CN113063834B - Method for visually detecting substance concentration based on mobile exchange interface - Google Patents
Method for visually detecting substance concentration based on mobile exchange interface Download PDFInfo
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- CN113063834B CN113063834B CN202110296012.3A CN202110296012A CN113063834B CN 113063834 B CN113063834 B CN 113063834B CN 202110296012 A CN202110296012 A CN 202110296012A CN 113063834 B CN113063834 B CN 113063834B
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Abstract
A visual method for detecting substance concentration based on mobile exchange interface mainly comprises: 1) different ligands perform displacement reaction on the surface of solid-phase substances (such as nanoparticles, cells and the like) to form optical signals; 2) fixing a solid-phase substance in an electrophoresis channel by using gel (or self-filling and other modes), driving an object to be detected to move in the channel by using an electrophoresis mode, and replacing a ligand on the surface of the solid-phase substance to form a mobile exchange interface; 3) recording the interface moving condition to establish a standard curve of the interface migration rate, the interface width and the substance concentration, and calculating the substance concentration (or ligand replacement reaction rate) of the solid phase substance surface reaction. The method has the advantages of simple and convenient operation process, low cost, visualization and no need of depending on expensive instruments.
Description
Technical Field
The invention relates to the fields of analytical chemistry, nano science, biotechnology and the like, in particular to a method for visually detecting substance concentration based on a mobile exchange interface.
Background
The theory of mobile interfaces has been proved to have the potential of detecting trace substances through the research of theories and applications for many years, and related work applies for patents in the aspect of mobile interface detection (an electromigration acid-base titration device for measuring acid-base concentration, China, 2010-12-18, ZL 201010596012.7; a visual biosensor device for measuring total protein concentration, China, 2012-05-09, ZL 201210142985.2; a high-flux protein titration method based on mobile reaction interface electrophoresis, China, 2013-03-20, ZL 201310089084.6; an anti-leakage electrophoresis titration electrode liquid flowing exhaust device and a use method thereof, China, 2014-09-18, ZL201410478864.4 and the like).
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.ZL, 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, biosen.Bioectron, 2016,77,284-291) is developed, and a related patent for chip electrophoresis titration (a method for qualitatively and quantitatively detecting the adulteration in milk products, Chinese, 08-28, ZL 201510542254.0; quantitative analysis of the adulteration degree of milk products, 2015 03-2015-38, and 201510140100.9) is applied. In order to further explore the application of QDs visualization, the application of an electrophoretic titration technology in the aspect of measuring the content of ligands on the surface of Nanoparticles (NPs) is exploited, a technology for detecting the content of the ligands on the surface of quantum dots by a mobile neutralization interface electrophoresis technology is developed, and related patents are applied (a method for rapidly detecting the content of the ligands on the surface of nanoparticles, China, 2019-04-30, ZL 201910363510.8).
Although the mobile reaction interface has a certain application in the aspects of protein and nucleic acid content detection and the like, and can successfully determine the ligand content on the surface of the quantum dot, the research on the mobile interface formed by ligand exchange on the surface of a substance has not been deeply carried out, and particularly, the research and application for detecting the concentration of an object to be detected based on the ligand exchange reaction of the electrophoresis technology are relatively lacked.
Disclosure of Invention
The invention provides a method for visually detecting the concentration of a substance based on a mobile exchange interface, which can quickly, conveniently and accurately determine the concentration of the substance to be detected.
The purpose of the invention is realized by the following technical scheme:
a method for visually detecting substance concentration based on a mobile exchange interface comprises the following steps:
(1) in the electrophoresis channel, an object to be detected with given concentration and a ligand on the surface of a solid phase substance are subjected to exchange reaction to form a visual mobile exchange interface, and a standard curve is established according to the relation between the mobile exchange interface parameter and the concentration of the object to be detected;
(2) and (3) under the same condition, carrying out electrophoresis on a substance with unknown concentration, recording mobile exchange interface parameters, and substituting the parameters into the standard curve established in the step (1) to obtain the concentration and surface density information of the substance.
Further, the solid phase material can be, but is not limited to, nanoparticles, microparticles, cells, viruses, functionalized gels.
Furthermore, the ligand on the surface of the solid-phase substance can be a single ligand or a collection of multiple ligands, the analyte can replace the ligand on the surface of the solid-phase substance, and the ligand performs replacement reaction on the surface of the solid-phase substance to form an optical signal.
Furthermore, the solid-phase substance is fixed in the electrophoresis channel in advance, the object to be detected is driven to move in the electrophoresis channel in an electrophoresis mode, and the ligand on the surface of the solid-phase substance is replaced to form a mobile exchange interface.
Further, the solid phase substance is fixed in the electrophoresis channel by adopting a gel or a self-filling mode.
The specific method comprises the following steps:
preparing a solution mixed with a ligand-coated solid-phase substance, mixing the solution with a gel solution, and transferring the mixture to an electrophoresis channel, or filling the mixture to the electrophoresis channel in a self-filling manner;
step two, respectively injecting the prepared gel to the positions marked by the capillary tubes, standing for gel polymerization, or standing to an equilibrium state after self-filling;
filling an object to be detected in a sample area of the electrophoresis tube, filling buffer solution in a buffer area, adding equal amount of electrode buffer solution in electrode solution pools at two sides of an electrophoresis channel, respectively connecting electrodes in a cathode solution pool and an anode solution pool with a power supply, and performing electrophoresis;
under the action of an electric field, the object to be detected migrates into a gel area, the object to be detected and a ligand on the surface of the solid-phase substance undergo a displacement reaction to form a mobile exchange interface, and after electrophoresis is carried out for a period of time, a power supply is turned off to stop the electrophoresis;
and step five, recording the interface migration process of electrophoresis by using equipment during electrophoresis to obtain the interface distance.
The capillary tube has a length of 10-100mm and an inner diameter of 75-600 μm. The capillary electrophoresis micro device is placed in an optical device, an electric field is applied, the released fluorescent signal is captured by the optical device, the whole migration process is recorded, and finally the average migration distance (speed and width) of the interface can be obtained through data processing.
The optical devices include, but are not limited to: CCD, smart phone. The interface migration needs to determine the interface position through data processing according to the interface signal, and further calculates parameters such as the average migration distance (speed, width) of the interface of the object to be detected in the sample.
Further, the voltage applied by electrophoresis is 10-50V or 60-120V, and the total electrophoresis time is less than 10 min.
Further, the adopted gel is polyacrylamide gel, the mass volume concentration is 15-45%, the crosslinking degree is 2-6%, the background electrolyte solution is tris-HCl buffer solution, and the pH value is 4-10; in the detection, a fluorescent substance is used as an indicator, and a fluorescent signal is quenched or restored in the process of electrophoretic titration.
Further, after the electrophoresis is started, the distance of the mobile exchange interface is obtained by recording the starting point and the end point at given time, when the interface completely appears and is marked as the starting point of the mobile exchange interface, the interface position is determined according to the extreme point after the derivation of the signal intensity distribution curve, and the interface migration rate v is calculatedMEBThe derivative peak width of the interface signal intensity distribution curve is the interface width, and the relationship between the width w of the interface and the velocity vMEBCan be expressed as:
wherein, f (c)A,cBAnd k) is a parameter related to experiments, and is calculated by specific experiment parameters, wherein k is a coefficient related to the reaction rate of the displaced ligand of the object to be detected.
Compared with the prior art, the method for detecting the substance concentration based on the mobile exchange interface visual detection technology has the characteristics of low detection consumption, low cost, simple and convenient operation, simple sample pretreatment, modularization, automation and the like, can realize quantitative analysis of the concentration of the substance to be detected without depending on expensive instruments, and particularly has high detection speed, and the whole detection process can be completed within 10 minutes.
Drawings
FIG. 1 is a schematic diagram of an MEB experiment, which is divided into MEB-A (Mobile switching A interface) and MEB-B (Mobile switching B interface);
FIG. 2 is a comparison of experimental, theoretical and numerical results for a mobile switching interface-quenching interface;
FIG. 3 is a comparison of results of mobile switching interface-recovery interface experiments, theoretical calculations and numerical calculations.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
The invention provides a method for visually detecting substance concentration based on a mobile exchange interface, wherein a visual mobile exchange interface is formed by the exchange reaction of a substance to be detected with given concentration and a solid phase substance (including but not limited to NPs, microparticles, cells and viruses) surface ligand in an electrophoresis channel; establishing a standard curve according to the relation between the interface distance (speed, width and the like) and the concentration of the object to be detected; and then carrying out electrophoresis on the substances with unknown concentration under the same condition, and substituting the recorded mobile exchange interface distance (or parameters such as speed, width and the like) into the standard curve to obtain the concentration and surface density information of the substances, thereby completing the visual detection of the corresponding detection objects.
The application detection reaction includes but is not limited to: the reaction between the analyte A and the ligand B on the surface of the solid-phase substance, the exchange reaction between the known concentration of the substance A and the content of the ligand B to be detected on the surface of the solid-phase substance, and the other known concentration.
Detectable target substances include, but are not limited to: a substance A to be detected which has specific reaction with the ligand B, the ligand B with unknown concentration, the reaction rate of the ligand replacement reaction and the like.
The method comprises the following steps:
i) and (3) uniformly mixing the solid-phase substance containing the ligand B and the polyacrylamide gel mother liquor, injecting the mixture into a capillary, waiting for solidification, placing the capillary into a capillary electrophoresis microdevice, and loading a sample (to-be-detected object) at the cathode end.
The gel adopted in the detection of i) is polyacrylamide gel, the mass volume concentration is 15-45%, the crosslinking degree is 2-6%, and the background electrolyte solution is tris-HCl (pH 4-10) buffer solution.
In the detection of i), a fluorescent substance is used as an indicator, and a fluorescent signal is quenched or recovered in an electrophoretic titration process.
In the i) detection, the sample is loaded at the cathode end, can be loaded in an electrophoresis tube, and can also be directly loaded in a cathode liquid pool.
ii) placing the capillary electrophoresis microdevice in an optical device, capturing the released fluorescent signal through the optical device after applying an electric field, recording the whole migration process, and finally obtaining the average migration distance (speed and width) of the interface through data processing.
The ii) voltage is applied but not limited to 10-50V for forming a quenching interface (or 60-120V for forming a recovery interface).
Said ii) optical devices include, but are not limited to: CCD, smart phone.
And ii) interface migration needs to determine the interface position through data processing according to the interface signal, and further calculates parameters such as the average migration distance (speed, width) of the interface of the object to be detected in the sample.
The present invention will be described in detail with reference to specific examples.
This example was carried out under the premise of the following inventive technical scheme, and detailed embodiments and specific procedures were given, with 5,5 '-dithiobis-2-nitro-benzoic acid (DTNB), i.e., Ellman's reagent, trimercaptopropionic acid (MPA) as a reaction substance, and Quantum Dots (QDs) as a signal conversion substance (indicator), but the scope of the present invention is not limited to the following examples.
1. Solution preparation:
the quenching interface in the mobile exchange interface (MEB) is exemplified by quenching QDs fluorescence by replacing the QDs surface ligand MPA with DTNB, and the recovery interface is exemplified by recovering QDs fluorescence by replacing the QDs surface ligand TNB with MPA.
Background and electrode buffer solution: both the background and electrode buffer solutions used tris-HCl as buffer solution. Tris solutions up to 100mM are individually adjusted to pH 4-10 with 0.01-1M hydrochloric acid as buffer solutions for capillary gel electrophoresis.
Gel mother liquor: preparing 15-45% acrylamide mother liquor, diluting with pure water to required concentration before use, shaking and storing in a refrigerator at 4 ℃.
Sample solution: preparing a DTNB standard solution with the concentration of 10-100 μ M, diluting the DTNB standard solution with a buffer solution, preparing an MPA standard solution with the concentration of 1-10000 μ M in the same way, and diluting the MPA standard solution with the buffer solution.
Preparation of QDs hydrogels: QDs were added to background buffer Tris-HCl (pH 4-10) for uniform distribution and then mixed with the gel stock. Subsequently, APS and TEMED were incorporated into the homogeneous mixture. Finally, the mixture was transferred to the designated area within the capillary and stored for later use in the experiment.
2. And (3) detection process:
mobile exchange interface (MEB) detection is performed at a given capillary label region. Fluorescent quantum dot particles (i.e., [ MPA-QDs ]) fluoresce under a given excitation light.
After preparation of [ MPA-QDs ], the sample area was filled with DTNB solution and the concentration and pH of the background buffer was kept constant throughout. Then, under the driving of the electric field, DTNB in the sample region enters the gel region (i.e., the detection region) and reacts with MPA-exchange on [ MPA-QDs ] (with fluorescence) to form [ TNB-QDs ] (without fluorescence), thereby forming an MEB-Q interface. At the same time, the migration of the quenching interface is recorded by the device.
Thereafter, the capillary system was washed with background buffer, the sample area was filled with MPA solution, and the concentration and pH of the background buffer was kept uniform throughout. Then, under the drive of an electric field, MPA in the sample area enters a gel area (namely a detection area) and generates ligand competition with TNB-on [ TNB-QDs ] (non-fluorescence) and displacement to obtain [ MPA-QDs ] (fluorescence), thereby forming an MEB-R interface. Likewise, the migration of this restoration interface is also recorded by the optical device.
Finally, in the data processing stage, the recorded video is processed by using a self-programming sequence to obtain information (fig. 2A, B and fig. 3A, B) such as interface distance (speed, width) of the mobile switching interface, and the interface related data of the object to be measured is substituted into a standard curve (fig. 2C, D and fig. 3C, D) obtained by measuring according to a standard solution to obtain the corresponding concentration of the object to be measured.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the 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 visually detecting substance concentration based on a mobile exchange interface is characterized by comprising the following steps:
(1) in the electrophoresis channel, an object to be detected with given concentration and a ligand on the surface of a solid phase substance are subjected to exchange reaction to form a visual mobile exchange interface, and a standard curve is established according to the relation between the mobile exchange interface parameter and the concentration of the object to be detected;
(2) under the same condition, carrying out electrophoresis on a substance with unknown concentration, recording mobile exchange interface parameters including interface migration time, interface migration distance, interface migration rate and interface width, and substituting into the standard curve established in the step (1) to obtain the concentration and surface density information of the substance;
fixing a solid-phase substance in an electrophoresis channel in advance, driving an object to be detected to move in the electrophoresis channel in an electrophoresis mode, and replacing a ligand on the surface of the solid-phase substance to form a mobile exchange interface;
after electrophoresis begins, the distance of the mobile exchange interface is obtained by recording the starting point and the end point at given time, when the interface completely appears and is marked as the starting point of the mobile exchange interface, the interface position is determined according to the extreme point after derivation of the signal intensity distribution curve, and the interface migration rate is calculatedv MEBThe derivative peak width of the interface signal intensity distribution curve is the interface widthwIn relation to speedv MEBCan be expressed as:
wherein the content of the first and second substances,f(c A, c B, k) Is a parameter relevant to the experiment, is calculated by a specific experiment parameter,kthe coefficient related to the reaction rate of the analyte displacing ligand.
2. The method of claim 1, wherein the solid phase material is selected from the group consisting of nanoparticles, microparticles, cells, viruses, and functionalized gels.
3. The method according to claim 1, wherein the ligand on the surface of the solid phase substance can be a single ligand or a collection of ligands, the analyte can displace the ligand on the surface of the solid phase substance, and the ligand performs a displacement reaction on the surface of the solid phase substance to form the optical signal.
4. The method for visually detecting the concentration of a substance based on the mobile exchange interface as claimed in claim 1, wherein the solid-phase substance is immobilized in the electrophoresis channel by means of gel or self-filling.
5. The visual detection method for substance concentration based on mobile switching interface of claim 1, wherein the specific method is as follows:
preparing a solution mixed with a ligand-coated solid-phase substance, mixing the solution with a gel solution, and transferring the mixture to an electrophoresis channel, or filling the mixture to the electrophoresis channel in a self-filling manner;
step two, respectively injecting the prepared gel to the positions marked by the capillary tubes, standing for gel polymerization, or standing to an equilibrium state after self-filling;
filling an object to be detected in a sample area of the electrophoresis tube, filling buffer solution in a buffer area, adding equal amount of electrode buffer solution in electrode solution pools at two sides of an electrophoresis channel, respectively connecting electrodes in a cathode solution pool and an anode solution pool with a power supply, and performing electrophoresis;
under the action of an electric field, the object to be detected migrates into a gel area, the object to be detected and a ligand on the surface of the solid-phase substance undergo a displacement reaction to form a mobile exchange interface, and after electrophoresis is carried out for a period of time, a power supply is turned off to stop the electrophoresis;
and step five, recording the interface migration process of electrophoresis by using equipment during electrophoresis to obtain the interface distance.
6. The method for visually detecting the concentration of a substance based on the mobile exchange interface as claimed in claim 5, wherein the capillary has a length of 10-100mm and an inner diameter of 75-600 μm.
7. The method for visually detecting the concentration of a substance based on the mobile exchange interface as claimed in claim 5, wherein the voltage applied by electrophoresis is 10-50V or 60-120V, and the total time of electrophoresis is less than 10 min.
8. The method for visually detecting the concentration of a substance based on the mobile exchange interface as claimed in claim 5, wherein the adopted gel is polyacrylamide gel, the mass volume concentration is 15-45%, the crosslinking degree is 2-6%, and the background electrolyte solution is tris-HCl buffer solution, the pH value is 4-10; in the detection, a fluorescent substance is used as an indicator, and a fluorescent signal is quenched or restored in the process of electrophoretic titration.
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