CN107488208B

CN107488208B - Isoelectric focusing and separation method of carrier-free ampholyte

Info

Publication number: CN107488208B
Application number: CN201710580746.8A
Authority: CN
Inventors: 吴志勇; 谢颂芳; 牛纪成; 方芳
Original assignee: Northeastern University China
Current assignee: Northeastern University China
Priority date: 2017-07-17
Filing date: 2017-07-17
Publication date: 2020-06-05
Anticipated expiration: 2037-07-17
Also published as: CN107488208A

Abstract

The invention discloses an isoelectric focusing and separation method of carrier-free ampholyte, belonging to the field of protein sample separation and analysis. The method comprises the steps that a paper channel, an acidic solution, an alkaline solution, an amphoteric sample, two electrodes and a direct current power supply form a loop, the two electrodes are respectively inserted into the acidic solution and the alkaline solution, the two ends of the paper channel are respectively contacted with the acidic solution and the alkaline solution after the paper channel is wetted by the acidic solution or the alkaline solution, the two electrodes are connected with the direct current power supply through leads, and the amphoteric sample is directly loaded into the acidic solution or the alkaline solution or onto the paper channel. Compared with the prior art, the method can realize the rapid focusing and separation of the amphoteric sample without the aid of a free carrier amphoteric electrolyte, and the separated components are easy to recover and convenient to be used with other analysis methods. When a volatile weak acid and volatile weak base system is adopted, the compatibility with a mass spectrum is better.

Description

Isoelectric focusing and separation method of carrier-free ampholyte

Technical Field

The invention belongs to the field of protein sample separation and analysis, and relates to an isoelectric focusing and separation method of carrier-free ampholyte.

Background

Isoelectric focusing (IEF) is one of the modes of electrophoretic separation and concentration for amphoteric components with isoelectric points, and is widely used for separation and purification of complex proteins and characterization of isoelectric points. On a separation channel with a pH gradient, under the action of an electric field, the amphoteric components migrate to the same pH position as the pI of the components according to the difference of isoelectric points, so that focusing and separation are realized. The methods for forming a pH gradient in the prior art are mainly divided into two types: one is by means of a free Carrier Ampholyte (CA), which is a mixture of a class of amphiphilic compounds, and upon application of a voltage, the mixture of a series of amphiphilic compounds with close isoelectric point spacing migrates under the influence of an electric field, forming a corresponding pH gradient within which the amphiphilic molecules are focused. However, the carrier ampholytes are not only expensive, but also interfere with other analytical methods such as mass spectrometry. The other is based on IPG (immobilized pH gradient) gel strip, acrylamide is taken as a matrix, and amphoteric monomers with different pK are utilized to form a polymer with certain pH gradient after crosslinking. However, the commercially available IPG strip is not only expensive, but also takes a long time to complete the IEF. In order to avoid the use of carrier ampholytes, researchers have tried isoelectric focusing methods for carrier ampholytes, such as self-focusing, thermally induced pH gradients, and electrolytically formed pH gradients, but pH gradients are not quantitatively given, the system is complex, and the operation is cumbersome.

Since the first introduction of Paper-based analytical devices into the field of microfluidic analysis by the whitestandards group in 2007, Paper-based microfluidic analytical devices (PAD) have attracted considerable attention. The PAD has the advantages of low price, portability, less sample consumption, simple preparation, good biocompatibility and the like. The application range of the paper-based microfluidics at present covers a plurality of fields, such as food safety, environmental monitoring, clinical diagnosis and the like.

At present, a paper-based isoelectric focusing and separation method based on a carrier ampholyte has been proposed. Since the method utilizes the carrier ampholyte, an isoelectric focusing method is adopted on a paper-based analysis device to realize rapid focusing and separation of an ampholyte sample. Therefore, this method is not only costly, but also the carrier ampholyte interferes with other analytical methods (e.g., mass spectrometry) and is not conducive to mass spectrometric characterization of the separated and recovered components.

In view of the foregoing, there is a need for a method of isoelectric focusing and separation that can achieve rapid focusing and separation of amphoteric samples, easy recovery of separated components, and favorable mass spectrometry without the aid of a carrier ampholyte.

Disclosure of Invention

Technical problem to be solved

In order to solve the above problems in the prior art, the present invention provides an isoelectric focusing and separation method which can realize rapid focusing and separation of an amphoteric sample, easy recovery of separated components and favorable mass spectrum characterization without the aid of a free carrier amphoteric electrolyte.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

a method for isoelectric focusing and separation of carrier-free amphoteric electrolyte comprises the steps that a paper channel, an acidic solution, an alkaline solution, an amphoteric sample, two electrodes and a direct current power supply form a loop, the two electrodes are respectively inserted into the acidic solution and the alkaline solution, the two ends of the paper channel are respectively contacted with the acidic solution and the alkaline solution after being wetted by any one of the acidic solution and the alkaline solution, the two electrodes are connected with the direct current power supply through leads, and the amphoteric sample is directly loaded into the acidic solution or the alkaline solution or onto the paper channel.

As a preferable embodiment of the method for isoelectric focusing and separation of a carrier-free ampholyte, the concentration of the acidic solution is 0.1mM-1.0M and the concentration of the basic solution is 0.1 mM-1.0M.

As a preferable scheme of the isoelectric focusing and separation method of the carrier-free ampholyte, the acidic solution is a volatile weak acid HAc solution or a HCOOH solution, and the alkaline solution is a volatile weak base NH₃·H₂And (4) O solution.

As a preferable scheme of the isoelectric focusing and separation method of the carrier-free ampholyte, the anode of the two electrodes adopts a platinum electrode, and the cathode adopts a platinum electrode.

As a preferable scheme of the isoelectric focusing and separation method of the carrier-free ampholyte, the paper channel is a separation channel made of a fiber material.

As a preferable scheme of the isoelectric focusing and separation method of the carrier-free ampholyte, the paper channel is a glass fiber membrane, and the length of the paper channel is 5-100mm, and the width of the paper channel is 100 μm-10 mm.

As a preferable embodiment of the method for isoelectric focusing and separation of the carrier-free ampholyte, the electric field intensity applied to the paper passage is in the range of 3 to 300V/cm.

(III) advantageous effects

Compared with the prior art, the invention has the following advantages:

1. according to the invention, a free or fixed carrier ampholyte in a certain range is not needed, the rapid focusing and separation of a complex ampholyte sample (protein sample) are realized on a paper-based analysis device by adopting an isoelectric focusing method of a carrier ampholyte, the separated components are easy to recover, the cost is reduced, the interference generated when the carrier ampholyte is combined with other subsequent analyses (such as mass spectrometry) is avoided, and the mass spectrometry characterization of the separated and recovered components is facilitated.

2. The amphoteric sample is directly loaded into the acidic anode solution or the alkaline cathode solution or on a paper channel, and has two advantages: on one hand, the amphoteric sample can be electrified, and the migration of amphoteric sample components under the action of an electric field is facilitated after the direct-current voltage is applied; on the other hand, the operation can be simplified.

Drawings

FIG. 1 is a schematic view showing the structure of a paper-based analyzer in example 1 of the present invention;

FIG. 2 is a graph showing the results of focusing separation and the current change with time during focusing separation of a mixed sample of phycocyanin and bovine hemoglobin by means of isoelectric focusing in example 1 according to the present invention;

FIG. 3 is a graph showing a pH distribution on a paper path and a signal intensity distribution on the paper path after completion of focusing in example 1 of the present invention;

FIG. 4 is a signal intensity distribution diagram on a paper path in example 1 of the present invention;

FIG. 5 is a SDS-PAGE profile and a mass spectrum focusing on a fraction to be recovered in example 1 of the present invention;

FIG. 6 is a graph illustrating the controllability analysis according to the present invention.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

Example 1

This example proposes a method for isoelectric focusing and separation of phycocyanin and bovine hemoglobin mixed samples on an open paper-based analytical device shown in fig. 1 without the aid of a carrier ampholyte, in which a paper channel, an acidic solution, an alkaline solution (exemplified by a volatile weak acid and a volatile weak alkaline solution), an amphoteric sample, two electrodes, and a dc power supply are used to form a loop.

Specifically, HAc solution with a concentration of 10mM, pH 4.3, is added into the anode reservoir, and the HAc solution contains mass volume fractions (i.e. the mass of HEC and the volume fraction of HAc solution)The volume ratio of the liquid, m/V) is 0.2% of HEC (wherein HEC is hydroxyethyl cellulose, belongs to nonionic surfactant, and is used for inhibiting electroosmotic flow. The surface of the paper channel has silicon hydroxyl groups, and the negative charge causes the solution on the paper channel to be positively charged, and the solution has the tendency of moving to the negative pole as a whole, which is called electroosmotic flow. In electrophoretic analysis, when electroosmotic flow is large, a surfactant is often used for inhibition. ) A platinum electrode was inserted as an anode. Adding mixed sample containing phycocyanin 0.5mg/mL and bovine hemoglobin 1.0mg/mL into cathode liquid storage tank, wherein the matrix of the mixed sample is NH with concentration of 10mM₃·H₂O solution (pH 9.8) with a platinum electrode inserted as cathode. The paper channel is wetted by the HAc solution, and the two ends of the wetted paper channel are respectively wetted with the HAc solution and NH₃·H₂The O solution is contacted, and the positive electrode and the negative electrode are connected with a direct current power supply through leads to form a loop. The paper channel was a glass fiber membrane with dimensions of 35mm in length and 3mm in width. And applying a direct current voltage with the electric field intensity of 100V/cm, and forming a certain pH gradient on the paper channel under the action of the electric field. The purpose of the direct wetting of the paper channels with the anode's HAc solution is to complete the circuit. Of course, the paper channel can be made of other fiber materials to form the separation channel.

The principle of the invention is as follows: the paper channels were wetted with an acidic supporting electrolyte and immersed at both ends in an acidic anolyte supporting electrolyte solution and an alkaline catholyte solution, respectively. After applying DC voltage, OH of cathode liquid storage tank^-Migration to the anode end, H of the anode pool⁺The materials migrate to the cathode, and neutralization reaction occurs when the materials meet, wherein a neutralization interface starts from the cathode end and gradually moves to the anode end; cathode terminal to the area of the neutralizing interface due to OH^-The migration forms regions of decreasing basicity, the species present being predominantly OH^-. Area of anode end to neutralization interface due to H⁺Will form a zone of decreasing acidity, the species present being predominantly H⁺. At the neutralization reaction interface, the substance existing is mainly H₂O, neutral. H⁺Not only migrate to the paper path under the influence of an electric field, but also electricityThe seepage will also pump it up the paper channel so the neutralization reaction will not proceed to the anode reservoir. As the neutralization reaction produces water with low conductivity, the current is reduced, the electromigration of the charged species is reduced, and eventually the system reaches equilibrium and a stable pH gradient is formed across the paper path.

Phycocyanin (isoelectric point about 4.45) and bovine hemoglobin (isoelectric point about 6.8) samples were in a sample matrix (pH 10) above their isoelectric points and were negatively charged, migrating toward the anode under the influence of an electric field, and the charges were zero at pH positions that migrated close to their isoelectric points, and focused there, allowing the separation of samples of different isoelectric points.

Fixing the mobile phone on an eyepiece of a stereoscopic microscope by using a fixing clamp, observing the whole focusing process by using professional photographing software Camera FV-5, setting the photographing interval to be 10s, and focusing and separating the result as shown in figure 2, wherein when the photographing time is 590s, phycocyanin and bovine hemoglobin are focused and separated simultaneously. From the current-time curve, the current drops and is maintained at a very low level, about 30 μ A, from the photographing time 350s, and the system can exist stably for a long time.

The pH information on the paper channel and the separation degree of phycocyanin and bovine hemoglobin were obtained by the following operation methods. Specifically, the experiment was performed without the carrier ampholyte on the open paper-based analysis device shown in fig. 1 by isoelectric focusing and separation of ampholyte components, in the case of a sample blank, and the other experimental conditions were as described above. And (5) representing the pH information on the paper channel at the photographing time of 590s by using a precise pH test paper at an interval of 5 mm. As a result of five replicates, it was found that the pH formed on the paper passage ranged from 4.3 to 9.8 (see FIG. 3). When the focus image of the phycocyanin and bovine hemoglobin at 590s obtained in fig. 2 was placed in this gradient, it was found that the pH at which phycocyanin was focused was about 4.5 and close to its isoelectric point of 4.45, and that the pH at which bovine hemoglobin was focused was about 6.5 and close to its isoelectric point of 6.8. The focused image was processed by ImageJ to obtain a signal intensity profile on the paper path (see fig. 4). Calculation formula based on signal intensity distribution graph and separation degree R on paper channel

In the formula, t_R2: retention time, t, of the latter component in two adjacent peaks_R1: retention time of the former component in two adjacent peaks, W₂、W₁: the width of the bottom of the two adjacent peaks is known as t_R2＝1435，t_R1＝958，W₂＝133，W₁The degree of separation R of phycocyanin and bovine hemoglobin was calculated to be 3.53, indicating that both had been completely separated (when the degree of separation in the chromatogram was 1.5, both fractions were considered to be completely separated).

After focusing is completed, the phycocyanin and the bovine hemoglobin focused on the paper channel are recovered, and the recovery process is as follows: 1. cutting a focusing belt on the paper channel at the photographing time of 590s shown in FIG. 2, and placing the focusing belt in two centrifugal tubes; 2. adding deionized water into each centrifugal tube, and oscillating and dissolving; 3. and (4) placing the two centrifuge tubes into a centrifuge for centrifugation, and reserving the supernatant after centrifugation to obtain the recovered sample. The recovered samples of phycocyanin and bovine hemoglobin were subjected to isoelectric focusing again on the open paper-based analytical device shown in fig. 1, and phycocyanin and bovine hemoglobin were separated by focusing, which proved that the sample recovery could be achieved by the method of this example.

To verify that the established method can separate mixed proteins, the paper channel is cut into five equal parts (as shown in FIG. 5 a), the recovered components are placed in an EP tube, and then the recovered components are dissolved by shaking with a sample buffer solution and denatured by heating for SDS-PAGE (SDS-PAGE for short). As shown in FIG. 5b, lane 1 is a low molecular weight protein marker, lanes 2 to 6 are sequentially recovery components from lanes 1 to 5, lane 7 is a mixture of phycocyanin and bovine hemoglobin.No. 2, 5 and 6 should have no protein band according to the pH distribution on the paper channel and the pI of the model protein used,

lanes

3 and 4 should have phycocyanin and bovine hemoglobin.A case where phycocyanin contains α and β subunits and both have molecular weights of about 18kDa and 19kDa, respectively; bovine hemoglobin contains four subunits, 15-15 molecular weights, and 15-one-three-two subunits, and four of the same molecular weights of the same as the total protein, and the same as the protein, and the protein, respectively.

As can be seen from the SDS-PAGE profile of FIG. 5b, protein bands appeared in only the fractions recovered No. 2 and No. 3, and no protein band appeared in the other fractions recovered. Therefore, when the matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF/TOF) is carried out to verify that the mass spectrum compatibility of the established system is good, only the recovery components No. 2 and No. 3 are selected for analysis. Acetic acid and ammonia water are volatile components and are mass spectrum friendly electrolytes. When the method is combined with mass spectrometry, sample introduction detection can be directly performed without desalting treatment.

The recovered components No. 2 and No. 3 are placed in an EP tube, are dissolved by shaking with distilled water, supernatant is taken by ultrasonic waves and then is mixed with matrix SA (3, 5-dimethoxy-4-hydroxycinnamic acid), MALDI-TOF/TOF is detected in a linear mode, for the convenience of comparison, MALDI-TOF/TOF analysis is also carried out on a protein sample (solvent is water) which is not subjected to PAD-CAF-IEF, the existing literature indicates that phycocyanin contains two subunits α and β, the molecular weights are respectively 18.188kDa and 19.220kDa, as shown in figure 5c, dark and light spectral lines are respectively the spectra of the phycocyanin sample and the recovered component No. 2, and MALDI-TOF/TOF measurement results show that the phycocyanin and the recovered component No. 2 both contain two subunits 18.192kDa and 19.289, indicate that both are the same substances, and correspond to literature values.

Examples 2 to 10

Similar to the isoelectric focusing and separation method of example 1, examples 2-10 were prepared by adjusting the concentration of anolyte HAc solution (0.1mM-1M), catholyte NH₃·H₂Separating phycocyanin and bovine hemoglobin by using parameters such as concentration of O solution (0.1mM-1M), length and width of paper channel (length 5-100mM, width 100 μ M-10mM), and electric field intensity of direct current voltage (3-300V/cm). Examples 2-10 procedures for the separation of phycocyanin and bovine hemoglobin are specifically shown in Table 1.

Table 1 examples 2-10 procedures for the isolation of phycocyanin and bovine hemoglobin.

As can be seen from Table 1, the isoelectric focusing and separation method described in example 1 was performed by adjusting the concentration of the anolyte HAc solution (0.1mM-1M), catholyte NH, without changing other parameter conditions₃·H₂The concentration of the O solution (0.1mM-1M), the length and width of the paper channel (the length is 5-100mM, the width is 100 mu M-10mM, and the large size can separate more kinds of amphoteric samples at one time), the electric field intensity of the applied direct current voltage (3-300V/cm) and other parameters can be used for aggregating and separating phycocyanin and bovine hemoglobin, and the protein components focused and separated are easy to recover and are beneficial to mass spectrum characterization.

The paper channel of the invention can be wetted by the anolyte HAc solution and can also be wetted by the catholyte NH₃·H₂And O solution is used for wetting, and the wetting function is to enable the whole circuit to form an electrically-conductive loop so as to facilitate the focusing separation of protein components on the paper-based analysis device by adopting an isoelectric focusing method.

The anolyte solution of the present invention may be volatile weak acid HCOOH solution, HF solution, H solution, in addition to the weak acid HAc solution described above₂S solution and H₂CO₃The following acidic solutions can of course also be used: such as perchloric acid HClO₄HI hydriodic acid and H sulfuric acid₂SO₄HBr, HCl and HNO₃Iodic acid, HIO₃Oxalic acid H₂C₂O₄Sulfurous acid H₂SO₃Phosphoric acid H₃PO₄Pyruvic acid CH₃COCOOH, nitrous acid HNO₂Citric acid C₆H₈O₇Hydrofluoric acid HF and malic acid C₄H₆O₅Gluconic acid C₆H₁₂O₇Carbonic acid H₂CO₃Lactic acid CH₃CH (OH) COOH, benzoic acid C₆H₅COOH, acrylic acid CH₂CHCOOH, propionic acid CH₃CH₂COOH, stearic acid CH₃(CH₂)₁₆COOH, hydrogen sulfate H₂S, hypochlorous acid HClO, boric acid H₃BO₃Silicic acid H₂SiO₃And acidic buffer systems, and the like.

The catholyte of the present invention employs NH as described above₃·H₂O solution, or other alkaline solution, such as potassium hydroxide KOH, sodium hydroxide NaOH, barium hydroxide Ba (OH)₂Calcium hydroxide Ca (OH)₂And alkaline buffer systems, and the like.

Further, in the method for isoelectric focusing and separation of amphoteric components on an open paper-based device shown in fig. 1 without the aid of a carrier ampholyte, by adjusting the concentrations (i.e., pH values) of acidic and alkaline electrolytes, a corresponding pH range is formed on the paper passage, and amphoteric molecules having isoelectric points in this pH range can be focused. As shown in fig. 6a, the anode supporting electrolyte was 10 mhac, pH 4.3, and HEC was contained in the HAc solution at a mass volume fraction of 0.2% and used to wet the paper channels; cathode is 0.5mgmL^-1Cytochrome C (pI-10.0) protein sample with 1M NH as sample matrix₃·H₂Solution O, pH 11.8, see example 1 for other experimental conditions. It is known that cytochrome C is focused on paper passages in the pH range of 4.3 to 11.8. Therefore, the anode is selected from an acidic supporting electrolyte, the cathode is selected from an alkaline supporting electrolyte, a certain pH range can be formed on a paper channel, and amphoteric molecules with isoelectric points in the pH range can be focused and separated.

We verified using the following experiment, as shown in FIG. 6b, that the anode supporting electrolyte was 1mM H₃PO₄Solution, pH 3.0, H₃PO₄HEC with the mass volume fraction of 0.2% is contained in the solution, and the solution is used for wetting a paper channel; cathode pool: 0.5mg mL^-1Phycocyanin and 1.0mg mL^-1Bovine hemoglobin, sample matrix 1mM NaOH, pH 9.4, other experimental conditions see example 1. It is known that when phosphoric acid and sodium hydroxide are used as the anode and cathode supporting electrolytes, respectively, phycocyanin and bovine hemoglobin are also separated in a focused manner.

When the volatile weak acid solution and/or the volatile weak base solution are/is selected, the mass spectrum is friendly, so that the mass spectrum characterization of the separated and recovered components is facilitated, and when the mass spectrum characterization of the separated components is carried out, the salt removal operation treatment can be omitted, and the mass spectrum is directly combined with mass spectrum analysis, so that the operation procedures are reduced.

The anolyte of the invention is preferably a volatile weak acid solution HAc solution, and the catholyte is preferably a volatile weak base NH₃·H₂And (4) O solution. The benefits of selecting a volatile weak acid weak base are: in one aspect, H can be provided separately⁺And OH^-(ii) a On the other hand, HAc and NH₃·H₂O is a volatile component, is a mass spectrum friendly supporting electrolyte and is beneficial to the recovery of a separation component and the characterization of a mass spectrum. When the separated components are subjected to mass spectrometry, the separated components can be directly combined with mass spectrometry without desalting operation treatment, so that the operation procedures are reduced, and interference on subsequent other analyses (such as mass spectrometry) in combination is avoided due to introduction of new substances.

In conclusion, the invention does not need to use a certain range of free or fixed carrier ampholytes, and adopts a carrier-free ampholyte isoelectric focusing method on a paper-based analysis device to realize the rapid focusing and separation of a complex ampholyte sample (a protein sample), thereby not only reducing the cost, but also avoiding the interference of the carrier ampholytes when the carrier ampholytes are used with other subsequent analyses (such as mass spectrometry).

In the present invention, the direct loading of amphoteric samples into acidic anode solutions or alkaline cathode solutions or on paper channels has two advantages: on one hand, the amphoteric sample can be electrified, and the migration of amphoteric sample components under the action of an electric field is facilitated after the direct-current voltage is applied; on the other hand, the operation can be simplified.

The technical principles of the present invention have been described above in connection with specific embodiments. The description is made for the purpose of illustrating the principles of the invention and should not be construed in any way as limiting the scope of the invention. Based on the explanations herein, those skilled in the art will be able to conceive of other embodiments of the present invention without any inventive step, which shall fall within the scope of the present invention.

Claims

1. A method for isoelectric focusing and separation of carrier-free amphoteric electrolyte is characterized in that a paper channel, an acidic solution, an alkaline solution, an amphoteric sample, two electrodes and a direct current power supply are adopted to form a loop, the two electrodes are respectively inserted into the acidic solution and the alkaline solution, the two ends of the paper channel are respectively contacted with the acidic solution and the alkaline solution after being wetted by any one of the acidic solution and the alkaline solution, the two electrodes are connected with the direct current power supply through leads, and the amphoteric sample is directly loaded into the acidic solution or the alkaline solution or onto the paper channel; the amphoteric sample contained no carrier ampholyte.

2. The method for isoelectric focusing and separation of a carrier-free ampholyte according to claim 1 wherein the concentration of the acidic solution is 0.1mM to 1.0M and the concentration of the basic solution is 0.1mM to 1.0M.

3. The method for isoelectric focusing and separation of a carrier-free ampholyte according to claim 2 wherein the acidic solution is a volatile weak acid HAc solution or HCOOH solution and the basic solution is a volatile weak base NH 3-H₂And (4) O solution.

4. The method for isoelectric focusing and separation of a carrier-free ampholyte according to claim 3 wherein the anode of said two electrodes is a platinum electrode and the cathode is a platinum electrode.

5. The method for isoelectric focusing and separation of a carrier-free ampholyte according to claim 4 wherein the paper channel is a separation channel made of fibrous material.

6. The method for isoelectric focusing and separation of a carrier-free ampholyte according to claim 5 wherein the paper channel is a glass fiber membrane, the paper channel having a length of 5 to 100mm and a width of 100 μm to 10 mm.

7. The method for isoelectric focusing and separation of a carrier-free ampholyte according to claim 6 wherein the electric field intensity applied to the paper path is in the range of 3 to 300V/cm.