KR20030040397A - Method and device for electronic control of the spatial location of charged molecules - Google Patents

Abstract

PURPOSE: A method and a device for electronically controlling the spatial location of charged molecules are provided which are useful in molecular biological reactions, such as nucleic acid hybridizations, nucleic acid amplification, sample preparation, antibody/antigen reactions, clinical diagnostics, and biopolymer synthesis. CONSTITUTION: A method of controlling the spatial location of charged molecules in a reaction unit having a central electrode surrounded by a plurality of outer electrodes on a substrate, wherein the unit being filled with medium for providing frictional resistance to the motion of the molecules, comprises the steps of: (1) placing a charged molecule in the reaction unit; (2) applying predetermined magnitudes of the first voltages to the central electrode and a set of at least one outer electrode selected from a plurality of outer electrodes to generate an electric field; and (3) applying predetermined magnitudes of the second voltages to the central electrode and a set of at least one outer electrode selected from a plurality of outer electrodes to rotate the electric field for controlling the spatial location of the charged molecules, wherein the net charge, generated by the voltage applied to the selected electrodes, across the unit is maintained at zero.

Description

Electrical apparatus and method for controlling spatial position of charged molecules {Method and device for electronic control of the spatial location of charged molecules}

Electrophoresis is the movement of charged molecules in an electric field and has been used as an analytical technique to separate and identify charged particles, ions or molecules.

The most frequently studied molecules are biological molecules such as proteins and DNA fragments, which are typically polymer electrolytes. Separation is the most frequently used electrophoresis in the life sciences. The molecules are separated by their different charge and frictional resistance properties. The motility (U) of the electrophoresis of the charged molecules in the electric field (E) is defined as the ratio of the velocity of the center of mass (Vg) to the externally applied electric field (Vg = UE). Exercise is faster.

When a mixture comprising several molecular species is introduced into an electrophoretic separation medium, when an electric field is applied, different charged components migrate out of the system at various rates, leading to analysis of the mixture. Bands appear depending on the properties of the components. The exact location (and therefore the time the components appear at the end of the medium opposite the point of introduction) is influenced by the pH, the strength of the ions, the type of the ions, the interaction of the polymer electrolyte with the surrounding medium, and the medium is the Or a buffer solution of a gel, such as a crosslinked gel. Crosslinked gels and polymer solutions can affect separation by the action of size or sieve. Thus, electrophoresis can be classified into two basic types, including (1) free solution and (2) gel electrophoresis. The most frequently used gel media is based on polyacrilamide (known as PAGE) and agarose gels.

Experimentation of free solution and gel electrophoretic separation combinations provides excess information such as the number and relative amounts of components in the mixture. When components are clearly identified, for example by methods such as antigen-antibody binding, clear identification is made for a given component. As a result, electrophoresis has become the cornerstone of macromolecular analysis in biotechnology.

U.S. Patent No. 5,126,022 (Solan invention, assigned to Soan Technology I & C) discloses a typical method and apparatus for moving charged molecules. This method requires a plurality of different electric fields being applied to the medium, such as a buffer solution, in order to move the molecules in the medium in an accurate manner. This technique can be used to move charged particles together and to separate them from each other to perform complex reactions and / or separation schemes.

WO 99/62622 (Arnold, William, Michael Invent, Transfered to Industrial Research Limited) discloses a method of concentrating and positioning particles in a medium by applying a voltage to an array of electrodes. In this method, the array of electrodes is spaced apart so that electrode elements, such as fingers interdigitated, are provided in rows, such as two comb teeth, to support the particles in the medium. Arnold et al. Did not indicate that this method provides a way to conduct biological reactions by concentrating particles into specific regions. This method provides a means for locating particles in a particular region for the purpose of separating or identifying the particular particles from the remaining buffer components.

U. S. Patent No. 6,017, 696 (Michael J. Heller invention, assigned to Nanogen I & C) discloses molecular biological analysis and electronic stringency control methods for diagnostics. In this method, molecules are transported to the micro position by the force of electrophoresis so that the speed of biological analysis can be significantly improved. This method also takes advantage of the electric field for stringency control so that various biological assays, including DNA hybridization, can be performed quickly.

However, in this way, negatively and positively charged molecules cannot be manipulated simultaneously. This requires the polarity of the electrodes to alternate from negative to positive in order to manipulate the charged particles. For example, the polymerase chain reaction (PCR) is an interaction between positively charged ions such as K ²⁺ , Mg ^2+, and a slightly positively charged Taq polymerase and a negatively charged DNA molecule. need. For PCR, the Nanoogen method cannot simultaneously manipulate all the reactants needed for the reaction. This is because the DNA molecules must be transported into the reaction unit to be fixed to the electrodes, and then the polarity of the electrodes must be changed to wash away the DNA molecules that are not immobilized. The polarity of the electrode must again be changed to transfer Taq polymerase to the reaction unit. This process is time consuming and it is not effective to repeat it for DNA extension.

These features limit the design of devices for biological analysis because of the complex process described above. The functionality is further limited in relation to new biological assays or new diagnostic techniques in which stringent control must be manipulated between negatively charged molecules and positively charged molecules.

It is known to separate and store ions by applying an electric field to a trap volume. U. S. Patent No. 6,124, 592, assigned to Sequiner, Glenn, and Nikaido et al., Discloses a method for separating and storing ions by utilizing the kinetic properties of the ions. In this way, ions are separated according to their kinetic properties by applying an electric field to the trap volume. The ions then move to equilibrium positions in the trap volume due to differences in motility and changes in electric field. Ions can be charged either positively or negatively, and the movement of the ions depends on the magnitude and direction of the applied electric field.

In this way, the ions cannot be localized to accelerate the desired reaction, such as hybridization. By applying an electric field, the ionized sample in the trap volume cannot be directed to a particular test site. Thus this method can only be used with ion mobility spectrometry (IMS).

WO 97/34689 (Petting et al., Assigned to University College of North Wales) discloses an apparatus for carrying out chemical, physical or physicochemical reactions between particles suspended in a medium of liquid. In this method, the particles move using dielectrophoresis or traveling wave dielectrophoresis.

There are several inventions related to the use of electrophoresis in biochips such as lab-on-a-chip. One of the reasons for inventing biochips using electrophoresis is the fact that many biological reactions require manipulation of two opposite charge molecules. Conventional inventions have difficulty in manipulating two opposite charged molecules for biological reactions.

This difficulty was solved through the geometry of the electrode arrangement and the pattern of applied voltage in the reaction unit. In the reaction unit invented by the inventor, a single central electrode is surrounded by a plurality of outer electrodes to simultaneously manipulate two oppositely charged molecules.

The present invention relates to the design and use of an addressable micro-electronic system capable of actively controlling the spatial position of charged particles in a microscopic format. The control is based on the force of electrophoresis and allows the selective separation and transfer of species of specific molecules. One embodiment of this method is in the control of biochemical reactions. In particular, such reactions include molecular biological reactions such as nucleic acid hybridization, nucleic acid amplification, sample preparation, antibody / antigen reactions, clinical diagnostics, and synthesis of biological polymers.

1 is the geometry of the electrode arrangement in the reaction unit.

2 is a pattern of application of electricity and rotation of the electric field.

3 is a clockwise movement of the electric field.

4 is the trajectory of molecules in a moving electric field.

5 is a reaction unit with an outer electrode ring.

6A is a schematic diagram of an electric field without an outer ring.

6b is a schematic diagram of an electric field with an outer ring.

7 is a cross section of a reaction unit made using microlithography.

8 is a matrix type apparatus with 50 reaction units.

9 is a schematic diagram of the reaction unit structure.

10A is a step of forming the binding layer (I).

10B is a step of forming the binding layer (II).

11 is a schematic diagram of a micromachined device.

The apparatus of the present invention and related methods enable molecular bioreaction and diagnostic reactions to be carried out under complete electronic control. This method and apparatus describe controlling the spatial position of charged molecules by applying an electric field to a reaction unit in which multiple electrodes are arranged. Control of the spatial position of the charged molecule may place the molecules necessary for biological analysis in a limited region so that the analysis speed can be improved.

According to a first aspect of the invention, there is provided a method of controlling the spatial position of charged molecules in a reaction unit having a central electrode surrounded by a plurality of outer electrodes on a substrate, the reaction unit having a frictional resistance to the motion of the molecules. Filled with a medium that provides the method,

(1) placing charged molecules into said reaction unit;

(2) applying a first voltage of a predetermined magnitude to a set of central electrodes and at least one outer electrode selected from the plurality of outer electrodes to generate an electric field;

(3) applying a second voltage of a predetermined magnitude to a set of central electrodes and at least one outer electrode selected from the plurality of outer electrodes to rotate the electric field controlling the spatial position of the charged molecule; and,

The net charge across the unit, generated by the voltage applied to the electrodes selected here, remains at zero.

According to a second aspect of the invention, there is provided a method of controlling the spatial position of charged molecules in a reaction unit having a plurality of electrodes on a substrate, the reaction unit being filled with a medium providing frictional resistance to the movement of the molecules. The method is

(1) placing charged molecules in the reaction unit;

(2) applying a first voltage of a predetermined magnitude to a set of at least two electrodes selected from the plurality of electrodes to generate an electric field; And,

(3) applying a second voltage of a predetermined magnitude to a set of at least two different electrodes selected from the plurality of electrodes to rotate the electric field controlling the spatial position of the charged molecule,

The net charge across the reaction unit, generated by the voltage applied to the electrodes selected here, remains at zero.

According to a third aspect of the invention, there is provided an apparatus for controlling the spatial position of a charged molecule, which is

Board;

A central electrode mounted on the substrate;

A plurality of outer electrodes on the substrate, the outer electrodes surrounding the center electrode; And,

At least one reaction unit having a medium providing frictional resistance to the charged molecules,

The medium covers the center electrode and the outer electrode on the substrate,

The position of the molecules is thereby controlled by rotating the electric field generated by varying the magnitude of the voltage applied to the electrode, and the net charge across the reaction unit, generated by the voltage applied to the selected electrode, remains at zero.

According to a fourth aspect of the invention, there is provided an apparatus for controlling the spatial position of a charged molecule, which is

Board;

A plurality of electrodes on the substrate; And,

At least one reaction unit having a medium providing frictional resistance to the charged molecules,

The medium covers the electrodes on the substrate;

The position of the molecules is thereby controlled by rotating the electric field generated by varying the magnitude of the voltage applied to the electrode, and the net charge across the reaction unit, generated by the voltage applied to the selected electrode, remains at zero.

According to a fifth aspect of the invention, there is provided an apparatus for reaction between molecules, which is

Board;

A center electrode on the substrate; And,

A plurality of outer electrodes surrounding the central electrodes on the substrate;

Wherein the center electrode is separated from the outer electrode at a distance between 1 and 100 microns, and the reaction unit having the binding entities for reaction is disposed on the center electrode; And,

And a grounded outer ring electrode surrounding the reaction unit to reduce the spread of the electric field generated by the voltage applied to the electrode over the reaction unit.

The concept and embodiments of the invention are described in two parts. The first part is a method of controlling the spatial position of molecules, and the second part is about the design and manufacture of a chip for this method.

Definition of terms used in the present invention

The term " negative dominant bias " indicates that the polarity of the center electrode is set to negative. In the present invention, the net charge of all electrodes is maintained at or close to zero.

The term "positive dominant bias" indicates that the polarity of the center electrode is set to positive charge. In the present invention, the net charge of all electrodes is maintained at or close to zero.

The term "nucleic acid hybridization" includes all hybridization reactions between deoxyribonucleic acid (DNA), ribonucleic acid (RNA), polynucleotides, and derivatives of nucleic acids including oligonucleotides and all natural and artificial forms. Means that.

"Polymerase chain reaction (PCR)" means a method of amplifying DNA fragments for analysis. The cycle begins by denaturing the DNA by heating (separating the double strands of the DNA molecule into single strands of DNA). Next, a primer (fragments of DNA complementary to a particular sequence) is bound to a particular portion of every single strand of DNA during cooling. This step is called annealing. In the final step, the temperature is raised again and a specific enzyme, a so-called Taq polymerase, is used to add complementary base bases along the remaining single stranded DNA template to the DNA from the ends of the primers. , Make multiple copies of the DNA fragment between the two primers. The entire process is repeated multiple times (cycles) to replicate a large amount of desired DNA region. Under ideal conditions, the amount of DNA doubles in each cycle, resulting in an increase of 2 ³⁰ (approximately trillion times) in the amount of DNA compared to the original sample.

“Self-Assembled Monolayers (SAMs)” refers to regular monolayers, naturally formed by adsorption of a surfactant with a particular affinity for a headgroup of substrate.

"Traping" refers to the spatial location control of charged molecules (positive or negative charges) by the presence of an externally applied electric field. This limitation of spatial location prevents the charging molecule from entering or exiting the designated site while the electric field is in place.

Chapter 1. Spatial position control method of charged molecule

Simultaneously manipulating two oppositely charged molecules with positive and negative charges can provide great advantages in biological analysis, where most biological reactions are between negatively charged and positively charged molecules. This is because it requires interaction.

The inventors have realized a method of simultaneously controlling the spatial position of two oppositely charged molecules in a reaction unit. The special geometry of placing the electrode in the reaction unit allows two oppositely charged molecules to be manipulated and the two charged molecules to be placed into a specific region where the desired biological reaction occurs. The geometry for this invention comprises a single central electrode surrounded by three or more outer electrodes in the reaction unit. 1 shows a typical geometry with a single central electrode 1 surrounded by eight outer electrodes 2 in a reaction unit as one preferred embodiment. Initially, a first voltage of a predetermined magnitude is applied to a set of at least two electrodes selected from a plurality of electrodes to generate an electric field, wherein the net charge across the electrodes of the reaction unit is maintained at or close to zero. The applied voltage can be positive or negative and is oriented in a manner that depends on the charge of the molecule to be trapped. Next, a second voltage of a predetermined magnitude is applied to the set of at least two outer electrodes selected from the plurality of outer electrodes to rotate the electric field controlling the spatial position of the charged molecule. The application of a voltage to this electrode can continue to trap the charged molecule. The rotation can be made clockwise or counterclockwise and is performed by the regular progression of the electrode voltage, where the voltage at a given electrode is set equal to the value at the adjacent electrode during the preceding time interval. 3 shows an example of electric field rotation generated by applying a positive voltage and a negative voltage to two outer electrodes, respectively.

In this method, a voltage can be applied to the central electrode to assist in trapping charged molecules. Applying a voltage to these electrodes to maintain the net charge at or near zero can include a bias of positive or negative charge dominance at the central electrode. 2 shows the pattern of the electric field generated by applying a voltage to the electrode. To control the trajectory of the molecules, alternating biases of positive and negative bias dominance may be required. The resulting electric field is also constructed to create an electronic trap, in which charged molecules from the electronic trap cannot enter the trap and in which the amplitude and rotational frequency of the electric field separate ions of electrophoretic kinetic ions. Can be adjusted. In one preferred embodiment, there are many possible combinations of setting the voltage for each electrode, such that the center electrode is set at 0.1 V, the outer electrode is set at -2 V, and the other outer electrode is set at 1 V. exist. The rotational frequency also affects the spatial position of the charged molecule. For example, the rotation frequency must be greater or the outer electrode must be closer to the center electrode at a lower voltage.

1. Parameters for controlling the spatial location of molecules

1) electrical charge

In order to keep the net charge of the electrode in the reaction unit at or close to zero, it is very important to set the value of the appropriate voltage for each electrode. For a round electrode with radius R and applied voltage V, the total charge Q on the electrode is given by the equation Q = 8xVx Rx πxε. This assumes that the charge distribution on the electrode surface is given by σ = Q / (2 × π × R × SQRT (R ² -ρ ² ), where ρ is the distance from the center of the electrode and ε is the free space Electrodes that are apparently of different radius but kept at the same voltage will have different amounts of electrical charge, according to this equation, when the central electrode is placed at a specified voltage, one or more of the outer electrodes To be balanced, they need to be at opposite voltages Due to the difference in the radius of the center electrode and the outer electrode, the balance voltage generally needs to be several times higher than the voltage of the center electrode. The magnitude of the voltage at the center electrode is in the range of 0.01 to 0.1 V, although its value depends highly on other factors.

For the geometry of a square or other electrode, the equation for calculating the total charge will be slightly different. However, the charge will still depend on the size of the electrode. In addition, the presence of an electric field due to other electrodes in the system will alter the charge distribution on the electrode surface. This makes the assumptions related to σ less accurate since the externally applied field at the surface of the electrode becomes less uniform. However, this does not change that the total charge depends on the size of the electrode.

2) Outer Ring

The electric field from a given reaction unit spreads in a way that can interfere with the electric field in any adjacent reaction unit. Inclusion of a surgical ring electrode is necessary to reduce this spreading effect of the electric field. Surgical ring electrodes, when grounded, help to prevent the unfolding of the electric field of the reaction unit. Mathematically, the grounded outer ring represents an additional boundary condition for the solution of the Laplace equation, which in this case is the basic differential equation governing the behavior of the electric field. By placing this additional boundary, the electrical potential and, ie, the electric field, are forced to go to zero at the position of the ring electrode. This effect extends vertically above the electrode surface. This does not force the potential to zero at all points on the electrode, but serves to reduce the potential. 5 shows a schematic diagram of a reaction unit surrounded by an outer electrode ring 3. 6A and 6B show the effect of the outer ring. FIG. 6A shows a schematic diagram of an electric field without a grounded outer ring and FIG. 6B shows a schematic diagram of an electric field with an outer ring. The presence of the outer electrode ring obviously reduces the spread of the electrical potential, minimizing interference with adjacent units. In this case, independent and simultaneous control of the spatial position of charged molecules across multiple reaction units can be achieved. This arrangement of reaction units can be used for high material processing screening or analysis of biological events such as DNA hybridization or polymerase chain reactions.

3) electrode

In order to precisely control the electric field so that the desired biological reaction can occur, the number of electrodes must be optimized. Depending on the desired reaction, the number of electrodes may preferably range from 3 to 30. However, the number of electrodes is not limited because point electrodes can be used for the present invention. In a preferred embodiment, a single center electrode is surrounded by eight outer electrodes. Internal electrodes in the central electrode but still electrically isolated from it may also be required for precise control of the electric field for biological reactions. These inner electrodes can provide a more precise way of controlling the spatial location of the molecule for the desired biological reaction.

4) rotation frequency

The electric field can be rotated by alternating alternating voltages applied to a set of electrodes, since each electrode can be controlled independently using a computer program. The rotation frequency may range from 0.001 Hz to 1,000 Hz depending on the magnitude of the voltage applied to the electrode. The rotational frequency must be low enough that the molecule can keep pace with the electric field as it changes. In a preferred embodiment, the frequency is in the range of 0.01 Hz to 10 Hz. The rotational speed of the molecule is directly proportional to the strength of the electric field, such that the time it takes for the molecule to move from one electrode to the next depends on the distance between the electrodes as well as the applied voltage. The weaker the electric field, the slower the rotation should be. At low electrode voltages, the rotational frequency must be low, or the outer electrodes must be close together. Such a change in geometry may require more outer electrodes or smaller center electrodes.

In a preferred embodiment, the dimensions of each electrode and the number of electrodes are summarized in Table 1 below.

Table 1. Dimensions of the electrode

Center electrode radius 100 μm Inner / Outer Electrodes 10 μm Radius to center of outer electrode 130㎛ Radius to center of inner electrode 50 μm Radius to outer ring 200 μm Width of outer ring 20 ㎛ Distance between two adjacent response units 1,000-2,000㎛

Table 2. Values of each parameter in the reaction unit

Minimum number of outer electrodes 3 Maximum number of outer electrodes 30 Minimum number of inner electrodes 0 Maximum number of inner electrodes 8 The maximum voltage of any electrode ± 10 volt Minimum voltage for stimulation 0.01 volt Rotational frequency 0.01 Hz Rotating frequency 10 Hz

5) geometric

Placement of electrodes in the reaction unit is one of the important factors in the present invention. For the purposes of the present invention, the arrangement of the electrodes must make a closed system, such as circular, square, or diamond shaped. In one preferred embodiment, the geometry is in the form of a toroid.

6) Other

The ion concentration of the ion concentration-buffer solution may affect the mobility of the molecule through interaction with the electric field generated by the electrode.

Some probes, such as probe-DNA molecules or proteins, have an intrinsic electric charge, either negative or positive. This can affect the electric field even though the effect is small compared to the externally applied electric field.

In addition to the present invention, any probe molecule, including but not limited to DNA fragments, antibodies, and enzymes, may be immobilized on a central electrode for biological reactions. 7 shows a cross section of a reaction unit with an electrode to which the probe is to be fixed. Many methods of immobilizing probes on electrodes have been studied, which vary widely in chemical mechanisms, ease of use, probe surface density, and immobilization ability. (Para N. Lehman, Nucleic Acid Research, 1999. Vol. 27, No. 2, pages 649-655) Among the most promising solid phase immobilization methods for the present invention are methods using polyacrylamide supports and self-assembled monolayers ( SAMs). They may have a thiol group (-SH) that can be bonded to an electrode or probe. The main advantages of these supports are high probe capacity, low nonspecific binding levels or relatively high thermal stability. Moreover, it is relatively easy to manipulate the density of the probe needed to normalize the hybridization properties of the probe array. The immobilization layer of polyacrylamide or self-assembled monolayers acts as a permeation layer to enable diffusion of the gas generated by electrolysis. It must have a permeation layer pore limit characteristic, which prevents or inhibits large bonding objects, reactants, and analytes from physically contacting the electrode. The penetrating layer must also physically restrain the active electrode surface from the bonding entity.

Once the infiltration layer is formed, the alternating of positive and negative charge bias dominance causes the charged molecules to be localized at high concentrations around the central electrode to facilitate the desired biological reaction.

Chapter II. Design and manufacture of devices for trapping molecules

1. Overview

The device can be designed to have as few reaction units as one reaction unit or as hundreds or thousands of reaction units. In general, complex devices with multiple reaction units are manufactured using microlithography techniques. Manufacturing is done on silicon or other suitable substrate materials such as glass, silicon dioxide, plastics or ceramics. The design of such microelectronic chips can be considered as multiple arrays or complex analysis devices. Devices with a small number of reaction units or large reaction units are manufactured using micromachining techniques.

The reaction unit may be of any shape and may preferably be circular, square or square. The size of the reaction unit depends on the size of the electrode disposed in the reaction unit. Making reaction units smaller than the resolution of the microlithography method will require techniques such as electron beam lithography, ion beam lithography, or molecular beam epitaxy.

While small reaction units are suitable for assay and diagnostic type applications, large reaction units are suitable for applications such as, but not limited to, pre-scale biopolymer synthesis, sample preparation, electronic distribution of reactants.

The electrodes can be of any size, preferably ranging from less than 1 micron to several centimeters, with 5 microns to 100 microns being the most preferred size range for devices made using microlithography techniques, and 100 microns. To 1 millimeter is the most preferred size range for devices made using micromachining techniques. The electrode can be any shape, and is preferably round or square.

The geometry of the electrode arrangement in the reaction unit may be of any shape, preferably round, square or square. In a preferred embodiment, one electrode is centered and surrounded by the other electrode.

The number of electrodes arranged in the reaction unit is three or more, preferably 3 to 30 electrodes. (In chapters 1-3 of Chapter I, it is stated that "the number of electrodes may be in the range of 3 to 30, depending on the desired reaction." This is exactly in accordance with Table 2.)

After the reaction unit, including the electrode, is made by using microlithography and / or micro-machining techniques, chemical modification, polymerization, or other micrography manufacturing techniques can be used to create specialized fixation and penetration layers. do. These critical layers isolate the bonding objects from the metal surface of the electrode. This isolation enables DC mode, where each electrode under the reaction unit surface (1) causes or influences free field electrophoretic transfer of molecules into the reaction zone where the reaction can occur. (2) can concentrate molecules into the reaction zone where the reaction can occur, (3) can continue to function actively under the application of DC mode after the molecules have been locally concentrated into the reaction zone, and (4 Electrochemical reactions and products should not adversely affect any biological reactions.

1. Design Parameters (Microlithography)

Reaction Unit Manufacturing

As one preferred embodiment of the present invention, FIG. 1 shows the basic geometry of electrode placement in a reaction unit made using microlithography. Nine electrodes E1-E9 are formed by the deposition of gold on the insulating matrix. One electrode is centered and surrounded by the other electrodes (outer electrodes). The number of outer electrodes is three or more, preferably 3-30. The insulator matrix separates the electrodes from each other. Insulator materials include, but are not limited to, silicon dioxide, silicon nitride, glass, resist, polyimide, rubber, plastic, or ceramic materials.

7 shows the basic properties of a reaction unit made by microlithography. The center electrode CE 1 and the outer electrode OEs 2 are formed on the matrix (base 4 and insulator 5), and the penetration layer PL 6 and the bonding layer BL are formed. (7) is incorporated on the CE.

Thiols, such as alkanethiols such as OH-((CH) ₂ ) _6-10 -SH in the permeation layer, provide a base on which the probe is fixed. The permeation layer provides a gap between the electrode CE and the bonding layer BL and allows solvent molecules, small counter-ions, and electrolysis reaction gases to pass freely to and from the electrode. In the permeation layer can contain substances which can reduce the negative physical and chemical effects of the electrolysis reaction, which are redox reaction trapping materials, such as palladium for H ₂ and iron compounds for O ₂ and peroxides. It includes, but is not limited to. The thickness of the penetrating layer for a device made by microlithography can range from approximately 1 nanometer (nm) to 100 microns (μm), with 2 nm to 10 μm being most preferred.

The spacing between the electrodes is determined by the ease of manufacture, the requirements for detector analysis between the electrodes, the nature of the electric field required for trapping, and the number of electrodes required for the device. Specific spacing between the electrodes is necessary for the function of the device. This is because a complex electric field pattern or dielectric boundary is needed to selectively move, separate, fix or align certain molecules in the space or medium between any of the electrodes. The device achieves this by controlling the spatial location of the molecule in a restricted region around the probe anchored to the thiol in the binding layer. The force of free field electrophoresis provides for the rapid and direct transfer between any spatial locations on the device and between all spatial locations, or from any bulk solution to any electrode, to any positive electrode. do.

8 shows a matrix type apparatus with 50 reaction units. The 50 reaction unit devices are of good design and fit into standard microelectronic chip package configurations. Such a device is fabricated on a silicon chip substrate that is approximately 1.5 cm x 1.5 cm with a central portion of approximately 750 μm x 750 μm containing 50 reaction units. The number of reaction units on the matrix can range from 10 to 10,000 depending on the application as described above (therefore, the dimensions of the electrodes disposed on the reaction units may vary in response to the application of such a device). ).

FIG. 9 shows a schematic diagram of the structure of the reaction unit to be deposited on the matrix shown in FIG. 8.

Penetration and bonding layer construction

After manufacture, the central electrode on the device is transformed into a special penetration and bonding layer. This is an important feature of the present invention. The aim is to create an intermediate penetration layer with selective diffusion properties on the electrode and an adhesion surface layer with optimal bonding properties.

The binding layer provides a base for binding various target probes such as DNA primers. The thickness of the bonding layer for devices made by microlithography can range from 0.5 nm to 5 μm, with 1 nm to 500 nm being most preferred. Ideally, certain bonding layers consist of (1) self-assembled monomolecular layers (SAM) or (2) polyacrylamide gels. Both the SAM or the gel provide a gap for the binding of large molecules on the center electrode of the reaction unit.

(1) SAM

Alkanthiol SAMs are provided by sulfur's affinity for gold and the relatively strong lateral interactions arising from van der Waals forces between the chains. This lateral interaction can be controlled by changing the length of the hydrocarbon chain. Gold has a crystal structure of (111) plane.

Optimally, the bonding layer has functionalized positions of 10 ⁵ to 10 ⁷ per square micron (μm ² ) for immobilization of specific bonding entities. The immobilization of the binding objects must not protectively coat or insulate the surface to prevent the underlying microelectrode from functioning. The functional device is kept accessible to the solvent (H ₂ O) molecules, and counter ions (eg Na ⁺ and, Cl ⁻ ) and electrolysis gas (eg O ₂ and, H ₂ ) are generated. Some portion (~ 5% to 25%) of the actual metal microelectrode surface is required.

The intermediate penetrating layer is also designed to allow diffusion. In addition, the penetrating layer should have pore limiting properties that prevent or prevent large bonding entities, reactants, and analytes from physically contacting the micro-electrode surface. The penetrating layer keeps the active micro-electrode surface physically separate from the bonding layer of the electrode.

This design allows the electrolysis reactions required for electrophoretic transfer to occur on the micro-electrode surface, but avoids the negative electrochemical effects on the binding entity, reactants and analytes.

The permeation layer may be designed to include materials that look for negative substances (H ₂ , O _2, free radicals, etc.) generated in the electrolysis reaction. The sub-layer of the penetrating layer can be designed for this purpose.

Various designs and techniques can be used to make the permeable layer. Typical designs include (1) physical adsorption, (2) chemical modifications, and (3) genetic engineering modifications.

The placement of linear alkanethiol molecules in the vertical direction from the metal electrode surface can be formed by directly immobilizing the linear alkanethiol directly to the metal electrode surface, with minimal crosslinking between the vertical structures. Ideally, these molecules have two functions, one terminal end suitable for covalent bonds to the metal electrode surface and the other terminal end suitable for covalent bonds of the binding entity.

Physical adsorption involves the probe molecules under investigation being immobilized directly through hydrophobic or electrostatic interaction onto the unmodified substrate surface. Such a process is simple in its entirety and does not involve manipulation of probe molecules. However, fixing the probe molecules directly onto the metal surface has some disadvantages. That is, some proteins are denatured and later deactivated when in direct contact with the metal surface. The bond between the protein and the metal surface is not stable. While proteins are unspecific on the metal surface, being fixed randomly and in multiple orientations can result in non-reproducibility of results, and lamination in multiple layers can present problems. And random orientation of the active site of the protein can interfere with the binding of the analyte.

An alternative to physical adsorption is provided by chemical modification of the probe. There are a variety of chemical manipulation processes that result in the formation of covalent bonds between the protein and the substrate surface under undenatured conditions. Compared with the results obtained using the direct fixation method, the chemical regulation of making self-assembled biolayers offers many important advantages, such as: Regeneration and stability of the protein monolayer on the substrate when exposed to a wide range of conditions; The density of fixed species and the possibility of controlling the environment; Generation of uniform structure; Higher coverage of the substrate surface; And a reduction in the number of possible random directions the protein can take on the surface.

One preferred process for making monomolecular layers based on chemical modifications includes components containing sulfur. The strong affinity of the constituents (thiols, thioethers and disulfides) containing sulfur for gold and other precious metals is such that they can form covalent bonds with a second functional group, ie a biomolecule. If present, it means that they can be good tagging molecules for protein modification and subsequent self-assembly.

Two different approaches are used for chemical fixation of the probe, which is (1) protein modification and (2) substrate modification.

By protein modification, the protein of interest is tagged in a solution with the appropriate sulfur-containing monomolecules, for example, by reaction with certain reaction groups present on the surface of the biomonomeric layer and the ε amino group of the lysine residue. do. The altered protein then self-assembles onto a gold-coated substrate through half of the sulfur site. Modification of the substrate relies on modifying the substrate by first self-assembling molecules containing sulfur. The protein is subsequently bound to the newly formed SAM through reaction with specific sites on the surface of the biomonolayer. 10A shows a schematic process of binding DNA comprising alkanediol to the electrode surface. 10B shows the process of forming a protein binding layer. The protein first reacts with the alkanethiol, and then the alkanethiol containing the protein is bound to the electrode surface. A third alternative for immobilizing proteins and enzymes on gold coated substrates is provided by the recent utility of genetic engineering techniques. The use of genetic engineering offers the opportunity to create specific immobilization sites on the protein surface and allows the formation of even more regular SAMs of the protein. Using genetic engineering techniques, one can overcome the problem of multiple and random orientation of proteins on gold, which is potentially still present in chemical transformation. The enzyme can be immobilized in its fully active form after the application of site-directed mutagenesis, which introduces the immobilization site into a specific position. The surface coverage of the recombinant dihydrofolate reductase mutant on the gold-coated substrate, lacking both natural cysteines, is one cysteine applied directly to the C-terminus. 4 times lower than that obtained with similar mutants with residues.

In order to obtain a high quality monolayer structure, it is important to allow sufficient time for the formation of SAM. After lamination of the monomolecular layer, the surface is exposed to extensive buffer cleaning to remove all protein material that is not self-assembled on the surface, which may have been stacked as a multimolecular layer.

(2) polyacrylamide gel

Polyacrylamide gels can be used as the base for the binding of large molecules on the central electrode of the reaction unit. For example, polyacrylamide gels having a high density of hybridizable oligonucleotides can be prepared by acrylamide modification. Oligonucleotides with 5'-terminal acrylamide modifications can be effectively copolymerized with acrylamide monomers to form thermally stable polyacrylamide copolymers comprising DNA (Para En. Lehmann et al. Nucleic Research). 1999, Vol. 27, No. 2, 649-655). Since the present invention does not cover such modifications, and many products are commercially available for the gels of the present invention, such as those of MOIC Technologies INC, the description will not be further described.

Design and manufacture of micromachined devices

This section describes how to use micromachining techniques (eg drilling, milling, etc.) or non-lithographic techniques to manufacture the device. 11 shows a schematic diagram of a micromachined device. In general, these devices are relatively larger than those produced by microlithography (> 100 microns). Such devices can be used for analytical applications as well as for preliminary applications such as the synthesis of biopolymers, the preparation of samples, and the distribution of reagents. Storage locations can be made in a three-dimensional format (eg a tube or cylinder) to carry large quantities of engagement objects (eg a tube or cylinder). Such devices can be manufactured using a variety of materials, including, but not limited to, plastics, rubber, silicon, glass (eg with microchannels, with microcapillaries, etc.) or ceramics. Low fluorescent materials are more ideal for analytical applications. In the case of a microfabricated device, the connection circuit and the large electrode structures can be printed onto the material using standard circuit board printing techniques known to those skilled in the art.

Application of the present invention

Once the device has been associated with specific binding entities, multi-step and complex analyzes of various biological types can be performed on the device. The apparatus of the present invention can electronically provide active and dynamic control over a number of important reaction parameters. This electronic control leads to new physical mechanisms that control the reaction and significant improvements in reaction rate, specificity, and sensitivity. The improvement in these parameters comes from the possibility of controlling the device electronically and directly affects the following: (1) Rapid delivery of reactants or analytes to specific reaction units containing immobilized specific binding entities. (2) The reaction rate increases due to the concentration of reactants or analytes together with certain binding entities on a particular microelectrode surface. (3) Rapid and selective removal of unreacted and nonspecifically bound components from the microelectrode. (4) Small amount of sample required for biological analysis since each reaction unit can use samples in series. Each reaction can use the same sample as in the previous reaction, since the original sample is not modified by the response to certain reactions. Therefore, each reaction unit is controlled independently, enabling multiple analyzes with a small amount of sample material. (5) Some components necessary for biological reaction can be fixed to the central electrode if necessary. (6) The product (s) produced by the biological reaction can be removed from the binding layer for subsequent reaction. For example, in PCR, cloned DNA can be separated from the binding layer and other extensions can be made using the same binding initiator sequence.

The self-addressed devices of the present invention can rapidly perform multi-step and / or complex reactions and processes in a variety of microformats, for example immobilized target DNA / probe DNA, immobilization DNA and RNA hybridization processes and assays in conventional formats such as probe DNA / target DNA, immobilized capture DNA / target DNA / probe DNA; Multiple or complex hybridization reactions in series or parallel mode; Analysis of restriction fragments and overall DNA / RNA fragment size; Restriction enzyme reactions and molecular biological reactions such as assays, ligase reactions, kinase reactions, and DNA / RNA amplification; Antigen / antibody reactions including large or small antigens and hapten; Diagnostic assessments such as, for example, hybridization assays (including hybridization in situ), genetic analysis, genetic fingerprinting, and immunodiagnostics; Preparation of samples, storage, selection and analysis of cells; Biomolecular conjugation (ie, sharing of nucleic acid, enzymes, proteins or agents with a reporter group comprising a fluorescent, chemiluminescent, calorimetric label, and label of a radioisotope, and Non-covalent labeling; for example, synthesis of biological polymers such as binding synthesis of peptides or oligonucleotides; synthesis of water-soluble synthetic polymers such as carbohydrates or linear polyacrylates; and macromolecules and nano Syntheses and manufactures of structures (particles of nanometer size and structures), including but not limited to.

Nucleic acid hybridization or PCR can be used as the main example of the present invention because of their importance in diagnostics and because they are characterized by one of the more difficult types in binding (affinity) reactions. This is especially true when they are performed in format multiple format, where each individual hybridization reaction requires conditions of different stringency.

The apparatus and methods of the present invention allow nucleic acid hybridization to be performed in a variety of conventional and novel formats. The ability to allow the device to electronically control the reaction parameters greatly enhances nucleic acid hybridization assays, and in particular the performance of the device to provide electronic stringency control (ESC) to each individual microelectrode on the array. In essence, this allows individual hybridization reactions (in a single reaction unit) on a covalent arrangement to be performed like a single tube analysis.

The term "nucleic acid hybridization" is meant to include all hybridization reactions between natural and artificial forms, as well as derivatives of nucleic acids, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), polynucleotides, and oligonucleotides. do.

Conventional polymerase chain reaction (PCR) formats can be performed with the disclosed apparatus as well as with multiple array or matrix formats. As an example, an Active Programmable Electronic Matrix (APEX) device, such as NanoChip®, developed by Nanoogen for DNA amplification and analysis is designed, manufactured, and used in the following manner: . An array of reaction units with microelectrodes is initially manufactured using microlithographic (or micromachining) techniques. The number of reaction units and the geometry of the electrode arrangement on the reaction unit are determined by the end use. The device is rapidly addressed in a serial fashion with a mixture of alkanthiols comprising oligonucleotides as DNA initiators. In this case, the oligonucleotides are 3'-terminal oligonucleotides ranging from 6-mers to 100-mers, and larger polynucleotides can be immobilized if necessary. The thiol functional group of the alkanethiol allows for covalent bond attachment to certain micro electrode immobilized surfaces (FIG. 10A). Such specific groups of oligonucleotides can be readily synthesized on conventional DNA synthesizers using conventional techniques. The synthesis of each particular oligonucleotide is initiated from a ribonucleotide controlled pore glass (CPG) support. Thus, the 3'-terminal position includes ribonucleotides, which are readily converted to dialdehyde derivatives by periodic acid oxidation after synthesis and purification. The hydroxyl group of the alkanethiol comprising the oligonucleotide will readily react with the basic amine functional group on the surface of the electrode.

Yes

These examples illustrate the ability of the present technology to control the spatial position of charged particles in the reaction chamber.

The reaction chamber consists of ten independently addressable gold electrodes embedded in an insulating material. The insulator and electrode surfaces are covered with a permeation layer and buffer solution commonly used in biological experiments.

The electrodes are arranged as a single, large, round electrode in the center. In this embodiment the radius of the center electrode is 35 microns. The central electrode is surrounded by eight outer electrodes. These outer electrodes are also round and uniformly positioned around the center electrode. The radius of the outer electrode is 5 microns. The distance between the middle of the center electrode and the middle of the outer electrode is 45 microns. This leaves a 5 micron gap between the central conductive surface and the outer electrode. With eight evenly spaced outer electrodes, the angle between the outer electrodes is 45 degrees with respect to the middle of the center electrode. This causes the center to center distance between the outer electrodes to be 34.4 microns. The resulting gap between the conductive portions of the outer electrodes is 24.4 microns. The outer electrodes are surrounded by a ring of conductive material, the inner edge of which is located 70 microns from the middle of the center electrode. The ring is 10 microns wide and always stays zero bolted. The voltage of the outer electrode changes depending on the operation performed in the reaction unit.

In performing the simulation, the buffer was taken to have the following characteristics. The viscosity was the same as that of pure water, 0.891x10 ^-3 kg / mxs, the temperature was 25 ° C, the dielectric constant 78.3, the pH was 8.3, the total ionic strength was 0.256M. The particles were taken to have motility of 3 x 10 ^-4 cm ² / (volt x sec).

An electric field was generated by applying a known voltage to the electrodes described above to induce motion in the charged particles present in this buffer. The outer electrodes can be numbered from 1 to 8 starting at the 12 o'clock position and moving clockwise. The voltage of the electrode 1 is set to V1 which is a specific value, and the electrode 2 is set to V2. Repetition of this pattern of rotating the electric field results in circular motion of nearby charged particles. This can be achieved by setting electrode 1 to -10 volts, electrode 4 to 10 volts, and all other electrodes to zero volts. In this configuration, the negatively charged particles are attached to the electrode 4 while the positively charged particles are preferentially immobilized on the electrode 1. By regularly converting the voltage at the two non-zero electrodes, the electric field rotates clockwise, so that the charged particles are immobilized at the new position of the electrode of opposite polarity. In this way the particles can be drawn in a circle around the center electrode.

The voltage used in this rotation affects the pattern of motion of the particles. Increasing the voltage allows the electric field to stretch further into the volume beyond the chamber into the volume above the reaction unit, but the grounded ring prevents this from spreading along the surface of the chip. Since the electric field spreads further due to the higher voltage, increasing the voltage can help attract charged particles from a greater distance. This can be used to increase the local concentration of charged particles in proximity to the center electrode. Larger voltages may result in faster movement of the particles. The voltage of the particles is equal to the product of their motility and electric field. Due to the geometry of the system, the lines of the electric field are not straight, but rather form arcs extending between the electrodes. This arc shape is also visible in the trajectory of the charged particles. The height at which particles move above the surface of the chip increases with increasing voltage. 4 shows the trajectory caused by rotating the voltages on the electrodes 1, 2, 3, 4 to -4, -1, -1, 10 volts. All electrodes are grounded. Setting electrode 2 to -1 volts causes any negatively charged particles proximate such surface of the electrode to be pushed off prior to rotation of the electric field. This is advantageous because it ejects particles with the same value of -4 volts more powerfully and at a higher height. This may not be desirable for some applications because it increases the overall distance that particles must travel between the electrodes. The increase in such distance may affect the frequency at which rotation must be performed at such frequency. In other words, if the electric field is rotated in such a way that the particles can match this rotation, care must be taken to reveal the overall path length of the particles. The voltage at the electrode leading to the 10 volt value should be at least slightly negative. This is because large positive voltages attract negative particles. If such values are reversed and replaced by zero, particles close to the surface of the electrode are screened on a large scale from the electric field of the adjacent electrodes. Positioning the electrode to a negative value pushes the negative particles off the surface, allowing the particles to interact more strongly with the electric field from adjacent electrodes. Without this (as in the first example with only + / 1 volt electrodes), most of the particles attracted by each electrode trapped close to the surface of the electrode until the oppositely charged electrode rotated to that position. It will remain trapped. At this point, the particles will be strongly rejected and pushed away over the surface of the chip. This effect is also controlled by the thickness, density and material properties of the penetrating layer.

The geometry of the electrode also affects the orbits of the particles. The greater the distance from the middle of the center electrode to the middle of the outer electrode, the lower the rotational frequency should be for particles of the same mobility. This is simply due to the increased path length between the outer electrodes and the corresponding decrease in the electric field generated with the given voltage at the two outer electrodes. This increased length can be compensated for by increasing the voltage at the outer electrode. Such an increase enables the use of higher rotational frequencies, but results in a path consisting of larger arcs. The geometry of the electrode and the relationship between the rotational frequency and the voltages are highly correlated. Other considerations may impose limitations on one or more of these values, such as the sensitivity of the biochemical process to an externally applied electric field generated by the electrode. For example, in applications where a larger electric field cannot be tolerated, it is necessary to reduce the radius or rotational frequency of the electrode. With the geometry and voltage described above, a rotation frequency of 1 Hz to 4 Hz results in the same trajectory as shown in FIG. Particles in such orbits are referred to as trapped. They can remain indefinitely in these orbits until needed. Located close to the central electrode, to which all targets are bound, the trapped particles can be made very rapidly available for reactions.

The present invention can be used for controlling the spatial position of charged particles in the fields of biology, medicine and the like.

Claims (50)

A method of controlling the spatial position of charged molecules in a reaction unit having a central electrode surrounded by a plurality of outer electrodes on a substrate, the reaction unit being filled with a medium that provides frictional resistance to the motion of the molecules, (1) placing a charged molecule into said reaction unit; (2) applying first voltages of a predetermined magnitude to a set of at least one outer electrode selected from the center electrode and the plurality of outer electrodes to generate an electric field; And, (3) applying second voltages of a predetermined magnitude to at least one set of outer electrodes selected from the center electrode and the plurality of outer electrodes to rotate the electric field controlling the spatial position of the charged molecule; A method of controlling spatial position of a charged molecule, characterized in that the net charge across the reaction unit generated by the voltage applied to the selected electrodes is maintained at zero. A method of controlling the spatial position of charged molecules in a reaction unit having a plurality of electrodes on a substrate, wherein the reaction unit is filled with a medium that provides frictional resistance to the movement of the molecules, (1) placing a charged molecule into said reaction unit; (2) applying a first voltage of a predetermined magnitude to a set of at least two electrodes selected from the plurality of electrodes to generate an electric field; And, (3) applying a second voltage of a predetermined magnitude to a set of at least two other electrodes selected from the plurality of electrodes to rotate the electric field controlling the spatial position of the charged molecule; A method of controlling the spatial position of a charged molecule, characterized in that the net charge across the reaction unit generated by the voltage applied to the selected electrodes is maintained at zero. The method of claim 1, Space number control method of the charged molecule, characterized in that the number of the outer electrode is 3 to 30. The method of claim 2, The number of electrodes is 3 to 30, the space position control method of the charged molecule, characterized in that. The method of claim 1, Probe molecules are fixed to the center electrode space position control method of the charged molecule, characterized in that. The method according to claim 1 or 2, The voltages applied to the electrodes are -10V to + 10V, the spatial position control method of the charged molecule, characterized in that. The method of claim 5, The probe is a spatial position control method of the charging molecule, characterized in that selected from DNA, RNA, enzymes or proteins. The method of claim 1, The center electrode may have one or more inner electrodes electrically insulated from the center electrode while in the center electrode. The method according to claim 1 or 2, The electric field generated by the electrode is caused to rotate in a clockwise or counterclockwise manner, the rotation being carried out by the regular advancement of the electrode voltage, where the voltage at the given electrode is the value of the electrode proximate to the previous time interval. The space position control method of the charging molecule, characterized in that set as. The method according to claim 1 or 2, A method of controlling spatial position of a charged molecule, wherein the amplitude of the electric field and the rotational frequency are modulated in a manner that separates ion species having different electrophoretic motility. The method according to claim 1 or 2, The rotational frequency of the electric field is 0.001 Hz to 1000 Hz method of controlling the spatial position of the charged molecule. The method according to claim 1 or 2, The generated electric field is configured to create an electronic trap and charged molecules cannot enter the trap through it. The method according to claim 1 or 2, Wherein the electric field can be temporarily reconstructed in such a way that a particular charged molecule can enter the trap. The method according to claim 1 or 2, A method of controlling spatial position of a charged molecule, wherein the charged molecule is selected from DNA, RNA, protein or enzymes involved in a biological reaction. The method of claim 14, Biological responses include nucleic acid hybridization, nucleic acid amplification, sample preparation, antibody / antigen reactions, clinical diagnostics, or biopolymer synthesis. The method according to claim 1 or 2, A method of controlling the spatial position of a charged molecule, characterized in that the temperature in the reaction unit is gradually increased or decreased. The method according to claim 1 or 2, Wherein the pH in the reaction unit is gradually increased or decreased. The method according to claim 1 or 2, A method of controlling the spatial position of charged molecules, characterized in that the ion concentration in the reaction unit is gradually increased or decreased. The method according to claim 1 or 2, Wherein the medium is a common buffer solution or a porous material. The method of claim 1, A method of controlling spatial position of a charged molecule, characterized in that a permeation layer and a bonding layer are formed on the central electrode. The method of claim 20, And the penetration layer is a self-assembled monomolecular layer of an alkane thiol or a porous material. The method according to claim 1 or 2, And said electrode is an electrically conductive material selected from gold, silver, copper or aluminum. The method according to claim 1 or 2, And said substrate is an electrically insulated material selected from glass, silicon or ceramic. The method of claim 1, Wherein the arrangement of the outer electrodes is a closed system. The method of claim 2, The method of controlling the spatial position of a charged molecule, wherein the arrangement of the electrodes is a closed system. Board; A central electrode mounted on the substrate; A plurality of outer electrodes surrounding the central electrode on the substrate; And, At least one unit having a frictional resistance to the charging molecule, the medium covering the central and outer electrodes on the substrate; The molecular position is controlled by rotating the electric field generated by varying the magnitude of the voltage applied to the electrode, Wherein the net charge across the reaction unit generated by the voltage applied to the selected electrode is maintained at zero. Board; A plurality of electrodes on the substrate; And, At least one reaction unit having a frictional resistance to the charged particles and a medium covering the electrode on the substrate; The position of the molecule is controlled by rotating the electric field generated by varying the magnitude of the voltage applied to the electrode, The space position control device of the charged molecule, characterized in that the net charge across the reaction unit generated by the voltage applied to the selected electrode is maintained at zero. The method of claim 26 or 27, And an outer ring surrounding the outer electrode on the substrate, the outer ring reducing the spread of the electric field generated by the voltage applied to the electrodes across the reaction unit. . The method of claim 26 or 27, Spatial position control device of the charged molecule, characterized in that the number of the plurality of electrodes is 3 to 30. The method of claim 26 or 27, The geometry of the electrode arrangement is a spatial position control device for charged molecules, characterized in that it is a closed system such as a circular, square or diamond shape. The method of claim 26, Probe molecules are fixed to the center electrode space position control device, characterized in that. The method of claim 31, wherein The probe is a spatial position control device of the charging molecule, characterized in that selected from DNA, RNA, enzymes or proteins. The method of claim 26, And wherein the center electrode can have one or more inner electrodes that are in the center electrode and electrically insulated from the center electrode. The method of claim 26 or 27, The electric field generated by the electrode is caused to rotate in a clockwise or counterclockwise manner, the rotation being carried out by the regular advancement of the electrode voltage, where the voltage at the given electrode is the value of the electrode proximate to the previous time interval. Spatial position control device of the charged molecule, characterized in that the same as set. The method of claim 26 or 27, Wherein the amplitude of the electric field and the rotational frequency are modulated in such a way as to separate ionic species having different electrophoretic motility. The method of claim 26 or 27, The electric field produced is configured to create an electronic trap, and the charged molecule space position control device, characterized in that the trap cannot enter through it. The method of claim 26 or 27, Wherein the electric field can be temporarily reconstructed in such a way that a particular charged molecule can enter the trap. The method of claim 26 or 27, A method of controlling spatial position of a charged molecule, wherein the charged molecule is selected from DNA, RNA, protein or enzymes involved in a biological reaction. The method of claim 38, Biological reactions include nucleic acid hybridization, nucleic acid amplification, sample preparation, antibody / antigen reactions, clinical diagnostics, or biopolymer synthesis. The method of claim 26 or 27, A method of controlling the spatial position of a charged molecule, characterized in that the temperature in the reaction unit is gradually increased or decreased. The method of claim 26 or 27, Wherein the pH in the reaction unit is gradually increased or decreased. The method of claim 26 or 27, Spatial position control device of charged molecule, characterized in that the ion concentration in the reaction unit is gradually increased or decreased. The method of claim 26 or 27, And the medium is a common buffer solution or a porous material. The method of claim 26, An infiltration layer and a bonding layer are formed on the central electrode. The method of claim 44, And the penetration layer is a self-assembled monomolecular layer of an alkane thiol or a porous material. The method of claim 26 or 27, And the electrode is an electrically conductive material selected from gold, silver, copper or aluminum. The method of claim 26 or 27, And said substrate is an electrically insulated material selected from glass, silicon, or ceramic. The method of claim 26, The arrangement of outer electrodes is a spatial position control device of charged molecule, characterized in that the closed system. The method of claim 27, The arrangement of electrodes is a spatial position control device of charged molecules, characterized in that the closed system. Board, A center electrode on the substrate, A reaction unit having a plurality of outer electrodes surrounding the center electrode on the substrate, The central electrode is separated by a distance of 1 to 100 microns from the outer electrodes, a penetration layer having a binding entity for reaction is disposed on the central electrode, A grounded outer ring electrode surrounds the reaction unit to reduce spreading of the electric field generated by the voltage applied to the electrode across the reaction unit.