CN111235224A - Accurate biomolecule modification method and device based on magnetophoretic separation - Google Patents

Abstract

The present invention provides a method for precise modification of biomolecules based on magnetophoretic separation, comprising the following steps: (1) providing a glass nanoporous sensor, the glass nanoporous sensor comprising a glass tube on which a metal conductive layer is deposited; The nanopore sensor is connected with a power supply; (2) a separation template is provided, and at least one groove is arranged on the separation template; (3) the magnetic beads are evenly distributed on the separation template, and a magnetic field is independently placed on each groove of the separation template. Beads; (4) provide a solution containing biomolecules, put them into the glass tube of the glass nanopore sensor, turn on the circuit, when the biomolecules pass through the glass tube to generate a blocking current signal, stop applying voltage, and a single biomolecule binds to the magnetic On the bead surface, the next magnetic bead is assembled in the same way to obtain a magnetic bead with a single biomolecule bound to it. The invention realizes the precise assembly of a single biomolecule and a magnetic bead, the assembly method is simple, and the assembly efficiency is high.

Description

A method and device for precise modification of biomolecules based on magnetophoretic separation

technical field

The invention relates to the field of single-molecule detection, in particular to a method for precise modification of biomolecules based on magnetophoresis separation.

Background technique

The rise of molecular biology has made the research mode of life science go deep into the molecular level, and people have been expecting to directly study basic life processes at the single-molecule level. The study of DNA molecules is the basis of molecular biology research, so single-molecule manipulation also begins with the study of DNA. Currently, commonly used single-molecule manipulation techniques include optical tweezers, magnetic tweezers, glass microneedles, AFM probes, and Stokes drag. In the process of applying these single-molecule manipulation techniques to control DNA, it is inevitable to indirectly control DNA through magnetic beads. Optical tweezers are optical traps formed by focusing a laser beam to generate radiation pressure to capture magnetic beads to control DNA displacement on the magnetic beads. Magnetic tweezers use an applied gradient magnetic field to control magnetic beads to control DNA. Both glass microneedles and AFM probes use mechanical linkage to adsorb magnetic beads to control DNA. Of course, glass microneedles or AFM probe tips can also be chemically modified to bind DNA to achieve the purpose of DNA control.

As mentioned above, magnetic beads are used indirectly in the application of single-molecule manipulation techniques. However, taking streptavidin magnetic beads as an example, in the process of linking modified biotin DNA experiments, all streptavidin magnetic beads All elements will interact and bind with biotin on DNA, which means that several DNAs will be bound on one magnetic bead, which is very unfavorable for DNA single-molecule research. The ideal measurement of DNA molecular mechanical properties, DNA molecular sequencing, and DNA-protein interaction research on this basis requires that there is only one DNA single strand on the magnetic beads, so as to avoid other DNA molecules on the magnetic beads from affecting the experimental results. influences. Therefore, it will be of great significance for molecular biology to study how to bind single biomolecules on single magnetic beads.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the problems that the existing magnetic beads are difficult to combine with a single biomolecule, and provide a method for precise modification of biomolecules based on magnetophoresis separation, which realizes the precise assembly of a single biomolecule and a magnetic bead, and avoids the need for magnetic beads. The effect of other biomolecules on the experimental results is simple and the assembly efficiency is high.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

A method for precise modification of biomolecules based on magnetophoretic separation, comprising the following steps:

S1. Provide a glass nanoporous sensor, the glass nanoporous sensor includes a glass tube, a metal conductive layer is deposited on the glass tube, and the glass nanoporous sensor is connected with a power supply;

S2, provide a separation template, the separation template is provided with at least one groove;

S3, evenly distributing the magnetic beads on the separation template, and placing a magnetic bead independently on each groove of the separation template;

S4. Provide a solution containing biomolecules, put it into the glass tube of the glass nanopore sensor, and turn on the circuit. When the biomolecules pass through the glass tube to generate a blocking current signal, stop applying voltage, and a single biomolecule binds to The surface of the magnetic bead is then assembled in the same way to obtain a magnetic bead with a single biomolecule bound to it.

There is no sequence between step S1 and step S2.

Optionally, in the step S1, the material of the metal conductive layer includes but is not limited to gold, silver, aluminum, platinum, etc., and the deposition process may adopt physical vapor deposition, atomic layer deposition, electroplating, and the like.

Optionally, in the step S1, the inner diameter of the glass tube < the diameter of the magnetic beads, which can effectively prevent the magnetic beads from being sucked into the glass tube.

Preferably, the inner diameter of the glass tube can be 200 nm-10 μm, preferably 300 nm-10 μm, more preferably 400 nm-10 μm, more preferably 500 nm-10 μm, more preferably 800 nm-10 μm, more preferably 1 μm-10 μm, and It is preferably 5 μm to 10 μm, and the inner diameter of the glass tube is not limited to the above range, and may be other values as long as the inner diameter of the glass tube<the diameter of the magnetic bead is satisfied.

Optionally, in the step S2, the diameter of the groove is denoted as D, and the diameter of the magnetic bead is denoted as d, where D>d.

Preferably, in the step S2, D<1.5d.

Optionally, in the step S2, the number of grooves on the separation template may be one, two, three or more, and the number of grooves can be designed according to requirements.

Optionally, in the step S4, the biomolecules include antigen molecules and/or DNA molecules, and the antigen molecules include but are not limited to streptavidin, digoxigenin, and the like.

The present invention also provides a magnetic bead bound with a single biomolecule modified according to the above-mentioned method for precise modification of biomolecules.

The present invention also provides a device for precise modification of biomolecules based on magnetophoretic separation, comprising a separation mechanism for separating magnetic beads, the separation mechanism has a separation cavity and a magnet located outside the separation cavity, and the separation cavity is provided with a separation A template, the liquid inlet of the separation cavity is communicated with a liquid supply container, the liquid outlet of the separation cavity is communicated with a collection container, and the separation template is provided with a groove for adsorbing magnetic beads.

Optionally, a glass nanopore sensor for transporting single biomolecules to the surface of the magnetic bead is also included, the glass nanopore sensor comprises a glass tube, and an electrode layer is provided on the outer surface of the glass tube.

Optionally, the solution in the glass tube and the electrode layer are each connected to a power source through a wire, and after a voltage is applied to the power source, the current can be turned on to realize the screening of biomolecules.

Optionally, it also includes a motion platform for controlling the movement of the glass nanopore sensor, and the motion platform controls the glass nanopore sensor to move in the X, Y, and Z directions.

The present invention has at least the following beneficial effects:

1. Realize the precise assembly of single biomolecules and magnetic beads. In ideal DNA molecular mechanical properties measurement, DNA molecular sequencing, and DNA-protein interaction research on the basis of DNA single-molecule research, the assembly method provided by the present invention realizes the precise assembly of a single biomolecule and a magnetic bead, Avoid the influence of other biomolecules on the magnetic beads on the experimental results.

2. The assembly method is simple and the assembly efficiency is high. The method for precise assembly of a single biomolecule and a magnetic bead provided by the present invention is simple and convenient to operate, and the magnetic bead separation template can be used to realize the simultaneous assembly of multiple magnetic beads and biomolecules, thereby improving the assembly efficiency.

Description of drawings

FIG. 1 shows a process flow diagram of a method for precise modification of biomolecules based on magnetophoretic separation in an embodiment of the present invention.

FIG. 2 is a schematic diagram of a glass nanopore sensor required for the method for precise modification of biomolecules based on magnetophoretic separation in an embodiment of the present invention.

FIG. 3 is a schematic diagram of a separation template required for the method for precise modification of biomolecules based on magnetophoretic separation in an embodiment of the present invention.

FIG. 4 is a schematic diagram of the magnetophoretic separation technology presented in step 4) of the method for precise modification of biomolecules based on magnetophoretic separation in the embodiment of the present invention.

FIG. 5 is a schematic diagram of the structure presented in step 4) of the method for precise modification of biomolecules based on magnetophoretic separation in the embodiment of the present invention.

FIG. 6 is a schematic diagram of the structure presented in step 5) of the method for precise modification of biomolecules based on magnetophoretic separation in the embodiment of the present invention.

FIG. 7 is a schematic diagram showing the blocking current detection presented in step 5) of the method for precise modification of biomolecules based on magnetophoretic separation in the embodiment of the present invention.

FIG. 8 is a schematic diagram showing the working of the motion platform in the method for precise modification of biomolecules based on magnetophoretic separation in an embodiment of the present invention.

Component label description: 1, glass nanopore sensor; 2, separation template; 3, magnetic beads; 4, biomolecules; 5, motion platform; 6, liquid supply container; 61, liquid inlet pipe; 7, pump; 8, collection Container; 81. Liquid outlet pipe; 9. Magnet; 10. Glass tube; 11. Electrode layer; 12. Separation mechanism; 13. Separation cavity; 20. Groove.

Detailed ways

The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1

As shown in FIG. 1 , this embodiment provides a method for precise modification of biomolecules based on magnetophoretic separation, which includes the following steps:

S1. Manufacture of glass nanoporous sensor: provide a capillary glass tube, use the laser melting method to pull out the conical nanoporous channel, and cut out the conical nanoglass tube of the desired shape, that is, the glass tube 10;

S2, depositing an electrode layer 11 on the outer surface of the tapered nanopore channel to form a glass nanopore sensor 1;

S3. Provide a separation template;

S4, using the magnetophoretic separation technology to evenly distribute the magnetic beads on the separation template, and place a magnetic bead on a single groove of the separation template;

S5. Through the glass nanopore sensor, a single biomolecule is transported to the surface of the magnetic bead whose surface is infiltrated. Depending on the number of blocked currents, precise assembly of individual biomolecules with magnetic beads can be achieved.

The method for precise modification of biomolecules based on magnetophoretic separation of the present invention will be described in detail below with reference to the specific drawings.

First, step S1 is performed, the tapered nanopore channel can be drawn by a laser needle puller, the inner diameter of the capillary glass tube 10 can be 200 nm˜10 μm, and the inner diameter of the capillary glass tube is smaller than the diameter of the magnetic bead. In this embodiment, the inner diameter of the capillary glass tube is about 500 nm.

Next, step S2 is performed to deposit conductive metal materials on the outer surface of the capillary glass tube, specifically gold, silver, aluminum, platinum, etc.; the deposition process can use physical vapor deposition, atomic layer deposition, electroplating, and the like. In this embodiment, the deposition material on the outer surface of the capillary glass tube is silver, and the deposition process is physical vapor deposition. The structure of the fabricated glass nanoporous sensor 1 is shown in FIG. 2 .

Next, step S3 is performed, and a plurality of circular grooves 20 are evenly distributed on the separation template, which may be distributed in an array such as 10×10, 20×20, etc. The groove diameter D ranges from 1 μm to 10 μm, the groove diameter D is larger than the magnetic bead diameter d but less than 1.5d, and the depth is 0.5d to 1.5d, so that one groove can only accommodate one magnetic bead. In this embodiment, there are six circular grooves 20 of 2×3 evenly distributed on the separation template 2 , and the diameter D of the grooves 20 is 6 μm, as shown in FIG. 3 .

Next, step S4 is performed, as shown in FIG. 4 and FIG. 5 , the magnetic beads are evenly distributed on the separation template 2 using the magnetophoretic separation technology, and a magnetic bead is placed on a single groove on the separation template, and the diameter d of the magnetic bead can be 1μm～10μm. In this embodiment, the diameter d of the magnetic beads 3 is 5 μm.

As shown in FIG. 4 , the separation mechanism 12 has a separation cavity 13 and a magnet 9 located outside the separation cavity 13 . The magnet 9 is located below the separation cavity 13 . bottom, so that the magnetic bead solution in the separation chamber 13 can immerse the separation template 2, the liquid supply container 6 is filled with the magnetic bead solution, and the liquid supply container 6 is connected to the liquid inlet of the separation chamber 13 through the liquid inlet pipe 61, and the liquid inlet pipe 61 is provided with a pump 7. The pump 7 can be a micro-injection pump specifically, which provides power for the flow of the solution. The liquid outlet of the separation chamber 13 is connected to the collection container 8 for collecting the solution through the liquid outlet pipe 81. There are grooves 20 for attracting magnetic beads.

The specific method of magnetophoresis separation is as follows: use a micro syringe pump to transport the magnetic bead solution, the magnetic beads will be adsorbed on the separation template under the action of the gradient magnetic field when passing through the separation template, and after a certain period of time, use deionized water or buffer solution to wash off excess Magnetic beads, which can be used to control gradient magnetic fields with electromagnets.

Finally, step S5 is performed, and the biomolecules in the present invention can only be charged biomolecules. When the biomolecule is negatively charged, the positive lead is attached to the silver coating, the lead is inserted over the molten silver, and the negative lead is placed in the solution in a glass tube. The buffer solution of biomolecules is PBS buffer. As shown in Figure 6, single biomolecules are delivered to the surface of a magnetic bead whose surface is infiltrated through a glass nanopore sensor, which can be moved using a nanomotion platform 5 (or a nanopositioning platform, purchased from 3 Yingjing Control (Tianjin) Instrument Equipment Co., Ltd.). The single biomolecule can be an antigenic molecule such as streptavidin, digoxin, and other biomolecules.

The voltage is 100mV. The binding of streptavidin to the magnetic beads is achieved by physical and chemical methods. One method is to modify the surface of the magnetic beads with an aldehyde group. The aldehyde group and the amino group of streptavidin are combined. A nucleophilic addition reaction occurs between the beads to realize the introduction of streptavidin on the surface of the magnetic beads.

Driven by an electric field, biomolecules pass through the capillary glass tube and bind to the surface of the magnetic bead. When biomolecules pass through the capillary glass tube, the physical occupation of the molecule generates a blocking current signal. When a biomolecule passes through the capillary glass tube, a blocking current is generated. Signal, the application of voltage is stopped at this time, and the nanomotion platform moves to the next magnetic bead position, enabling the precise assembly of single biomolecules, using fluorescently labeled streptavidin, or using long DNA chains labeled with biotin, magnetic The streptavidin on the beads binds to biotin, and if the magnetic beads drag only one piece of DNA, only one streptavidin is bound to the magnetic beads. In this example, the biomolecule used is streptavidin. As shown in Figure 7, when a single streptavidin passes through the glass nanopore channel, the blocking current will drop; the nanomotion platform moves as shown in Figure 8 Show.

In conclusion, the present invention provides a method for precise modification of biomolecules based on magnetophoretic separation, which realizes the precise assembly of single biomolecules and magnetic beads. In ideal DNA molecular mechanical properties measurement, DNA molecular sequencing, and DNA-protein interaction research on the basis of DNA single-molecule research, the assembly method provided by the present invention realizes the precise assembly of a single biomolecule and a magnetic bead, Avoid the influence of other biomolecules on the magnetic beads on the experimental results. In addition, the assembling method is simple and the assembling efficiency is high, and the magnetic beads are used to separate the template, so that multiple magnetic beads and biomolecules can be assembled at the same time, and the assembling efficiency is improved.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (10)

1. A biomolecule precise modification method based on magnetophoretic separation is characterized by comprising the following steps:

s1, providing a glass nanopore sensor, wherein the glass nanopore sensor comprises a glass tube, a metal conducting layer is deposited on the glass tube, and the glass nanopore sensor is connected with a power supply;

s2, providing a separating template, wherein the separating template is provided with at least one groove;

s3, uniformly distributing magnetic beads on the separation template, and independently placing one magnetic bead on each groove of the separation template;

s4, providing a solution containing biological molecules, loading the solution into a glass tube of the glass nanopore sensor, conducting a circuit, stopping applying voltage when the biological molecules pass through the glass tube to generate a blocking current signal, binding single biological molecules to the surface of the magnetic beads, and then assembling next magnetic beads in the same way to obtain the magnetic beads bound with the single biological molecules.

2. The method for precisely modifying a biomolecule according to claim 1, wherein: in step S1, the material of the metal conductive layer is at least one selected from gold, silver, aluminum, and platinum.

3. The method for precisely modifying a biomolecule according to claim 1, wherein: in the step S1, the inner diameter of the glass tube is smaller than the diameter of the magnetic beads.

4. The method for precisely modifying a biomolecule according to claim 1, wherein: in the step S1, the inner diameter of the glass tube is 200nm to 10 μm.

5. The method for precisely modifying a biomolecule according to claim 1, wherein: in the step S2, the diameter of the groove is denoted as D, and the diameter of the magnetic bead is denoted as D, where D > D.

6. The method for precisely modifying a biomolecule according to claim 5, wherein: in step S2, D is less than 1.5D.

7. The method for precisely modifying a biomolecule according to claim 1, wherein: in step S4, the biomolecule includes an antigen molecule and/or a DNA molecule, and preferably, the antigen molecule includes streptavidin and digoxin.

8. The method for precisely modifying a biomolecule according to any one of claims 1 to 7, wherein the method is modified to obtain magnetic beads having single biomolecules bound thereto.

9. The utility model provides a device is decorated to accurate biomolecule based on magnetophoresis separation, its characterized in that, including separating mechanism (12) that is used for separating the magnetic bead, separating mechanism (12) have separation chamber (13) and are located magnet (9) in the separation chamber (13) outside, be equipped with separation template (2) in separation chamber (13), the inlet intercommunication of separation chamber (13) has liquid supply container (6), the liquid outlet intercommunication of separation chamber (13) has collecting container (8), be equipped with recess (20) that are used for adsorbing the magnetic bead on separation template (2).

10. The device for precisely modifying biomolecules according to claim 9, further comprising a glass nanopore sensor (1) for delivering single biomolecules to the surface of the magnetic beads, wherein the glass nanopore sensor (1) comprises a glass tube (10), and an electrode layer (11) is disposed on the outer surface of the glass tube (10).

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