CN110082419B - Method for rapidly detecting ligand content on surface of nanoparticle - Google Patents
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- 239000002105 nanoparticle Substances 0.000 title claims abstract description 60
- 239000003446 ligand Substances 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 45
- 238000004448 titration Methods 0.000 claims abstract description 42
- 230000005012 migration Effects 0.000 claims abstract description 23
- 238000013508 migration Methods 0.000 claims abstract description 23
- 230000008569 process Effects 0.000 claims abstract description 7
- 238000001962 electrophoresis Methods 0.000 claims description 68
- 239000000499 gel Substances 0.000 claims description 18
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 16
- 239000007853 buffer solution Substances 0.000 claims description 13
- 238000006386 neutralization reaction Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 11
- 230000002378 acidificating effect Effects 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 229910052697 platinum Inorganic materials 0.000 claims description 8
- 150000002500 ions Chemical class 0.000 claims description 5
- 238000004132 cross linking Methods 0.000 claims description 3
- 125000000524 functional group Chemical group 0.000 claims description 3
- 230000007704 transition Effects 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 239000012670 alkaline solution Substances 0.000 claims 1
- 238000012800 visualization Methods 0.000 abstract description 2
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 4
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 4
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- 238000004364 calculation method Methods 0.000 description 3
- 101100136092 Drosophila melanogaster peng gene Proteins 0.000 description 2
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- 238000005259 measurement Methods 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000002696 acid base indicator Substances 0.000 description 1
- 238000002479 acid--base titration Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
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- 239000000969 carrier Substances 0.000 description 1
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- 238000000576 coating method Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
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Abstract
The invention relates to a method for rapidly detecting the content of a ligand on the surface of a nanoparticle, which comprises the following steps: setting a control group without a sample and a detection group with the nano-particles to be detected, carrying out electrophoretic titration, measuring the interface migration distance of the control group and the detection group in the same time, and calculating the surface ligand content of the nano-particles of the detection group according to the difference of the interface migration distances. Compared with the prior art, the method has the advantages of simple and convenient operation process, short time consumption, visualization and no need of depending on expensive instruments.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for rapidly detecting the content of a ligand on the surface of a nanoparticle.
Background
The surface of Nanoparticles (NPs) has a high specific surface area and can bind to different ligands. The existence of the ligands can change the solubility, the specificity, the adsorption capacity to different substances, the surface charge number, the electrochemical activity and the like of the NPs in different solutions, and particularly, the NPs can realize adjustable bioactive functions by combining different macromolecules. Quantitative analysis of ligands adsorbed on NPs can help us to understand ligand exchange reactions, and binding of biological targets and carriers is often necessary in many applications, such as chemical detection.
Ligand content detection of NPs remains a significant challenge, and there are often problems with elemental analysis (j.aldana, y.a.wang, x.peng, jacs.2001,123,8844-8850, j.aldana, n.lavelle, y.wang, x.peng, jacs.2005; 127, 2496-2504) and optical methods (g.kaluyzzhne, r.w.murray, j.phys.chem.b.2005,109,7012-7021, k.e.knoes, d.b.tie, e.a.mcarthur, g.c.solomon, e.a weiss.cs.2009; 132,1041-1050, a.m.munro, i.jen-La, m.s.plaig, d.s.langer, phus.622009; 132,1041-1050, a.m.m.m.munro, i.jen-pent-nte, m.s.langer, d.s.gis.6224, etc., phj.12-7021, k.m.m.m.m.g. these methods are time-expensive and expensive methods are used for electrochemical resonance signals.
The carboxylic acid and the amine group similar to the protein are basic groups coated on the surface of the NPs, and the acid-base neutralization reaction is a quantitative and rapid chemical reaction and can accurately and rapidly detect the ligand coverage rate of the NPs containing acidic or basic groups. In order to rapidly quantify the protein content in the dairy product, an electrophoresis titration technology and a device (H.Y.Wang, C.Y.Guo, C.G.Guo, L.Y.Fan, L.Zhang, C.X.Cao, anal.Chim.acta,2013,774, 92-99; H.Y.Wang, J.Yang, J.Y.Dong, W.Zhang, L.Y.Fan, W.B.Zhang, C.X.Cao, Chinese J.anal.Chem.2012, 40,968-972 and the like) are developed based on a mobile reaction interface (MRB), and related patents (an acid-base titration device for determining acid-base concentration, an electromigration titration device for determining total protein concentration, a Chinese patent in 2011, a 2012 application number: CN201010596012.7, a visualization biosensor device for determining total protein concentration, a Chinese patent in years, an application number CN201210142985.2, an electrophoresis titration method based on a mobile reaction interface, an anti-leakage method, an electrophoresis titration method for determining total protein concentration, a high-flux titration method for a Chinese patent in 20136, an electrophoresis titration device for determining total protein concentration, an electrophoresis titration method for a Chinese patent in 2014, an electrophoresis titration method for determining total protein concentration, an anti-discharge liquid, and the like are applied. In addition, in order to further reduce the consumption of detection samples and shorten the detection time, a protein electrophoresis chip titration technology (H.Y.Wang, Y.T.Shi, J.Yan, J.Y.Dong, S.Li, H.Xiao, H.Y.Xie, L.Y.Fan, C.X.Cao, anal.chem.,2014,86,2888-2894, L.X.Zhang, Y.R.Cao, H.Xiao, X.P.Liu, S.R.Liu, Q.H.Meng, L.Y.Fan, C.X.Cao, biosens, bioelectrtron, 2016,77,284-291) is developed, and a related chip electrophoresis titration patent (a qualitative and quantitative integrated detection method for milk blending, CN201510542254.0, an electrophoresis method for determining the degree of milk blending, CN201510140100.9) is applied. Although the electrophoretic titration technology has been well applied to the aspect of protein content detection, the application of the electrophoretic titration technology to the aspect of ligand content determination on the surface of the nanoparticle is still blank, and especially, the absolute quantitative detection and analysis of the ligand content without depending on an internal standard are still difficult.
Disclosure of Invention
The invention aims to solve the problems and provide a method for rapidly detecting the ligand content on the surface of the nanoparticle.
The purpose of the invention is realized by the following technical scheme:
a method for rapidly detecting the content of a ligand on the surface of a nanoparticle comprises the following steps: setting a control group without a sample and a detection group with the nano-particles to be detected, carrying out electrophoretic titration, measuring the interface migration distance of the control group and the detection group in the same time, and calculating the surface ligand content of the nano-particles of the detection group according to the difference of the interface migration distances.
The method comprises the following specific steps:
(1) preparing high-crosslinking gel with indicator and dividing the gel into two parts, setting one part of gel without any sample as a control group, adding a nanoparticle sample to be detected into one part of gel as a detection group, and injecting the same amount of the nanoparticle sample into two electrophoresis channels of an electrophoresis titration instrument respectively;
(2) after the gels in the control group and the detection group are polymerized, adding electrode buffer solution into an electrophoresis channel of an electrophoresis titrator and electrode solution pools at two sides of the channel, and connecting a power supply to carry out electrophoresis;
(3) and in the electrophoresis process, the indicator indicates to form a visible moving neutralization interface, after a period of time, the power supply is turned off to stop electrophoresis, and the content of the ligand on the surface of the nanoparticle of the detection group is calculated according to the difference of the migration distances of the two groups of interfaces.
The content of the ligand on the surface of the nanoparticle is calculated by the following formula:
Δdrel＝dblank-dNPs
in the formula, the first step is that,
represents the ligand content on the surface of the nano-particles; c represents the concentration of the titration standard solution; dblankAnd dNPsThe transition distance, Δ d, of the neutralization interface in the same time period in the control group and the test group is shown separatelyrelRepresenting the difference in distance between the two interfaces.
When the ligand on the surface of the nano-particle to be detected is acidic, the indicator adopts an alkaline indicator, such as phenolphthalein, and color change is generated in the titration process. And (3) mixing the electrode buffer solution added into the cathode liquid pool in the step (2) with alkali liquor, wherein the concentration of OH-ions is 10-50 mM.
When the indicator in the electrophoresis channel is developed and moved to form a neutralization interface, taking t0Recording the interface position at the moment as an initial value, continuing electrophoresis for a period of time, and taking t1Recording the interface position at the moment as an end value, and the difference between the two interface distances is delta drelD is used as the migration distance of the neutralization interface in the same time for the control group and the detection groupblankAnd dNPsIt shows that the detection group is added with the nanoparticle detection sample, the acidic groups contained in the ligand on the surface of the NPs sample have a blocking effect on the interface movement (certain concentration of acid is present to neutralize OH-ions entering a channel during electrophoresis), the more NPs are added, the higher the concentration of the ligand in the gel is, the more obvious the blocking effect on the interface migration is, therefore, the migration distances of the two groups of interfaces are different, and the interface migration speed of the detection group is slower and the migration distance is small than that of the control group in the same time range, namely d isNPs<dblankSetting the difference of the moving distance of the interface between the control group and the detection group in the same time as deltadrel＝dblank-dNPsThe control group as the system parameter can be regarded as a constant after determining the condition of electrophoretic titration, and the difference of interface migration distance DeltadrelAnd a ligand concentration c containing acidic groupsAcidThere is a direct relationship, so the difference in migration distance between the two groups of neutralization interfaces is equivalent to the concentration of acid consumed by titration, i.e.:
wherein, Δ drel＝dblank-dNPsRepresents the difference between the control group and the detection group in the same electrophoresis time and the migration distance of the neutralization interface,
represents the concentration consumed by titration of the ligand containing the acidic functional group on the surface of the nanoparticles of the detection group,
represents the OH-ion concentration, cNPsIndicates the concentration of nanoparticles in the detection group, NrealThe ratio of ligand concentration to nanoparticle concentration is expressed.
Preferably, in the step (2), the ionic strength of the electrode buffer solution is not higher than 0.05-0.1M, the volume of the electrode buffer solution injected into the electrode solution pool is required to completely immerse the platinum electrode and the electrophoresis channel, and the volumes of the electrode buffer solutions in the two electrode solution pools are equal and the ionic strengths are the same.
Preferably, the power voltage applied in the method is 10-50V, the voltage magnitude has a direct relation with the electrophoresis distance, after the chip channel structure is determined, the voltage magnitude can be properly adjusted according to the electrophoresis distance, and the total electrophoresis time is less than 1-5 min.
The detection is carried out by adopting a detection device, the device comprises a power supply, a white light plate, an electrophoresis titrator, a digital microscope and a computer, wherein the electrophoresis titrator is placed on the white light plate and is connected with the power supply through a lead, the digital microscope is used for recording an interface migration process and is placed above the electrophoresis titrator and is connected with the computer, and software is arranged in the computer and is used for controlling the digital microscope to work.
Preferably, the electrophoresis titration apparatus comprises an electrophoresis channel, an electrode liquid pool at two ends of the channel and a platinum electrode arranged in the electrode liquid pool, and the power supply is connected with the platinum electrode through a lead.
Preferably, the electrophoresis titration apparatus has two or more electrophoresis channels, the cross section of the channel is selected from circular, rectangular or trapezoidal, the inner diameter or side length of the channel is 50-500 μm, and the length of the channel is 10-30 mm.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method has the advantages that the content of the ligand on the surface of the nano-particles is measured by the electrophoretic titration technology, absolute quantitative detection can be realized without depending on an internal standard, the blank of the electrophoretic titration technology in the measurement of the content of the ligand on the surface of the nano-particles is filled, the operation is simple and convenient, and the sample pretreatment is simple.
(2) The total time of the electrophoretic titration is short, the time consumption is less, and the mass determination of the ligand content on the surface of the nano particles is convenient.
(3) The indicator is used for indicating interface migration of the control group and the detection group, and the visual effect is realized, so that the operation is convenient.
(4) The instruments required by the adopted device are all conventional and common instruments, and the determination work can be carried out only through simple assembly.
Drawings
FIG. 1 is a schematic diagram of the calculation of ligand content of nanoparticles based on the difference in interface migration distance;
FIG. 2 is a schematic structural diagram of an apparatus for rapidly detecting the ligand content on the surface of a nanoparticle.
In the figure: 1-a power supply; 2-a white light board; 3-an electrophoretic titration instrument; 4-a digital microscope; 5-a computer; 3.1-electrophoresis channel; 3.2-electrode liquid pool; 3.3 platinum electrode.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Examples
Taking electrophoretic titration measurement of NPs surface-coated 3-mercaptopropionic acid (MPA) as an example, the method for rapidly detecting the MPA of the NPs surface ligand comprises the following steps:
1) preparing gel solutions added with indicators and dividing the gel solutions into two groups, wherein one group is used as a control group, the other group is added with NPs (N-phosphosilicate complexes) and is used as a detection group, after uniformly mixing, respectively injecting the same amount of gel solutions into an electrophoresis channel, and standing the gel, wherein the gel is high-crosslinking polyacrylamide gel and the phenolphthalein is added as an acid-base indicator;
2) after gel polymerization, at least 1.6mL of cathode and anode electrode buffer solutions are respectively added into an electrophoresis channel of an electrophoresis titration instrument and electrode solution pools at two sides of the channel, the ionic strength of the electrode buffer solution is about 0.1M/L, the concentration of OH-ions in the cathode buffer solution is not lower than 0.1mM/L, and a power supply is connected to apply 40V voltage to start electrophoresis;
3) in the electrophoresis process, the electromigration of OH < - > forms a neutralization interface in an electrophoresis channel, and a control computer records the interface migration process through a digital microscope;
4) after electrophoresis for a period of time, the power supply is turned off, and the MPA content of the ligand on the surface of the added NPs sample is calculated by recording the migration distance of the control group and the detection group in the same time.
The specific calculation steps are as follows: the amount of OH-ions entering the electrophoresis channel in a certain time by electromigration can be calculated according to the migration distance of the control group interface:
wherein, A represents the cross-sectional area of the electrophoresis channel, in the other electrophoresis channel, one part of the amount of OH-ion electromigration entering the electrophoresis channel in the same electrophoresis time of the detection group is indicated by the indicator, and the other part is consumed by MPA, which can be expressed by formula:
the two channels have equal amounts of OH-ions under the same conditions, and then:
cOH-dblankAt＝cR，OH-dNPsAt+COH-dNPsAt (3)
the amount of OH-ions consumed by the reaction is equal to the amount of MPA, so that the concentration of MPA can be used to represent the OH consumed by the reaction-The above equation is rewritten as:
when under given electrophoretic conditions, cOH -Known as a constant, the MPA content of the NPs surface coating can be calculated according to the migration distance of the control group and the detection group interface recorded by the electrophoretic titration and the formula (4), as shown in FIG. 1.
In a 30mm long capillary channel with an inner diameter of 500 μm, in cathode bufferOH -At 0.1mM/L, the neutralization interfaces of the blank group and the detection group move by 6.67mM and 5.18mM within 5min, respectively, and the ligand content on the surface of the nanoparticle is 28.72 μ M/L according to the calculation of the formula.
In order to realize the detection of the MPA content of the ligand on the surface of the NPs, the device adopted by the invention is shown in figure 2, and comprises a power supply 1, a white light plate 2, an electrophoresis titration micro-device 3, a digital microscope 4 and a computer 5, wherein the electrophoresis titration micro-device 3 is arranged on the white light plate 2 and is connected with the power supply 1 through a lead, and the digital microscope 4 is arranged right above the electrophoresis titration micro-device 3 and is connected with the computer. In the invention, the electrophoresis titration micro-device 3 comprises two electrophoresis channels 3.1, two electrode liquid pools 3.2 and two platinum electrodes 3.3 which are arranged in the electrolyte liquid pools 3.2, the inner diameter or side length of each channel is 50-500 mu m, and the length of each channel is 10-30 mm.
In the above examples, OH is passed-Ion titration of the acidic ligand, OH-Ion replacement by H+Ion, acidic ligand replacement for basic ligand the method is equally applicable.
The foregoing description of the embodiments is provided to enable one of ordinary skill in the art to make and use the invention, and it is to be understood that other modifications may be readily made to the embodiments and that the general principles defined herein may be applied to other embodiments without the use of inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (8)
1. A method for rapidly detecting the content of a ligand on the surface of a nanoparticle is characterized by comprising the following steps: setting a control group without a sample and a detection group containing the nano-particles to be detected, carrying out electrophoretic titration, determining the interface migration distances of the control group and the detection group within the same time, and calculating the surface ligand content of the nano-particles of the detection group according to the difference of the interface migration distances;
the method comprises the following specific steps:
(1) preparing high-crosslinking gel with indicator and dividing the gel into two parts, setting one part of gel without any sample as a control group, adding a nanoparticle sample to be detected into one part of gel as a detection group, and injecting the same amount of the nanoparticle sample into two electrophoresis channels of an electrophoresis titration instrument respectively;
(2) after the gels in the control group and the detection group are polymerized, adding electrode buffer solution into an electrophoresis channel of an electrophoresis titrator and electrode solution pools at two sides of the channel, and connecting a power supply to carry out electrophoresis;
(3) in the electrophoresis process, the indicator indicates to form a visible moving neutralization interface, after a period of time, the power supply is turned off to stop electrophoresis, and the content of the ligand on the surface of the nano-particles of the detection group is calculated according to the difference of the migration distances of the two groups of interfaces;
the content of the ligand on the surface of the nanoparticle is calculated by the following formula:
Δdrel＝dblank-dNPs
in the formula, the first step is that,
represents the ligand content on the surface of the nano-particles; c represents the concentration of the titration standard solution; dblankAnd dNPsThe transition distance, Δ d, of the neutralization interface in the same time period in the control group and the test group is shown separatelyrelRepresenting the difference in distance between the two interfaces.
2. The method as claimed in claim 1, wherein when the ligand on the surface of the nanoparticle to be detected is acidic, the indicator is alkaline indicator, and the electrode buffer solution added to the cathode liquid pool in step (2) is mixed with alkaline solution, OH-The concentration of the ions is 10-50 mM.
3. The method for rapidly detecting the ligand content on the surface of the nanoparticle as claimed in claim 2, wherein the specific method in step (3) is as follows:
when the indicator in the electrophoresis channel is developed and moved to form a neutralization interface, taking t0Recording the interface position at the moment as an initial value, continuing electrophoresis for a period of time, and taking t1Recording the interface position at the moment as an end value, and respectively using the transition distance of the neutralization interface in the same time of the control group and the detection group as dblankAnd dNPsShowing that the difference between the two interface distances is Deltadrel＝dblank-dNPsThe content of the ligand having an acidic functional group on the surface of the nanoparticle is determined by the following formula:
wherein,
representing the concentration of titrated consumption of the ligand having an acidic functional group on the surface of the nanoparticles of the detection group, cOH-represents OH-The ion concentration.
4. The method for rapidly detecting the content of the ligand on the surface of the nanoparticle according to claim 1, wherein the ion concentration of the electrode buffer solution in the step (2) is 0.05-0.1M, the platinum electrode and the electrophoresis channel are completely immersed by the electrode buffer solution injected into the electrode solution pool, and the volume of the electrode buffer solution in the two electrode solution pools is equal and the ion concentration is the same.
5. The method for rapidly detecting the ligand content on the surface of the nanoparticle as claimed in claim 1, wherein the power voltage applied during electrophoresis is 10-50V, and the total electrophoresis time is 1-5 min.
6. The method for rapidly detecting the ligand content on the surface of the nanoparticle according to claim 1, wherein the detection is performed by using a detection device, the detection device comprises a power supply (1), a white light plate (2), an electrophoresis titration instrument (3), a digital microscope (4) and a computer (5), the electrophoresis titration instrument (3) is placed on the white light plate (2) and is connected with the power supply (1) through a conducting wire, and the digital microscope (4) is placed above the electrophoresis titration instrument (3) and is connected with the computer.
7. The method for rapidly detecting the ligand content on the surface of the nanoparticle as claimed in claim 6, wherein the electrophoresis titration instrument (3) comprises an electrophoresis channel (3.1), electrode liquid pools (3.2) at two ends of the channel and platinum electrodes (3.3) built in the electrode liquid pools (3.2), and the power supply (1) is connected with the platinum electrodes (3.3) through conducting wires.
8. The method for rapidly detecting the ligand content on the surface of the nanoparticle as claimed in claim 7, wherein the number of the electrophoresis channels (3.1) of the electrophoresis titration instrument (3) is two or more, the cross-sectional shape of the channel is circular, rectangular or trapezoidal, the inner diameter or side length of the channel is 50-500 μm, and the length of the channel is 10-30 mm.
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