it is characterised in that described by zeta potential values
ζ show that the charge density σ of every bit formula is as follows：
<mrow>
<mi>&sigma;</mi>
<mo>=</mo>
<mn>2</mn>
<msqrt>
<mfrac>
<mrow>
<mi>N</mi>
<mi>&epsiv;</mi>
<mi>k</mi>
<mi>T</mi>
<mi>I</mi>
</mrow>
<mrow>
<mn>2000</mn>
<mi>&pi;</mi>
</mrow>
</mfrac>
</msqrt>
<mi>sinh</mi>
<mrow>
<mo>(</mo>
<mfrac>
<mrow>
<mi>e</mi>
<mi>&zeta;</mi>
</mrow>
<mrow>
<mn>2</mn>
<mi>k</mi>
<mi>T</mi>
</mrow>
</mfrac>
<mo>)</mo>
</mrow>
</mrow>
N in formula is Avogadro number, and k is the graceful constant of Bohr thatch, and ε is dielectric constant, and T is absolute temperature, and I is that ion is strong
Degree, e is quantities of charge.
A kind of 6. method for determining surface charge according to claim 1, it is characterised in that described fluorescence microscope pair
It is general fluorescence microscope in two-dimensional detection, is then fluorescent scanning microscope or confocal microscope for three-dimensional values.
7. a kind of method for determining surface charge according to claim 1, it is characterised in that if the powered property of determinand
It is unknown in advance, determinand is first determined into its zeta current potential with zeta potentiometers, to determine its potential value ζ sizes and powered property.
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| CN201710498660.0A CN107356571A (en) | 2017-06-27 | 2017-06-27 | A kind of method for determining surface charge |
| PCT/CN2018/090808 WO2019001265A1 (en) | 2017-06-27 | 2018-06-12 | Method for measuring surface charges |
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| CN201710498660.0A CN107356571A (en) | 2017-06-27 | 2017-06-27 | A kind of method for determining surface charge |
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| WO (1) | WO2019001265A1 (en) |
Cited By (7)
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| CN108254632A (en) * | 2017-12-22 | 2018-07-06 | 同济大学 | Based on SiO2The method that microballoon movable information analyzes its surface charge density |
| WO2019001265A1 (en) * | 2017-06-27 | 2019-01-03 | 暨南大学 | Method for measuring surface charges |
| CN109991129A (en) * | 2019-03-29 | 2019-07-09 | 中国海洋石油集团有限公司 | A method of measurement oxide surface chemical group number density |
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| CN108254632A (en) * | 2017-12-22 | 2018-07-06 | 同济大学 | Based on SiO2The method that microballoon movable information analyzes its surface charge density |
| CN108254632B (en) * | 2017-12-22 | 2020-07-28 | 同济大学 | Method for analyzing surface charge density of SiO2 microsphere based on motion information |
| CN109991129A (en) * | 2019-03-29 | 2019-07-09 | 中国海洋石油集团有限公司 | A method of measurement oxide surface chemical group number density |
| CN109991129B (en) * | 2019-03-29 | 2021-08-17 | 中国海洋石油集团有限公司 | Method for measuring number density of chemical groups on surface of oxide |
| CN112240977A (en) * | 2019-07-17 | 2021-01-19 | 香港中文大学 | Method for measuring effective maximum energy output of nano generator and data processing method |
| CN112345434A (en) * | 2020-10-23 | 2021-02-09 | 大连理工大学 | Micro-nano bubble internal pressure calculation method |
| CN112345434B (en) * | 2020-10-23 | 2022-02-15 | 大连理工大学 | Micro-nano bubble internal pressure calculation method |
| CN114235702A (en) * | 2021-12-21 | 2022-03-25 | 山东威高血液净化制品股份有限公司 | Separation membrane surface potential detection method and automatic detection device |
| CN114002290A (en) * | 2022-01-04 | 2022-02-01 | 苏州大学 | Transient photovoltage measurement system and method in particle-scale sample in-situ reaction |
| CN114002290B (en) * | 2022-01-04 | 2022-03-22 | 苏州大学 | Transient photovoltage measurement system and method in particle-scale sample in-situ reaction |
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