CN106525794B - A kind of measuring method of the adsorption/desorption rate constant of the neighbouring counter ion counterionsl gegenions of polyelectrolyte - Google Patents
A kind of measuring method of the adsorption/desorption rate constant of the neighbouring counter ion counterionsl gegenions of polyelectrolyte Download PDFInfo
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Abstract
The invention discloses a kind of measuring methods of the adsorption/desorption rate constant of counter ion counterionsl gegenions near polyelectrolyte.The measuring method includes the following steps: the polyelectrolyte 1) marked using fluorescent probe molecule D label polyelectrolyte；2) polyelectrolyte of label and the aqueous solution of fluorescent probe molecule A are prepared；Fluorescence resonance energy transfer can occur between fluorescent probe molecule D and fluorescent probe molecule A；3) the laser irradiation aqueous solution of fluorescent probe molecule D will be excited, fluorescence signal caused by fluorescent probe molecule D and fluorescent probe molecule A is divided to obtain two beam fluorescence signals, it detects two beam fluorescence signals and carries out auto correlation and cross correlation is analyzed to obtain correlation function, Pair correlation function, which is fitted, obtains polyelectrolyte nearby diffusion information of the counter ion counterionsl gegenions with main chain and the adsorption/desorption rate constant information relative to main chain.Measuring method of the present invention can measure in the space of fL magnitude, be particularly suitable for the research of biological sample or precious sample.
Description
Technical field
The present invention relates to a kind of measuring methods of adsorption/desorption rate constant, and in particular to a kind of polyelectrolyte nearby contends with
The measuring method of the adsorption/desorption rate constant of ion.
Background technique
Polyelectrolyte is that one kind can be dissolved in aqueous solution and release a large amount of charged ions, while make main chain oppositely charged
Macromolecule.Polyelectrolyte is in fields such as water treatment field, displacement of reservoir oil field, biomedicine field, new energy field, gas separation
Extensive effect is suffered from, while being also functionality and structural materials important in many life entities.Its viscosity, osmotic pressure etc.
Property with without electric polymer have apparent difference.The major reason for causing this difference is a large amount of counter ion counterionsl gegenions around it
Thus the long-range electrostatic interaction occurred.The dynamic property of research counter ion counterionsl gegenions is the weight for disclosing polyelectrolyte solution property
Want basis.
Whether traditional theory thinks that counter ion counterionsl gegenions are adsorbed on around the main chain of polyelectrolyte, but have relative to main chain for it
Movement, there are also argue (Angelini, T.E. for the influence that how to move, move by those factors；Golestanian,R.,
et al.Counterions between charged polymers exhibit liquid-like organization
and dynamics.Proc.Natl.Acad.Sci.2006,103(21),7962-7967).This is primarily due to this physics
Process occurs in micro-scale, either from time scale or detection all very small, traditional from distance scale
Method is difficult to play a role to this sad journey.The detection method mentioned in only some reports includes suitable using scattering, electronics
The methods of magnetic resonance, dielectric spectrum (Prabhu, V.M.Counterion structure and dynamics in
Polyelectrolyte solutions.Curr.Opin.Colloid Interface Sci.2005,10,2-8), obtain
Data perhaps only provide indirect evidence or the result provided not system, rely solely on these means to study polyelectrolyte
Solution has its theoretical developments very big difficulty.Therefore, develop new measurement counter ion counterionsl gegenions dynamic property in this field
Method it is very necessary.
Summary of the invention
The object of the present invention is to provide a kind of measurement sides of the adsorption/desorption rate constant of counter ion counterionsl gegenions near polyelectrolyte
Method, measuring method of the present invention have the resolution ratio of unimolecule rank, can directly obtain under equilibrium state counter ion counterionsl gegenions 2~
The dynamic property of 10nm scale.
The measuring method of the adsorption/desorption rate constant of counter ion counterionsl gegenions near polyelectrolyte provided by the present invention, including such as
Lower step:
1) polyelectrolyte, the polyelectrolyte marked are marked using fluorescent probe molecule D；
2) polyelectrolyte of the label and the aqueous solution of fluorescent probe molecule A are prepared；
Fluorescence resonance energy transfer can occur between the fluorescent probe molecule D and the fluorescent probe molecule A；
3) aqueous solution described in the laser irradiation of the fluorescent probe molecule D will be excited, to the fluorescent probe molecule D and institute
It states fluorescence signal caused by fluorescent probe molecule A to be divided to obtain two beam fluorescence signals, detects the two beams fluorescence signal
And carry out auto correlation and cross correlation is analyzed to obtain correlation function, the correlation function is fitted and obtains the poly- electrolysis
Diffusion information of the counter ion counterionsl gegenions with main chain and the adsorption/desorption rate constant information relative to main chain near matter.
In above-mentioned measuring method, the polyelectrolyte can be kayexalate, polyvinylpyridine and polypropylene
At least one of acid, the molecular weight of the polyelectrolyte can be 20k~240k.
In above-mentioned measuring method, the structural formula of the fluorescent probe molecule D (Alexa 488) as shown in formula I is maximum
Absorbing wavelength is 488nm, maximum emission wavelength 536nm；
In formula I, * indicates the group being connected with the polyelectrolyte；
Formula I.
In above-mentioned measuring method, the structural formula of the fluorescent probe molecule A (Atto 610) as shown in formula II is maximum
Absorbing wavelength is 615nm, maximum emission wavelength 634nm；
In formula II, * indicates the group being connected with the polyelectrolyte；
Formula II.
In above-mentioned measuring method, in the aqueous solution, the molar concentration of the polyelectrolyte of the label is less than 50nmol/
L, such as 30nmol/L；
The molar concentration of the fluorescent probe molecule A is less than 1000nmol/L, such as 200nmol/L.
In above-mentioned measuring method, it is rapid 3) in, the laser is generated by continuous wave laser；
The continuous wave laser can be argon ion gas laser.
In above-mentioned measuring method, in step 3), it is divided using grating.
In above-mentioned measuring method, in step 3), fluorescence signal is detected using photomultiplier detector (PMT), such as
The GaAsP detector of Zeiss company research and development.
In above-mentioned measuring method, in step 3), it can realize that auto correlation and cross correlation are analyzed by commercial software.
Measuring method of the present invention is based on following mechanism:
Due to counter ion counterionsl gegenions around polyelectrolyte chain with respect to main chain movement so that between two probe molecules away from
It can include this multidate information in the mutual relevance of two detected kind fluorescence intensity from constantly changing,
Characteristic time can react the speed of counter ion counterionsl gegenions absorption and desorption process.
Measuring method of the present invention has the advantage that
Firstly, measuring method of the present invention, fluorescence resonance energy transfer is combined with fluorescence correlation spectroscopy technology, can be obtained
Adsorption/desorption information of the counter ion counterionsl gegenions in small range, small time scale near main chain under to equilibrium state.This method can be with
The kinetic characteristic that counter ion counterionsl gegenions are disclosed from microcosmic point is conducive to probing into for polyelectrolyte theory.
Secondly, measuring method of the present invention, can measure, this unimolecule scale is ground in the extremely dilute solution of polyelectrolyte
The method of studying carefully can obtain high sensitivity not available for macroscopic measurement method.
Finally, measuring method of the present invention, can measure in the space of fL magnitude, it is particularly suitable for biological sample or treasure
The research of your sample.
Detailed description of the invention
Fig. 1 is the experimental provision light path schematic diagram that measuring method of the present invention uses.
Fig. 2 is the polyelectrolyte chain and the mixed correlation function of dyestuff counter ion counterionsl gegenions that the embodiment of the present invention 1 measures.
Fig. 3 is the relationship of the adsorption/desorption rate constant that the embodiment of the present invention 1 measures and additional salinity.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The measurement of counter ion counterionsl gegenions adsorption/desorption rate constant near embodiment 1, kayexalate
1, the preparation and dyeing of kayexalate
By the P of the concentrated sulfuric acid of 1.0mL and 0.5g2O5It is added in single port bottle, is uniformly mixed at a temperature of 40 DEG C.
(molecular weight is 30 × 10 to 30mg amino-terminated polystyrene3g·mol-1, purchase and be dissolved in 9mL's with Polymer Source)
In hexamethylene, 40 DEG C of sufficiently dissolutions are heated to, the concentrated sulfuric acid and P are then slowly dropped to2O5Mixed solution in, in 40 DEG C of water
It is vigorously stirred 30min in bath, stops stirring later, stands 1h and completes reaction.Flask is put into mixture of ice and water to cooling, use
Pipettor sops up the cyclohexane solvent on upper layer, and the trash ice of 3.0g is then added into single port bottle, the Off-white solid of viscous shape occurs,
Remove the concentrated sulfuric acid of lower layer, is then neutralized with sodium hydroxide solution until the pH of entire solution is 7.By polymer derived above
Solution is transferred in bag filter, is removed the salt in solution with ultrapure water dialysis, is changed 1 st pure water, and use conductivity meter within every 3 hours
The conductivity of dialyzate is detected until its conductivity is close with the conductivity of pure water.The solution that dialysis is completed is lyophilized to obtain white
Powder is kayexalate sample.
The kayexalate sample (5mg, 0.08 μm of ol) that sulfonating reaction obtains is dissolved in the 0.1M's that pH value is 8.6
In sodium bicarbonate buffer liquid (400 μ L), Alexa 488 (0.5mg, 0.8 μm of ol) is dissolved in the DMSO of 100 μ L, is then added
Into the buffer solution of kayexalate, it is protected from light and is stirred to react 10 hours.
The mixture that above-mentioned staining reaction is obtained passes through the polypropylene using the NaCl solution of 0.1mol/L as mobile phase
Acrylamide gel chromatographic column is separated off most of unreacted free dyestuff, the polystyrene through fluorescent marker that then will be obtained
Sodium sulfonate solution is placed in bag filter with ultrapure water dialysis until the conductivity of dialyzate and the conductivity value of pure water are close, then incite somebody to action
Kayexalate solution in bag filter is transferred in the ultra-filtration centrifuge tube that molecular cut off is 5k, and ultrapure water is added repeatedly
Ultrafiltration centrifugation 30 times or more, until can't detect fluorescence signal in filter liquor completely, finally the polyelectrolyte solution after ultrafiltration
The nylon leaching film for being 0.2 μm with aperture is filtered to remove possible dust and large granular impurity in solution, and obtained sample is placed in
It is saved under low temperature stand-by.
2, the measurement of the neighbouring counter ion counterionsl gegenions adsorption/desorption rate constant of kayexalate
Experimental provision of the invention be Zeiss company production commercialization confocal fluorescent micro imaging system
The schematic diagram of LSM780, light channel structure are as shown in Figure 1.By being added dropwise in the objective lens in optical microphotograph lens device, 20 μ L are pure
After water, by the sample cell of the mixed solution of the free dyestuff Atto 610 of the PSS and 200nmol/L of the label equipped with 30nmol/L
It is put in thereon.Adjusting the burnt pin hole position of copolymerization keeps signal best, and focus is adjusted to 30 μm of interface on coverslip.Setting difference
Collect the fluorescence signal between 490~530nm and 630~690nm.
The Autocorrelation Function and cross correlation function curve of two beam laser are as shown in Figure 2.It is risen or fallen by the curve
The corresponding characteristic time can know the dynamic property of the counter ion counterionsl gegenions corresponding characteristic time.It is fitted, can be obtained
Absorption and desorption rate constant to counter ion counterionsl gegenions are respectively 0.016 μ s-1With 0.030 μ s-1。
Fig. 3 is the pass of salt ionic concentration in the polyelectrolyte absorption of counter ion counterionsl gegenions nearby and desorption rate constant and solution
System, it can be seen that with the addition of additional salt ion, polyelectrolyte molecules absorption dyestuff counter ion counterionsl gegenions rate constant becomes smaller, and
The rate constant that dyestuff counter ion counterionsl gegenions are desorbed increases.
Claims (6)
1. the measuring method of the adsorption/desorption rate constant of counter ion counterionsl gegenions, includes the following steps: near a kind of polyelectrolyte
1) polyelectrolyte, the polyelectrolyte marked are marked using fluorescent probe molecule D；
2) polyelectrolyte of the label and the aqueous solution of fluorescent probe molecule A are prepared；
Fluorescence resonance energy transfer can occur between the fluorescent probe molecule D and the fluorescent probe molecule A；
3) aqueous solution described in the laser irradiation of the fluorescent probe molecule D will be excited, to the fluorescent probe molecule D and described glimmering
Fluorescence signal caused by light probe molecule A is divided to obtain two beam fluorescence signals, is detected the two beams fluorescence signal and is gone forward side by side
Row auto correlation and cross correlation are analyzed to obtain correlation function, are fitted that obtain the polyelectrolyte attached to the correlation function
Nearly diffusion information of the counter ion counterionsl gegenions with main chain and the adsorption/desorption rate constant information relative to main chain；
The structural formula of the fluorescent probe molecule D is as shown in formula I -1 or formula I -2:
The structural formula of the fluorescent probe molecule A is as shown in formula II:
In formula I -1, formula I -2 and formula II, * indicates the group being connected with the polyelectrolyte.
2. measuring method according to claim 1, it is characterised in that: the polyelectrolyte is kayexalate, gathers
At least one of vinylpyridine and polyacrylic acid.
3. measuring method according to claim 1 or 2, it is characterised in that: in the aqueous solution, the poly- electrolysis of the label
The molar concentration of matter is less than 50nmol/L；
The molar concentration of the fluorescent probe molecule A is less than 1000nmol/L.
4. measuring method according to claim 3, it is characterised in that: in step 3), the laser is produced by continuous wave laser
It is raw；
The continuous wave laser is argon ion gas laser.
5. measuring method according to claim 4, it is characterised in that: in step 3), be divided using grating.
6. measuring method according to claim 5, it is characterised in that: in step 3), examined using photomultiplier detector
Survey fluorescence signal.
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| CN105675560A (en) * | 2016-01-18 | 2016-06-15 | 中国科学院化学研究所 | Method for obtaining fluorescence emission spectrum information of single polymer molecule in shearing field |
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| CN102243176A (en) * | 2010-05-14 | 2011-11-16 | 中国人民解放军军事医学科学院毒物药物研究所 | Method for eliminating screened drug false positive by in vitro molecular level fluorescence method |
| CN105675560A (en) * | 2016-01-18 | 2016-06-15 | 中国科学院化学研究所 | Method for obtaining fluorescence emission spectrum information of single polymer molecule in shearing field |
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