WO2021052146A1

WO2021052146A1 - Label-free biochemical reaction detection method

Info

Publication number: WO2021052146A1
Application number: PCT/CN2020/112228
Authority: WO
Inventors: 谢彦博; 马昱; 孙淼
Original assignee: 西北工业大学
Priority date: 2019-09-18
Filing date: 2020-08-28
Publication date: 2021-03-25
Also published as: CN110702563A; CN110702563B

Abstract

A label-free biochemical reaction detection method, comprising: S100, filling a pipe (4) with an electrolyte solution; S200, selecting receptor molecule A (6) capable of reacting with target molecule B (7) and modifying receptor molecule A (6) on the inner wall of the pipe; S300, introducing a dispersed phase into the pipe (4) for same to form a dispersed phase area (3), and retaining the dispersed phase area (3) in the pipe (4); S400, measuring and recording a contact angle of the dispersed phase area (3) in the pipe (4) and the total resistance of the solution in the pipe (4) before reacting with the target molecule (7); S500, introducing a solution or fluid possibly containing target molecule B (7) into the pipe (4) and reacting with receptor molecule A (6); S600, repeating step S300 to step S400, comparing changes in the contact angle and resistance of the dispersed phase before and after the reaction; if the change is great, then the solution or fluid contains target molecule B (7); and if not, same does not contain target molecule B (7). This serves as an inexpensive, highly sensitive, quick, and label-obviating label-free detection method for a biochemical molecular reaction.

Description

A label-free chemical biological reaction detection method

Technical field

The invention belongs to the field of chemical biosensors, and specifically relates to a label-free chemical biological reaction detection method.

Background technique

There are chemical and biological reactions between many molecules or molecular clusters, such as the specific binding between antigen and antibody, which can be used for research in the fields of chemical analysis, biomedicine, and immunodiagnosis. Traditional methods used to detect such molecular reactions, such as fluorescence immunoassay and radioimmunoassay (RIA), generally have limitations such as cumbersome detection procedures, low detection efficiency, and high detection costs. Enzymes can be used instead of fluorescently labeled molecules or isotopic labels, and molecular reactions can be measured by detecting the activity of enzymes attached to antigens or antibodies, such as enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA) and so on.

With the development of microscopy and nano-processing technology, nano-channels made of materials such as silicon, polysilicon, and silicon oxide (glass) have begun to be used in immunoassays. Compared with traditional biochemical detection methods, the use of nanopores to detect molecular reactions is a physical detection method. There is an electric double layer on the inner wall of the nanopore, and the double electric layer composed of charged ions overlaps, which will affect the impedance in the pore. Since the molecules to be detected or the molecules formed after the reaction are often charged, the binding on the wall of the nanopore is accompanied by a change in the charge. The electrical measurement of the nanochannel before and after the reaction can detect a significant change in impedance. It is the realization principle of nanofluid detection. However, the solid nanochannel itself has some limitations, such as the high cost of preparation, the difficulty of manufacturing, the need to use a strong corrosive agent as an etching reagent, the operation is more dangerous, and it also pollutes the environment to a certain extent.

Summary of the invention

In view of the high cost, high technical barriers, slow detection time, and low detection sensitivity of traditional chemical and biomolecular reaction detection methods, the present invention proposes a label-free chemical and biological reaction detection method, which is low-cost, high-sensitivity, and rapid. , A label-free detection method for chemical and biomolecule reactions without labeling and low sample volume loss.

In order to achieve the above objectives, the present invention adopts the following technical means:

A label-free chemical and biological reaction detection method, including the following steps:

S100, make the pipe full of electrolyte solution;

S200, select the receptor molecule A that can react with the target molecule B, and modify the receptor molecule A on the inner wall of the pipeline;

S300: Pass the dispersed phase into the pipeline to form a dispersed phase area, and keep it in the pipeline;

S400: Measure and record the contact angle of the dispersed phase area in the pipeline, and the total resistance of the solution in the pipeline when the dispersed phase exists;

S500, the solution or fluid that may contain the target molecule B enters the pipeline and reacts with the receptor molecule A;

S600, repeat steps S300 to S400, and compare the contact angle and impedance changes of the dispersed phase before and after the reaction:

If the change is large, the solution or fluid contains the target molecule B; otherwise, it does not contain the target molecule B.

Compared with the prior art, the beneficial effects of the present invention are:

The detection method of the present invention can detect all molecular reactions that have changes in charge amount or changes in hydrophilicity and hydrophobicity. The changes in charge or changes in hydrophilicity and hydrophobicity before and after the reaction can be identified by the change in resistance or contact angle of the film channel, respectively, avoiding traditional detection methods. The marking process. Compared with solid nanochannels, on the one hand, the changes in hydrophilicity and hydrophobicity brought about by molecular reactions are used to make the detection highly sensitive; on the other hand, molecular modification and reaction in the pipeline is easier than directly in the narrow solid channel. Easy and fast. This detection method is also suitable for almost all molecular detection situations, which mainly depends on the type of reaction molecule selected. In theory, it can complete immune reaction detection, gas detection or any other detection work involving interface charge or contact angle changes. .

Furthermore, since the acquisition of the dispersed phase is simpler, it is only necessary to inject the dispersed phase into the cylindrical pipe connected with the solution to obtain the nanochannel, the main material required for detection is extremely low in cost, and no special processing steps are required for preparation. The modification and interaction of different molecules is on the inner wall of the cylindrical pipe, and the channel size can be on the order of micrometers or even larger. The modification operation process is more convenient and feasible, and it is easier to avoid the slow diffusion of molecules in the traditional nanochannel, and speed up The speed of detection of molecular reactions improves the detection efficiency.

Furthermore, it is possible to have changes in wettability and impedance at the initial stage of the reaction, so that chemical and biological reaction differences can be detected in the early stage of the reaction, and the time required for detection is shortened.

Furthermore, because the reaction occurs on the inner surface of the narrow pipe, it is possible to detect samples with a small content (as small as pL or even below), which reduces the loss of sample volume.

Furthermore, the impedance change based on the detection is not affected by the salt concentration of the environment, and there is no need for selective and pretreatment of the salt concentration of the liquid to be tested. For example, it can be directly used for blood detection (normal saline concentration), which simplifies the detection steps and improves the detection speed .

Furthermore, for any chemical or biological molecular reaction involving changes in charge or hydrophilicity and hydrophobicity, the detection can be achieved theoretically by this detection method, and it has the aforementioned detection advantages. Any receptor molecule and target molecule that can meet the requirement of infiltration change or impedance change at the interface can be detected.

Description of the drawings

Figure 1 is a schematic diagram of a thin film channel formed by the dispersed phase in a cylindrical pipe for molecular reaction detection.

Figure 2 is a schematic diagram of the functional surface of a cylindrical pipe, where receptor molecules and target molecules are combined on this interface (solid/liquid interface).

Fig. 3 is a schematic diagram of the change of the dispersed phase after the target molecule is detected by the thin film channel; where (a) is before the molecular reaction, (b) is after the molecular reaction; that is, before and after the molecular reaction, the contact angle of the dispersed phase in the channel changes.

Among them, 1 is a cylindrical pipe; 2 is an aqueous solution; 3 is a dispersed phase area; 4 is a pipe; 5 is a film channel formed by a solution; 6 is a receptor molecule modified at the interface between the pipe and the film channel; The target molecule of the receptor molecule reaction can come from the continuous phase or the dispersed phase; 8 is an instrument that can measure the impedance change, and records and monitors the impedance change before and after the reaction for reaction detection.

detailed description

The present invention will be further described in detail below in conjunction with the accompanying drawings:

In the method for detecting chemical or biomolecular reactions of the present invention, the reaction here includes any reaction that causes the disperse phase to cause impedance changes at the continuous phase interface. The steps are as follows:

Step 1: Insert one end of a section of cylindrical transparent pipe into a pool of electrolyte solution, and fill the pipe with detection solution;

Step 2: Select a pair of molecules that can react: receptor molecule A and target molecule B, and modify receptor molecule A by modifying the inner wall of the pipeline;

Step 3: Use the solution and gas to form bubbles in the cylindrical pipe 1, and control the pressure balance of the liquid at the two ports of the pipe to keep the bubbles in the pipe, as shown in Figure 1; or make other dispersed phases form one in the solution Disperse phase area and keep it in the pipeline;

Step 4: Measure and record the contact angle of the dispersed phase in the pipeline at this time, and the impedance of the solution in the pipeline when the dispersed phase exists before the molecular reaction;

Step 5: Let the solution or fluid that may contain the target molecule B enter the pipeline and react with the receptor molecule A for a period of time;

Step 6: Repeat steps 3 to 4 to compare the contact angle and impedance changes of the bubbles before and after the reaction. If there is a big difference, it proves that the solution or fluid contains the target molecule B; otherwise, it does not contain the target molecule B.

The present invention uses the "thin-film nanochannel" formed by the dispersed phase in the cylindrical pipe to detect a type of molecular reaction. Such molecular reaction will cause the change of the contact angle. By using the change of the contact angle before and after the reaction, electricity can be used. /Impedance detection and contact angle observation are used to measure the reaction, and finally achieve the purpose of detection.

Specifically, the precautions in each step are as follows:

The cylindrical pipe used in step 1 can be made of hard materials such as glass and plexiglass. The type of material is not limited. The material should be transparent or translucent to control the dispersed phase in the pipe and measure the contact angle; the length of the pipe is not limited, and the inner diameter of the nozzle Unlimited. One end of the pipe is immersed in a pool of aqueous electrolyte solution. The electrolyte used is generally a neutral strong electrolyte salt or buffer salt to maintain the pH balance. The other end of the pipeline can be connected to a pressure pump or immersed in a pool of solution to ensure that the pipeline can be filled with solution and meet the needs of subsequent injection of the dispersed phase.

The reaction molecule selected in step 2 is not limited, it can be any one or more pairs of chemical or biological molecules that can react. The selected molecule determines the final detection function of the membrane channel, such as the choice of crown ether and potassium ions, APTE With carbon dioxide, antigens and antibodies, viruses and virus antibodies, etc. The reaction here means that in a mixed system containing various molecules, one kind of molecule only reacts with a certain kind of molecule. The reaction of two molecules is accompanied by detectable significant changes such as changes in charge or hydrophilicity and hydrophobicity. For example, one molecule carries additional hydrolyzable chemical groups, or the hydrolyzable chemical groups of a pair of molecules can react with each other to become electrically neutral. , Or differences in the hydrophilicity and hydrophobicity of the two molecules, and so on. One of the molecules can be modified on the inner wall of the pipeline and remains stable, as shown in Figure 2.

The dispersed phase used in step 3 can be any dispersed phase that is not easily soluble in the solution, unless there are special requirements, such as the detection target molecule is the molecule of the dispersed phase. The length of the dispersed phase region formed by the dispersed phase is not limited. In order to form thin film nanochannels and avoid shrinking into a spherical shape, the length should not be shorter than the pipe diameter.

The contact angle measured in step 4 is the contact angle of the dispersed phase on the surface of the water phase, which can be recorded and measured with a camera (the small dispersed phase area in the micron level can be observed and measured with a microscope) for comparison after subsequent reactions. The impedance of the liquid film is measured, and after the measurement is completed, the dispersed phase is discharged from the pipe to prepare for molecular reaction.

In step 5, the solution that may contain the target molecule is injected into the pipeline to keep or make it flow. The specific reaction method is not limited, and the reaction conditions are not limited. The reaction time should be based on the premise that the two molecules can fully react to ensure that if there is the target molecule to be detected , The receptor molecule should have time to contact and bind.

Step 6: After the reaction is over, inject the dispersed phase into the pipeline again. After it is stable, observe and measure the contact angle, measure the impedance of the liquid film after the reaction, and compare the contact angle or the change of the liquid film impedance to confirm the existence of the detected target molecule, such as As shown in Figure 3(a) and Figure 3(b).

The present invention will now be further described in conjunction with the embodiments and drawings:

Take the reaction of biotin and avidin molecules as an example. First, PDMS and glass capillary are used to prepare a chip for making bubbles, and according to the aforementioned step 2, the HEPES solution containing 200nM FITC-SAv molecules is injected into the capillary with a pump to modify the inner wall. At the same time, it can also be observed with a Zeiss optical microscope that before and after the streptavidin reaction, the contact angle of the bubble in the capillary changes from 50° to 28°. The change in the contact angle brings about a change in the thickness of the liquid film, which leads to a change in the thickness of the liquid film. A more significant change in the conductivity of the liquid film.

By using different concentrations of phosphate solutions to generate bubbles, a computer program written in LabVIEW was used to control the Keithley 6482 picoammeter to measure the conductance of the bubble liquid membrane, and it was found that the membrane channel was detected in a high concentration solution in the range of 0.1×PBS～10×PBS Utilizing the wettability change of the capillary wall, the conductivity change rate before and after the reaction is up to 200 or more. The solid channel detection can only occur at low concentrations, and the sensitivity is about 4, which shows that liquid film sensing can increase the detection sensitivity by two orders of magnitude. When testing the concentration of streptavidin that the membrane channel can detect, it was found that a minimum of 10 nM streptavidin could be detected. The reaction between biotin and streptavidin molecules takes a certain amount of time. For 200nM streptavidin molecules, it takes more than 1 hour to complete the reaction using solid nanochannels until it can be detected. When using thin-film nanochannels to detect 200nM streptavidin molecules, the solution of streptavidin molecules is injected into the capillary at a speed of 35 mm/s, and the conductivity changes 60 times in 10 minutes, that is, a significant conductivity change can be detected .

Therefore, the present invention has the following advantages:

Low technical cost and economic cost: As the disperse phase is easier to obtain, you only need to inject the disperse phase into the cylindrical pipe connected with the solution to obtain the nanochannel. The main material cost for detection is extremely low, and no special preparation is required.的processing steps. The modification and interaction of different molecules is on the inner wall of the cylindrical pipe, and the channel size can be on the order of micrometers or even larger. The modification operation process is more convenient and feasible, and it is easier to avoid the slow diffusion of molecules in the traditional nanochannel, and speed up The speed of detection of molecular reactions improves the detection efficiency.

Fast detection time: There is a change in hydrophilicity and hydrophobicity at the beginning of the reaction, so that the difference caused by the reaction can be observed and measured. Therefore, the detection can be realized in the initial stage before the reaction reaches equilibrium, which shortens the time required for detection.

High sensitivity: Through the change of contact angle and thin film liquid, a large impedance difference can be generated to achieve high-sensitivity detection.

No need for labeling: The detection reaction is based on the contact angle or impedance change, and there is no need for any markers used to identify the reaction. This eliminates the cumbersome labeling process and makes the detection easier.

Applicable to various salt concentrations: The contact angle change based on the detection is not affected by the environmental salt concentration, and there is no need to pre-process the environmental salt concentration, which simplifies the detection steps and enhances the detection accuracy.

Low sample loss: This method can detect samples with a small content (as small as pL or even smaller), reducing the loss of sample volume.

Applicable to a variety of chemical and biological reaction detection: For any chemical or biological molecular reaction involving changes in charge or hydrophilicity and hydrophobicity, this detection method can theoretically be used to achieve detection, and it has the aforementioned detection advantages. The specific detection function depends on the selected receptor molecule and target molecule type. Switching to a different molecule can complete immune response detection, gas detection or any other detection work involving interface charge or contact angle changes.

The above content is only to illustrate the technical ideas of the present invention, and cannot be used to limit the scope of protection of the present invention. Any changes made on the basis of the technical solutions based on the technical ideas proposed by the present invention fall into the claims of the present invention. Within the scope of protection.

Claims

A label-free chemical and biological reaction detection method, which is characterized in that it comprises the following steps:

S100, make the pipe full of electrolyte solution;

S200, select the receptor molecule A that can react with the target molecule B, and modify the receptor molecule A on the inner wall of the pipeline;

S300: Pass the dispersed phase into the pipeline to form a dispersed phase area, and keep it in the pipeline;

S400: Measure and record the contact angle of the dispersed phase area in the pipeline, and the total resistance of the solution in the pipeline when the dispersed phase exists;

S500, the solution or fluid that may contain the target molecule B enters the pipeline and reacts with the receptor molecule A;

S600, repeat steps S300 to S400, and compare the contact angle and impedance changes of the dispersed phase before and after the reaction:

If the change is large, the solution or fluid contains the target molecule B; otherwise, it does not contain the target molecule B.
The label-free chemical and biological reaction detection method according to claim 1, wherein step S100 specifically includes inserting one end of a section of cylindrical pipe into a pool containing an aqueous electrolyte solution, and providing pressure at the other end to fill the pipe with electrolyte Solution.
The label-free chemical biological reaction detection method according to claim 1, wherein the receptor molecule A is modified on the inner wall of the pipeline by coating, immersion or any other feasible modification methods.
The method for detecting unmarked chemical and biological reactions according to claim 1, wherein the pipe is a cylindrical pipe, and the material of the pipe is a transparent or semi-transparent material.
The label-free chemical biological reaction detection method according to claim 1, wherein the electrolyte is a neutral strong electrolyte salt or a buffer salt.
The method for detecting a label-free chemical biological reaction according to claim 1, wherein the target molecule B and the receptor molecule A are chemical or biological molecules capable of reacting, and the reaction includes any accompanying charge or hydrophobicity, etc. The process that can cause impedance changes.
The method for detecting label-free chemical and biological reactions according to claim 1, wherein the dispersed phase is a dispersed phase that is not easily soluble in a solution.
The label-free chemical and biological reaction detection method according to claim 1, wherein the length of the dispersed phase area formed by the dispersed phase should not be shorter than the pipe diameter; the dispersed phase area can be any gas, liquid, solid, etc. Compatible substances.
The method for detecting a label-free chemical and biological reaction according to claim 1, wherein the contact angle is the contact angle of the dispersed phase on the surface of the water phase and changes before and after the reaction; the impedance of the liquid film before and after the reaction changes. Measure this change for qualitative and quantitative analysis to determine the progress of the reaction.