HK1031042A1 - Electromagnetic chips and electromagnetic biochips having arrays of individually addressable micro-electromagnetic units as well as its application - Google Patents

Description

Single-point gating type micro-electromagnetic unit array chip, electromagnetic biochip and application

Technical Field

The invention relates to a novel single-point gating type micro-electromagnetic unit array chip and an electromagnetic biochip. By using the device of the invention, the purpose of operating biomolecules (DNA, protein, cells and the like) modified by magnetic materials or micro devices or structures connected with paramagnetic particles can be achieved by controlling the on, the size and the off of electromagnetic fields of all micro areas on the surface of the chip, so as to realize the biochemical analysis and detection and clinical diagnosis with miniaturization, high speed and high flux. In addition, the invention also relates to a method for directionally manipulating biological molecules, chemical molecules and particle structures by using the electromagnetic biochip, and a biochemical reaction control process and a detection means related to the method.

Background

The biochip technology is a new technology which is completely open in the field of life science research in recent years, and has wide application values, such as researches on DNA mutation detection, DNA sequencing, gene expression, drug screening, disease diagnosis and the like. It integrates (integrates) and miniaturizes (rings) existing discrete biochemical analysis systems into chip-based devices, mainly by using microelectronics and micromachining techniques in the semiconductor industry or other techniques with similar effects. The recent scientific literature describes the use of many such devices.

The wide use of biochips can be seen by the following articles. "Rapid determination of single base mismatch interaction in DNA hybrids by direct electric field control", Sosnorkski, R.G, etc. (Proceedings of the National Academy of Sciences, USA, 94: 1119-; "Accurate sequencing by hybridization for DNA diagnostics and inductive Genetics", Dumanac, S. et al (Nature Biotechnology, 16: 54-58, 1998), "Quantitative pharmaceutical analysis of yeastomatous using a high molecular-coding sequence", Shoemaker, D.D. et al (Nature Genetics, 14: 450-; the use of biochips for gene expression monitoring can be seen in the following articles: "Genome-wide expression monitoring in Saccharomyces cerevisiae", Wodicka, L. et al (Nature Biotechnology, 15: 1359-1367, 1997), "Genome and serum analysis-variation on variation", Brown, P.O., Hartwell, L., "Toward Arabidopsis Genome analysis: (ii) monitoring expression profiles of 1400 genes using cDNA microarrays. ", Ruan, Y., etc. (The Plant Journal 15: 821-; "Selective antibiotic reagents on combinatorial oligonucleotide arrays", Milner, N.et al (Nature Biotechnology, 15: 537) 541, 1997), "Drug target identification and diagnosis of second target infection using DNA microarray", Marton, M.J. et al (Nature Medicine, 4: 1293) describe the use of biochips for Drug screening. "cytotoxic hybridization detection by light-generated DNA probes", Cronin, M.T. et al (Human Mutation, 7: 244-255, 1996), "Polypyrrole DNA chip on a silicon device: the use of biochips in clinical diagnosis is described by Example of hepatitis C virus genotyping, Livache, T.et al (Analytical biochemistry.255: 188-. From the above, it can be seen that the biochip is widely used.

Many chips immobilize biomolecules (e.g., oligonucleotides, cDNAs, and antibodies) on their surfaces. There are many ways to make such chips. For example: the light-guided in-situ synthesis method developed by Affymetrix is a method for synthesizing biomolecules at a fixed point on a chip, which is derived by combining an optical lithography method and a photochemical synthesis method. The chemical spraying method used by Incyte Pharmaceutical is to spray oligonucleotide probes synthesized in advance onto predetermined spots on a substrate to fabricate a biochip. The contact imprinting method used by Stanford university is a method of spotting cDNA probes onto a substrate by bringing a pipetting head into contact with the substrate by a high-speed precision robot. Washington university in Seattle obtained oligonucleotide probes by using four piezojets each containing four nucleosides A, T, G, and C on a substrate, ejecting them as needed, and synthesizing them in parallel. Hyseq corporation has developed a passive thin film device for genome sequencing.

Biochips can be broadly divided into two categories: active chips and passive chips. The passive biochip means that biochemical reactions performed on the chip are passively realized by virtue of the diffusion motion of sample molecules. In the active biochip, the reactant is actively moved and concentrated by an external force, and thus the reaction is closely related to not only the diffusion movement of the sample but also the force applied by others. Most of the chips, such as oligonucleotide-based DNA chips developed by Affymetrix and cDNA-based biochips developed by Incyte Pharmaceutical, belong to passive chips. The active and passive chips have some similarities in structure: they all employ different ligands (ligand) or arrays of ligand molecules immobilized on the chip surface. Here, the ligand (ligand) or ligand molecule refers to a biological/chemical molecule that can react with other molecules. For example, the ligand may be a single-stranded DNA which hybridizes to its complementary single strand of nucleic acid; the ligand can also be an antibody molecule which can specifically combine with a corresponding antigen molecule; the ligand may also be a particle having on its surface a plurality of molecules which are reactive with other molecules. By adding a labeling molecule, such as a fluorochrome molecule, the reaction between the ligand and other molecules can be monitored and quantified. Thus, an array of different ligands immobilized on the surface of a biochip can be used to perform and monitor reactions of a variety of reactant molecules.

Currently, many passive biochips do not take full advantage of the processes and methods in microfabrication and microelectronics. Therefore, it is difficult to integrate and miniaturize the bio-analysis system from the sample preparation to the result detection by the passive biochip. In addition, passive biochips have a number of other disadvantages, including: low sensitivity of analysis, long reaction time, inability to control temperature, pressure, electric field at individual sites (or units) constituting the biochip, and difficulty in controlling local concentrations of molecules.

On the other hand, by applying an external force to the molecule, such as microfluidic manipulation or electrical manipulation, multiple functions such as molecular manipulation, interaction, hybridization, and separation detection (e.g., PCR and capillary electrophoresis) can be accomplished by means of an active chip. However, many of these chips do not currently have the ability to be used at high throughput. The electronic biochip developed by Nanogen corporation can manipulate and control biomolecules by an electric field generated from a microelectrode, and has significantly improved reaction speed and detection sensitivity compared to passive chips, such as the electronic chips (see U.S. Pat. Nos.5,605,622, 5,632,957, 5,849,486). However, in order to effectively move biomolecules in suspension/solution using an electric field, the conductivity of the solution must be low. This greatly limits the choice of buffers in biochemical assays. Under conditions of low ionic strength, many enzymes and other biomolecules denature and non-specific binding may occur on the chip surface.

Disclosure of Invention

The invention aims to provide a micro electromagnetic unit array chip capable of realizing single-point gating. It can be used to magnetically orient biomolecules, chemical molecules, etc.

The invention also aims to overcome the defects of the existing biochip and provide a novel single-point gating type electromagnetic biochip, which is characterized in that the novel single-point gating type electromagnetic biochip is internally provided with a single-point gating type micro electromagnetic unit array chip, and the chip can achieve directional operation and directional release of biomolecules by controlling the on and off of electromagnetic fields of all units in the array and combining with the magnetic modification of the biomolecules, so that the biochemical analysis has the advantages of high sensitivity, short reaction time, no damage to the biomolecules, wide range of buffer solutions for reaction selection, good reproducibility of analysis results and the like.

It is a further object of the present invention to provide a method for the directed manipulation of biomolecules, chemical or pharmaceutical molecules and the like.

According to an aspect of the present invention, there is provided an electromagnetic chip having a single-point-gating type micro electromagnetic unit, including: a substrate; a plurality of micro-electromagnetic units on the substrate, each unit generating a magnetic field by applying a current; means for gating any one of the plurality of cells to generate a magnetic field therein.

According to another aspect of the present invention, there is provided an electromagnetic biochip having a single-point-gated micro-electromagnetic unit, comprising: a substrate; a plurality of micro-electromagnetic units on the substrate, each unit generating a magnetic field by applying a current; means for gating any one of the plurality of cells to generate a magnetic field therein; and the functional layer is coated on the surface of the chip and is used for fixing ligand molecules.

According to another aspect of the present invention, there is provided an electromagnetic biochip having a single-point-gated micro-electromagnetic unit, comprising: a substrate; a plurality of micro-electromagnetic units on the substrate, each unit generating a magnetic field by applying a current; means for gating any one of the plurality of cells to generate a magnetic field therein; a functional layer covering the surface of the chip; ligand molecules fixed on the functional layer.

According to another aspect of the present invention, there is provided an electromagnetic chip including an array of single-point-strobable micro-electromagnetic units, including: a substrate; an array of pits disposed in the substrate in rows and columns, each pit containing a core; the first layer of conducting wires are respectively positioned between the iron cores in each row; the second layer of wires are insulated with the first layer of wires and are positioned between the iron cores in each row and are vertical to the first layer of wires; a second layer of insulating material is deposited over the surface of the second layer of conductors and the surface of the core.

According to another aspect of the present invention, there is provided an electromagnetic chip including an array of single-point-strobable micro-electromagnetic units, including: a substrate; an array of pits disposed in the substrate in rows and columns, each pit containing a core; and a first layer of conductors, each of the first layer of conductors extending at least 90 ° around one of the cores.

According to another aspect of the present invention, there is provided a method of manipulating a magnetic bead, comprising: providing an electromagnetic chip, wherein the chip comprises a plurality of micro-electromagnetic units which can be gated by a single point; placing magnetic beads on the surface of the chip; the current applied to each of the micro-electromagnetic units is varied to change the magnetic field distribution on the chip surface, thereby changing the magnetic force applied to the beads and affecting the position, velocity and other kinematic properties of the beads.

According to another aspect of the invention there is provided a method of directing a reaction between a ligand and a target-like molecule, comprising the steps of: providing a unit comprising a plurality of single-point-selectable micro-magnetic cores; forming a functional layer for fixing ligand molecules on the magnetic core; modifying the ligand molecule such that the ligand molecule can be positioned by a magnetic field; depositing a solution containing modified ligand molecules on the functional layer; gating the magnetic core to form a magnetic field, positioning the ligand molecules on a predetermined position by the magnetic field, and fixing the positioned ligand molecules on the functional layer so as to generate a pattern of the immobilized ligand molecules; modifying the target-like molecules such that the target-like molecules can be localized by a magnetic field; depositing a solution comprising modified target-like molecules on the pattern of immobilized ligand molecules; the gate core forms a magnetic field that positions the modified target-like molecule adjacent to the predetermined immobilized ligand molecule to cause the predetermined target-like molecule and the predetermined ligand molecule to react. The present invention provides a novel active biochip, the surface of the biochip is arranged into an array by a plurality of magnetic units which can be single-point gated, and the magnetic force generated by the magnetic units is used for controlling and manipulating magnetically modified molecules and particles and promoting the molecular reaction on the biochip. Magnetic field forces have been widely used for various applications in biological, biochemical and biomedical applications. For example, one of them is cell sorting using a magnetic field, and the operation is required to be performed by selectively binding antibody-modified magnetic microparticles to a specific cell type. After the antibody has specifically bound to the cells, the bound magnetic beads can be used to selectively remove the cells from the cell mixture with magnetic microparticles (see Miltenyi, S. et al, "High gradient magnetic cell-separation with MACS." Cytometry 11: 231. sub.236, 1990). Still other examples are described in us 5,439,586 for a magnetic sieve for separating magnetically labelled particles from non-magnetic particles in a fluid, and 5,655,665 discloses a magnetic particle separator for microfluidic magnetic separation made by micro-machining.

The invention discloses an electromagnetic chip comprising a micro-electromagnetic unit array capable of single-point gating. An array refers to a plurality of micro-electromagnetic units. One or more arrays of micro-electromagnetic elements may be on an electromagnetic chip. After each unit is electrified, a magnetic field can be induced. Because of the single-point switching capability, the on-off control and the intensity adjustment of the magnetic field of each unit can be performed by controlling the existence and the magnitude of the current in the selected unit according to the intensity and the direction of the magnetic field. Magnetic particles or magnetically modified biomolecules or particles can be manipulated by changing the magnetic field on the surface of the chip (the embodiment section describes the magnetic modification method of biomolecules or particles in detail). The magnetic particles or molecules are guided to predetermined positions on the chip surface by the magnetic field. Chemical modification of the chip surface can result in a functional layer for ligand immobilization, such that affinity interactions between ligand molecules and magnetically guided molecules or particles occur or through specific biological/chemical reactions (the functional layer portion is described in detail in the examples section). Magnetic guidance and manipulation changes the local concentration of the reactants and thereby increases the rate of different biochemical and chemical reactions, thereby achieving the goal of improving analytical sensitivity. Since the ionic strength and buffer properties have little or no effect on the magnetic field, the buffer most favorable for biochemical reactions can be selected. Further, there is no adverse effect of generating a strong electric field that complicates analysis or reaction due to electrochemical reaction.

When current is applied to the micro-electromagnetic units processed and manufactured on the substrate material, each unit can generate an independent magnetic field. One example is a ring of conductive material on a ferromagnetic or ferrous core and connected to a current source through an electronic switch. This ring may be circular, elliptical, square, triangular or any other shape, the only prerequisite being the ability to form a current flow around the ferromagnetic body. If the loop is single-turn, it should be closed or nearly closed. The ring may also be a number of turns around the ferromagnetic body. The turns may be machined in the same layer of the microstructure or in the alternative, each turn representing a separate layer in the structure. The electrical conductor may be a deposit-formed electrical conductor, such as a metal structure formed by electroplating, sputtering or deposition, while the electrical conductor may also be formed by selectively doping the semiconductor layer. The array of micro-electromagnetic units may be arranged in a row and column configuration similar to that typically found in microelectronic devices. The rows and columns may be perpendicular to each other or may be disposed at other angles (e.g., 80 degrees).

The shapes and sizes of the micro-electromagnetic units in the chip can be uniform or different. The characteristic dimension of the cell may range from less than 1 micron to greater than 1 cm. The characteristic dimension is the diameter of a circular unit and the side length of a square unit. In general, large size units are required when large numbers of ligand molecules are present. The cells may be arranged in a regular repeating pattern (e.g., a rectangular square matrix) or in an irregular or random pattern.

The individual micro-electromagnetic units can be individually gated, so that only one unit can be energized to generate a local magnetic field at any time, or a plurality of units can be energized simultaneously to generate magnetic fields with different complexity. Gating a micro-electromagnetic element means passing an electric current through the element to generate a magnetic field in its vicinity. The magnitude and direction of the current can be adjusted to produce sufficient magnetic induction to attract and move magnetic particles or magnetically modified molecules. Cells that are not gated may have two conditions, one being in a completely "off" (i.e., zero magnetic field) condition, and the other being that the cells are unable to generate a magnetic field large enough to attract or move magnetic particles.

There are many ways to gate a single cell. For example, for a cell consisting of a ring of conductors, one end of the conductor is connected to a current source (in the form of an electrical switch) and the other end is connected to a current sink, so that current flows through the ring. As another example, cells in an array may be gated by connecting rows to current sources (in the manner of switches) and columns to current sinks (in the manner of switches). This activates the cells located at the intersection of the rows and columns.

The invention further discloses a method for manipulating magnetic beads on a magnetic chip. The magnetic particles may be suspended in some fluid (aqueous or non-aqueous) or in air, even in a vacuum. When a micro-electromagnetic unit is energized, magnetic particles in the vicinity of the unit are attracted to the surface of the unit by magnetic force. I.e. when the magnetic particle suspension is spread over the whole chip, gating a cell will only affect the magnetic particles directly adjacent to the cell. However, by sequentially energizing the cells, it is possible to move all the suspended magnetic particles that accumulate on the entire chip. Such coordinated movement is called "steering", which can be controlled by turning the units on or off in a predetermined sequence. The manipulation of the magnetic beads also includes changing the magnetic field distribution and the force acting on the magnetic particles by changing the direction of the current applied to the micro-electromagnetic units, thereby achieving the purpose of changing and controlling the position, speed and other motion characteristics of the magnetic particles. All or part of the units can be powered on synchronously according to different use requirements. It is of course also possible to energize the micro-electromagnetic units one by one.

The size of the magnetic particles or materials in the present invention can be in the range of nanometers to micrometers or even millimeters. The magnetic particles can be prepared using different materials and different processing methods, as long as the magnetic field generated by the chip described in the present invention can induce a sufficient magnetic dipole moment in the magnetic particles.

The invention further discloses a method for manipulating biomolecules/biological particles, chemical agent molecules, drug molecules or other forms of molecules and particles by using the electromagnetic chip. In general, the electromagnetic chip can be used to manipulate any magnetic particle. In order to control and manipulate the non-magnetic particles/molecules, they need to be magnetically modified. For example, the molecules may be attached to the surface of the magnetic particles by covalent attachment or physical adsorption. These biomolecules may be proteins (e.g., antibodies, antigens, receptors), nucleic acids (e.g., single-stranded DNA or RNA) or other molecules such as lipids and carbohydrates. The surface of the electromagnetic chip can be modified so as to fix the ligand molecules capable of interacting with the magnetic bead surface molecules. The aggregation of magnetic particles at specific locations, to which molecules of suitable ligands have been immobilised, under the action of magnetic field forces can be promoted, causing an interaction between them.

When the molecules diffuse and collide in solution, the two molecules (e.g., antibody + antigen, the particular DNA probe and its complementary single-stranded target-like molecular DNA) will meet or react. The efficiency and speed of the reaction depend on the local concentration of molecules participating in the reaction and the kinetic energy of the collisions between them. In many systems constructed from biochips, it is common for one molecule to be immobilized on the chip surface while another molecule is in solution above the chip surface. The reaction will only occur when the passively diffused molecules in solution meet the immobilized molecules. Only a small proportion of the molecules in solution actually meet by diffusion over a certain period of time. Thus, the reaction is slow and inefficient, severely limiting the sensitivity, efficiency and speed of biological/chemical analysis on a chip. In the electromagnetic chip of the present invention, molecules in solution are "actively" pulled into contact with molecules immobilized on the chip surface by magnetic forces. Thus, the reaction speed, efficiency and sensitivity are improved.

For a typical magnetic bead made of superparamagnetic material, it is associated with a magnetic fieldWhen acting, will induce magnetic dipole in the magnetic bead

In the formula V_pVolume of magnetic bead, x_pAnd x_mPolarizability, μ, of the magnetic beads and the surrounding medium, respectively_mIs the magnetic permeability of the medium and,is the magnetic field strength. The magnetic force acting on the magnetic particles is shown by the following formulaThe polarizability and magnetic field gradient depending on the magnetic dipole:

the symbols "●" and "" in the equation represent dot product and gradient operations, respectively. The magnetic bead velocity at equilibrium under the action of magnetic force and viscous drag is

Wherein r is the diameter of the magnetic bead, eta_mIs the viscosity coefficient of the surrounding medium. Thus, in order to obtain a sufficiently large magnetic steering force, the following factors must be considered:

(1) the polarizability of the particles must be as large as possible;

(2) the magnetic field strength must be as large as possible;

(3) the magnetic field gradient must be as large as possible.

According to an embodiment of the invention, a row-column array chip composed of single-point gating type micro-electromagnetic units comprises: the chip comprises a substrate, a pit array arranged in the substrate, iron cores arranged in each pit, a first layer of wires arranged in the substrate, a first insulating layer arranged on the surface of the substrate and covering the first layer of wires, a second layer of wires arranged on the surface of the first insulating layer, a second layer of wires arranged between the iron cores in each row and vertical to the first layer of wires, and a second insulating layer arranged on the top of the chip and covering the iron core array and the second layer of wires.

In another embodiment of the present invention, the electromagnetic biochip comprises a single-point-gated micro-electromagnetic unit array chip. The chip includes: the chip comprises a substrate, a pit array arranged in the substrate, iron cores arranged in each pit, a first layer of wires arranged in the substrate, a first insulating layer arranged on the surface of the substrate and covering the first layer of wires, a second layer of wires arranged on the surface of the first insulating layer, a second insulating layer arranged between the iron cores in each row and vertical to the first layer of wires, and a second insulating layer arranged on the top of the chip and covering the iron core array and the second layer of wires; an affinity thin layer (or functional layer) formed on the surface of the chip insulating layer for fixing ligand molecules, and a ligand bound to the fixed ligand molecule thin layer by magnetic field operation or other methods.

The functional layer is used for fixing ligand molecules. For example, the functional layer may be (but is not limited to) the following thin layers: a monolayer, a film layer, a glue layer, a non-dense or dense material layer. The functional layer may be an attachment layer (corresponding to the second insulating layer in the above example) adhered to the surface of the biochip. Alternatively, the functional layer may be formed by directly chemically modifying the surface molecules of the biochip. In the above example, the surface of the second insulating layer may be chemically modified to carry chemical groups or chemical molecules that can bind to ligand molecules. Ideally, the functional layer does not bind non-specifically to other molecules except the ligand molecules, and the specific binding to the ligand molecules is highly efficient. Reference is made to the examples section for a detailed description of functional layers.

A method for the directed manipulation of a biomolecule, chemical molecule or drug molecule according to a further embodiment of the present invention comprises the steps of:

a. preparing a micro-electromagnetic unit array chip which can be single-point gated as described above;

b. forming a thin layer for fixing ligand molecules on the surface of the chip;

c. carrying out magnetic modification or load (load) on the ligand molecules to be immobilized;

d. the magnetic field is generated by the appointed micro-electromagnetic unit through controlling the wire of the gating micro-electromagnetic unit array chip, so that the ligand molecules which are subjected to magnetic modification or load bearing are directionally fixed on the appointed sites of the fixed ligand molecule thin layer to form an affinity binding region;

e. carrying out magnetic modification on the target molecules to enable the target molecules to be combined with the magnetic micro-magnetic beads;

f. contacting the target sample molecular solution connected with the micro magnetic beads with the micro electromagnetic unit array chip combined with the ligand molecules;

g. alternately gating all units on the micro-electromagnetic unit array chip to enable the designated units to generate magnetic fields, so that the target molecules subjected to magnetic modification directionally approach to ligands for affinity binding on the designated units of the chip to perform affinity binding reaction;

h. the target-like molecules are separated from the microbeads, and the microbeads are removed.

In the above method, the ligand and the target molecule may be biomolecules, chemical molecules or drug molecules.

The method of the present invention can be used for hybridization detection of DNA molecules having specific sequences, affinity binding analysis of antibodies and antigens, etc., which are involved in drug screening, biochemical reaction control, biochemical monitoring, and clinical diagnostic applications.

The following describes the single-point gating type electromagnetic array chip, the electromagnetic biochip and the method for directionally manipulating molecules in the present invention in detail with reference to the attached drawings:

drawings

FIG. 1 is a schematic diagram of a chip with a micro-electromagnetic cell array capable of single-point gating.

FIG. 2 shows a single-point-strobable micro-electromagnetic cell array chip with magnetic particles being attracted to an energized electromagnetic cell.

FIG. 3 is a schematic diagram of a single-point gating type micro-electromagnetic unit biochip.

Figure 4 is a schematic diagram of a micro-electromagnetic chip having micro-electromagnetic elements distributed in a row and column array that can be gated at a single point.

Fig. 5 is a schematic cross-sectional view of the chip of fig. 4.

Fig. 6 is a schematic diagram showing the direction of current applied to gate the micro-electromagnetic unit (magnetize the core).

Figure 7 is a schematic diagram of a first set of wires used to form a micro-coil around a core.

Figure 8 is a schematic diagram of a second set of wires used to form a micro-coil around a core.

Fig. 9 is a schematic diagram of a third set of wires used to form a micro-coil around a core.

Fig. 10(a) is a schematic diagram of individual gating of each micro-electromagnetic unit by an electrical switch. Each unit is connected to power and ground by two series connected electrical switches. The switching of the two electrical switches is controlled by electrical signals applied to the row and column conductors.

The electronic switch shown in fig. 10(B) may be a bipolar transistor.

The electronic switch shown in fig. 10(C) may be a Metal Oxide Semiconductor Field Effect Transistor (MOSFET).

FIG. 11 is a schematic diagram of an electromagnetic biochip capable of single-point gating.

FIG. 12 is a schematic view showing the structure of the biochip of the present invention after a fluid cell and a window for optical detection are mounted thereon.

FIG. 13 schematically shows magnetic modification of a ligand or target-like molecule by a cleavable chemical linking arm.

FIG. 14 is a schematic illustration of the pick-up of frozen microparticles comprising ligand molecules and magnetic beads using a magnetic dispenser.

FIG. 15 is a schematic view showing the release of the frozen microparticles of FIG. 14 onto the surface of a biochip according to the present invention.

FIG. 16 shows thawing of the frozen microparticles of FIG. 14 comprising ligand molecules and magnetic beads.

FIG. 17 shows the separation of the magnetic beads from the ligand molecules of FIG. 16.

FIG. 18 shows the random movement of magnetically modified target-like molecules on the surface of the biochip of the invention.

Figure 19 is a schematic diagram illustrating the manner in which current flows through a plurality of wires on an electromagnetic core in a manner to excite a group of micro-electromagnetic elements (i.e., magnetize a group of cores).

Fig. 20 shows a different current pattern through the wire of the electromagnet chip. This current flow pattern can excite the micro-electromagnetic units that the current flow pattern shown in fig. 19 cannot excite.

FIG. 21 shows the immobilization of magnetically modified target molecules on the surface of an electromagnetic chip.

FIG. 22 is a schematic view showing the principle of cleaving magnetic fine particles from target-like molecules after the target-like molecules react with ligands on the surface of the biochip of the present invention.

Detailed Description

The following description is presented to enable any person skilled in the art to make and use the invention and is provided in the context of a preferred embodiment as contemplated by the inventor. Since the various underlying principles involved in the present invention are set forth in terms of creating a micro-electromagnetic device that can manipulate a variety of molecules and particles to accomplish a variety of specialized reactions, it will be apparent to those skilled in the art that various modifications can be made in specific steps.

Fig. 1 is a schematic diagram of an electromagnetic chip 10 according to the present invention. The chip 10 includes a plurality of micro-electromagnetic units 11, which may be fabricated on a substrate 16 of silicon, glass, silicon oxide, plastic, ceramic, or other dense and non-dense materials. The electromagnetic units 11 on the chip 10 are arranged in a 3 x 3 array. The electromagnetic unit 11 may induce a magnetic field when a current 15 is applied17 and can be selectively activated by a variety of means. Fig. 1 is a schematic diagram of 6 micro-electromagnetic units in 9 micro-electromagnetic units, which are excited by current to generate magnetic fields near the units. Note that the direction of the magnetic field depends on the direction of the current circulation.

In fig. 1, the electromagnetic unit 11 may be formed by annular electrical conductors (e.g., rings 15 as shown) and a center 19 around which the electrical conductors are insulated from the conductive loop. The rings can have a variety of geometries such as circular, helical, square, and square spiral. As known to those familiar with microetching and micromachining, these wires of different widths and thicknesses can be fabricated on silicon substrates using different photolithography and processing schemes (see, e.g., the Handbook of Microlithagy, Microchip and Microchip, volume 2: Microchip and Microchip, SPIE optical engineering Press, Bellingham, Washington, USA, 1997). These schemes involve a number of basic steps such as making a photolithographic mask, depositing metal, depositing insulators, coating photoresist, patterning a photoresist through a mask and developer, patterning a metal or insulating layer. The conductive lines may be a metallic material such as aluminum, gold, silver, tin, copper, platinum, palladium, or a non-metallic material such as carbon, a semiconductor material such as phosphorus doped silicon, or any other material capable of conducting electricity. In order to conduct currents up to several hundred milliamperes, the wire must have a cross-sectional area of several thousand square microns. The thickness and width of the conductive lines may vary between 0.1 to 500 μm and 1 to 500 μm, respectively. The wire may be single or multiple turns for each electromagnetic unit. Such as multiple turns, it may be necessary to use a multi-layer micro-machining scheme to fabricate the cells.

In one chip design of the invention, the gating of the micro-electromagnetic units is achieved by connecting the conductive loops to a current source via an electrical switch. The current flow in the conductive circuit can be controlled by changing the signal applied to the electrical switch, so that the electromagnetic unit is either switched on or off. In another chip design scheme, the gating of the micro-electromagnetic units can be realized by controlling the current on-off of the conductive loop through a mechanical switch. In both forms, the electromagnetic unit is connected with the switch, and various combinations of on/off gating states of the electromagnetic unit can be realized by controlling the switch to be turned on or off.

To increase the strength of the magnetic field induced by the current in the conductive coil, a core of ferrite or ferromagnetic material may be employed. In this case each electromagnetic unit comprises a magnetic core on a substrate, a single or multiple turn wire surrounding the magnetic core, means for applying a current from a current source to the wire. Thus, in fig. 1 the central area 19 of the electromagnetic unit 11 may be made of a ferrite material or a ferromagnetic material and electrically insulated from the current loop 15. Various methods known in the art may be used to deposit ferrite or ferromagnetic materials on a substrate (see, for example, the Ahn and Allen article, "A new toroid-viewer type integrated indicator with a multilevel medium and magnetic core", IEEE transactions on Magnetics 30: 73-79, 1994).

Fig. 2 is a schematic view of the guidance of a magnetic bead 21 towards an active electromagnetic unit. When a current 15 is applied, an induced magnetic field is generated near the cell17, which magnetic field exerts a magnetic action on the particles. As shown in equation 3, the magnetic force is greatly dependent on the magnetic field(and field intensity)) Distribution of (2). The distribution of the magnetic field can be controlled and varied by selectively gating the electromagnetic units. For example, a quadrupole magnetic field can be generated by simultaneously exciting four adjacent electromagnetic units with a current in an appropriate direction. The distribution of the magnetic field can be further changed by changing the magnitude and direction of the current applied to the micro-electromagnetic units. The change in the magnetic field in turn affects the magnitude of the magnetic force generated on the magnetic beads, and affects the position, velocity, and other kinetic parameters of the particles. For example, as shown in equations (2) and (3), the velocity of the particles can be increased by increasing the magnetic field strength and the magnetic force.

The electromagnetic biochip shown in FIG. 3 is identical to the chip shown in FIG. 1 except for the functional layer 42 located on the surface of the chip. The functional layer is used for immobilizing ligand molecules and may be a hydrophilic or hydrophobic monolayer, a hydrophilic or hydrophobic film, a hydrophilic or hydrophobic gel layer, a polymer layer, a non-dense or dense material and/or a combination of these materials. Molecular monolayers refer to monolayers (e.g., Langmuir-Blodgett films). For immobilization of the nucleic acid ligands, binding materials used in Southern blotting and Northern blotting, such as nitrocellulose or nylon, can be used. Proteins and polypeptides can be immobilized (e.g., hydrophobic) by a variety of physical or chemical means. For example, specific receptors such as antibodies or lectins may be added to the functional layer 42 in order to immobilize the protein or polypeptide ligand molecules. Depending on the ligand to be immobilized and the reactions and analyses to be carried out on the biochip, different molecules can be added to the functional layer 42 in order to achieve immobilization of the ligand. These molecules introduced into the functional layer 42 for immobilizing ligand molecules are referred to as functional groups. The functional group can be, but is not limited to, acetaldehyde, carbodiimide, succinimide ester, antibody, receptor, and lectin. These functional groups also include chemical groups or molecular sites formed by chemical modification of the chip surface. The method of using the electromagnetic biochip 10 shown in FIG. 3 will be described in detail later.

FIG. 4 is a schematic diagram of the micro-electromagnetic biochip 10 according to one embodiment of the present invention. The connection point 12 is electrically conductive with the array of electromagnetic units via a conductor 14. Fig. 5 gives a detailed schematic cross-sectional view of a single electromagnetic unit. The substrate illustrated in the drawings is a surface polished silicon substrate 16, although similar micro-electromagnetic chips can be fabricated on many substrates. The process of fabricating the electromagnetic biochip 10 of FIG. 4 is described in detail below. These processes are for illustration only. Those skilled in the art of microfabrication can easily modify these steps or processes to improve a partial processing method of a biochip having the same structure as that shown in FIG. 4. The conductive region is surface diffused (doped) with phosphorus to create a resistance with a doping concentration of 2-10 omega/□. The silicon dioxide insulating layer has a thickness of 1000 a-8000 * a and is produced by a thermal decomposition method described below.

Parallel conductive lines 18 are formed on the substrate 16 by phosphorus doping and photolithography, depending on the requirements of the micro-electromagnetic element array chip size and array density. The surface density of the phosphorus diffusion is adjusted so that the sheet resistance of the wire 18 is less than or equal to 10 Ω/□. Because the wires 18 are formed in the substrate 16, they do not undulate nor rise above the surface of the substrate 16.

After the first layer of wires 18 is formed, the chip is placed in a high temperature oven (e.g., 1000 ℃) and an insulating layer of silicon dioxide is grown to a thickness of 2000-4000 * a on the surface of the substrate 16. A first insulating layer 20 is then formed on the substrate 16 to cover the first layer of conductors 18.

And etching small holes for electroplating in the designated area between the first layer of wires by using a photoetching method. For example, a 10 μm deep array of plated pits 22 is etched by applying a KOH solution (30% w/w) to the surface of the silicon substrate 16. Each plating pit 22 is trapezoidal in cross-section with the smaller parallel side being adjacent the lower surface of the substrate 16. Another layer of silicon dioxide 24 having a thickness of 5000 a 5000 * is deposited on the surface of plating pit 22 and a layer of silicon dioxide at the bottom of plating pit 22 is removed by photolithography.

The small holes 22 are filled with a ferromagnetic material to form a magnetic core. First, the substrate 16 is placed in NiSO₄Heating the solution (200-400g/l) to 400-600 ℃ for 30 minutes under the condition of introducing nitrogen gas so as to form a nickel seed layer with the thickness of 1 μm at the bottom of the electroplating pit 22.

Magnetic core 26 in well 22 may be formed by electroplating according to the following steps and conditions: (1) placing the mixture at 20-40 ℃ in Fe/FeCl₂In solution (ratio 200: 500 g/l); (2) at 30-60 ℃ under FeNi/NiSO₄In solution (200: 400 g/l); (3) at 30-60 ℃ in FeCl₂In solution (10-60 g/l). Thus, an array of magnetic cores 26 is formed on substrate 16, wherein the surface of magnetic cores 26 is higher than the surface of first silicon dioxide insulating layer 20. The magnetic core 26 having a different composition structure is obtained by electroplating according to other conditions and steps. For example, to obtain a nickel (81%) -iron (19%) permalloy, the electroplating solution may comprise the following components: NiSO₄·6H₂O(200g/l)，FeSO₄·7H₂O(8g/l)，NiCl₂·6H₂O(5g/l)，H₃BO₃(25g/l) and Saccarin (3 g/l). At 5mA/cm²The plating speed of-0.3 μm/min can be obtained under the current density condition of (2). Other details regarding electroplating conditions can be found in a number of references (e.g., Romankiw and O' Sullivan, "Plating techniques" inHandbook of Microlithography，Micromaching and Microfabrication，Volume 2：Micromaching and Microfabrication，Editor：Rai-Choudhury P.，SPIE OpticalEngineering Press，Bellingham，Washington，USA，1997)。

After core array 26 is formed, a layer of Si having a thickness of approximately 5000 a 5000 * a₃N₄Insulating layer 28 is deposited onto magnetic core 26 and first insulating layer 20 at a temperature of 200-300 deg.c. Followed by sputtering a layer of conductive aluminum having a thickness of about 1.2 μm onto the Si₃N₄On the surface of the insulating layer 28. Second layer conductors 30 are formed between magnetic cores 26 perpendicular to first layer conductors 18 by photolithography and wet etching of aluminum. This forms an array of micro-electromagnetic elements consisting of an array of magnetic cores and a two-dimensional network of wires. The surface of aluminum wire 30 may be even or slightly higher than the surface of magnetic core 26.

Finally, a second Si thickness of about 4000 * a₃N₄An insulating layer 32 is deposited on the surface of the aluminum wire 30 at 300 deg.c. The insulating material at the ends of the first layer of wires 18 and the second layer of wires 30 is then removed by dry etching so that the ends of the wires are connected to the bond pads 12 by the conductors 14, thereby connecting the wires on the chip to external circuitry.

The conductive paths 18 and 30 of the array of micro-electromagnetic units are powered by a dc power source. Each element of the array of micro-electromagnetic elements can be controlled by selectively energizing different ones of the conductive paths 18 and 30. As shown in fig. 6, by selecting the direction of the current in channels 18 and 30 around core 26 so that a circular current is formed around core 26, a magnetic field is generated in the vicinity of the cell, that is, to magnetize one core in either column, the conductive channels 18 on the left and right sides of the column must be selected and the two channels are supplied with currents in opposite directions. Of course, this current will magnetize all the cores in the column to some extent. However, any predetermined cell on the column is also a cell on a row. When current flows in the conductive paths 30 on both sides of the row, all of the constituent cells in the row will have some degree of magnetization. But as shown in fig. 6, for the selected cell, there will be a loop around the periphery of the conductive path through which current flows. So that the magnetic field strength generated at the selected cells will be twice those of the non-selected cells.

When it is desired to increase the magnetic field strength of selected cells, multiple "turns" of conductive paths (e.g., microcoils) can be made to increase the current around the core. One or more further two-dimensional webs of conductive pathways may be formed over insulating layer 32 by a process similar to that used to form pathways 18 and 30, each web comprising two layers of conductive pathways similar to pathways 18 and 30, but insulated from each other and positioned to correspond to pathways 18 and 30, respectively.

The magnetic field strength of the selected cells can also be enhanced by fabricating microcoils around the core using micromachining techniques. Under a constant current, the magnetic force generated by the core is proportional to the number of turns of the micro coil. For those who work with microfabrication, they should know that many microfabrication techniques are available in the existing methods for fabricating the microcoil. The following methods are exemplary descriptions of this patent, but the fabrication method is not limited to this one. The micro-coil is fabricated in a similar manner to the conductive vias described above, using either doped conductive layers or metal conductive layers (e.g., aluminum), except that the conductive layers are connected through contact holes in a direction perpendicular to the chip surface. In making the first layer of conductive vias, rather than making straight vias around magnetic core 26, vias 34 are made to almost completely surround the conductive vias of magnetic core 26 as shown in fig. 7. This layer of conductive vias may be implemented using a phosphorus diffusion process as used in the fabrication of the column conductive vias 18. An insulating layer 20 is then applied over the conductive vias. As shown in fig. 8, a second layer of conductive vias 36 is deposited over the insulating layer 20, which may be formed by sputtering and etching as described above for the row conductive vias 30. Prior to sputtering, the insulating layer 20 is first etched to form a vertical connection hole 35 to connect the microcoil passages 34 and 36. The connection points 35 are arranged such that the tails of the conductive paths 34 of the first layer of the microcoil correspond to the beginning of the conductive paths 36 of the second layer of the microcoil. The second layer of microcoil channels 36 is also covered with an insulating layer 20 and the above process is repeated to form a third layer of conductive channels 38, as shown in fig. 9. Vias 38, 34 are connected to the array and conductors 14 and pads 12 (not shown). The key point is that each layer of conductive vias forms a single turn on the microcoil, and each microcoil is formed of an initial "column" layer 34 and a final "row" layer 38, where the annular conductive vias 36 can be made in multiple layers as desired. Note that the gap 40 between each adjacent conductive layer is slightly offset, and this offset arrangement is necessary to ensure that the via 35 always corresponds to the tail end of the next conductive layer and the head end of the previous conductive layer. Similarly, the microcoil can be implemented by the method (doping) used to initially fabricate the column channels 18. Depending on the requirements of the device being designed. One method of doping in silicon is to deposit a layer of polysilicon over the insulating layer 20 and then use a lithographically guided doping to create the conductive channel. After all the microcoil layers except the "row" layer 38 have been fabricated, the etching process is used to create the pits 22, the electroplating process is used to fabricate the magnetic core 26, and finally the "row" conductive layer 38 and the insulating cover layer are fabricated to complete the structure fabrication.

The benefit of the microcoils is that each core can produce a much stronger magnetic force (proportional to the number of turns in the microcoil) than a single turn coil, especially when one cell is row gated and magnetized, the other cores will have little or no magnetization.

Fig. 10 shows the principle of switching on micro-electromagnetic cells, where in fig. 10(a) each cell 41 is connected between a common power supply 43 and ground 45 by series switches 37 and 39. The switch 37 can be controlled by an electrical signal (current or voltage) applied to the row conductor 30. The switch 39 may be controlled by applying an electrical signal on the column conductor 18. Unit 41 is configured to pass current therethrough (i.e., current flows from power supply 43 through solenoid unit 41 to ground 45 only when the two series switches are open. the switch can be implemented using a bipolar transistor as shown in FIG. 10(B) or a MOSFET as shown in FIG. 10 (C). The switch can be turned on or off by applying a potential to the base of the bipolar transistor or the gate of the MOSFET. Unit 41 can be configured as a single turn square as shown in FIGS. 10(A) and (C) or as a multiple turn square as shown in FIG. 10 (B). these transistors can be configured in a manner similar to that described above for the electromagnetic array and can be integrated on the same substrate as the conductive paths Resistance and resistance of the conductive loop). In the above example the substrate material is silicon, but other materials, such as glass, silicide, ceramics or even plastics, may be used as the substrate. The substrate may also be a dense or non-dense substance. Similarly, the materials for forming the insulating layers 20, 28, and 32 are not limited to those exemplified above, and they may be plastic, glass, photoresist, rubber, ceramic, or the like. The conductive layer may be aluminum, gold, tin, copper, titanium, platinum, carbon, a semiconductor, or a composite thereof. Other configurations of micro-coils and conductive channels are similarly possible. The above-described method of manufacturing the magnetic core by electroplating is also merely an example. The magnetic core may be fabricated by electron beam evaporation, ion sputtering, or other deposition methods in microfabrication. Specifically, the core may be formed by depositing a series of ferrite or electromagnetic materials by e-beam evaporation, ion sputtering or other methods. The invention comprises various micro electromagnetic units which can be single-point gated and are manufactured on a substrate. By using the chip, the biomolecule chemical reagent and the drug molecule can be mobilized and controlled under the action of a magnetic field.

After the micro-electromagnetic array chip is fabricated, the surface of the insulating layer 32 can be chemically modified or covered with a thin film. This layer, referred to herein as the functional layer 42, is used to immobilize ligand molecules. As shown in fig. 11, the functional layer 42 may be a hydrophilic or hydrophobic monolayer, a hydrophilic or hydrophobic membrane, a hydrophilic or hydrophobic gel layer, a polymeric layer or a composite of these materials, as described in connection with fig. 3. The functional layer 42 may also be composed of a dense or non-dense substance. Specific molecules, such as antibodies, may be incorporated into the functional layer 42 for the purpose of immobilizing ligand molecules, ultimately depending on the specific ligand to be immobilized on the chip surface and the assay or reaction to be performed. These molecules embedded in the functional layer are molecules of the immobilized ligand, which are called functional groups. The nitrocellulose or nylon as the ligand-immobilized material for immobilizing nucleic acid may be a functional layer made of a material used in Southern blot transfer and Northern blot transfer, such as polylysine, agarose gel, hydrogel, polyacrylamide gel, etc. To immobilize proteins and polypeptides, antibodies or other protein molecules may be embedded in the functional layer 42 to serve as functional groups.

After the functional layer is formed, the ligand molecules 44, which are magnetically modified or supported (explained below), can be immobilized on the functional layer 42 by binding to different functional groups provided. FIG. 11 depicts a direct binding reaction such as binding of an antibody to an antigen, however, the immobilization reaction is not limited to this reaction. The position of the ligand molecules on the functional layer 42 can be precisely controlled by the magnetic field generated by the electromagnetic unit, i.e. in most cases if a single electromagnetic unit 26 is magnetized, the ligand molecules will be immobilized on the functional layer immediately above the unit. It is well known that the polarity of the electromagnetic field is determined by the direction of the current circulating around the electromagnetic unit. The electromagnetic units may be polarized to different polarities depending on the different current directions (clockwise or counterclockwise). Therefore, when two adjacent electromagnetic units are induced to have the same or opposite polarities, the total magnetic field formed by the superposition of the magnetic fields generated by the two electromagnetic units will determine the magnitude of the magnetic force acting on the magnetically modified ligand molecules and determine the position where the ligand molecules are immobilized. And if each electromagnetic unit is gated according to a certain time sequence, the magnetic field distribution on the chip can be adjusted and the magnetic force acting on the magnetic modification molecules can be changed. A fluid reservoir 46 is constructed on the surface of the chip 10 for accommodating affinity ligand molecules, reagents and reagents, and also for transporting liquids. FIG. 12 is an example of a biochip equipped with a fluid cell. The fluid reservoir 46 may be made of plastic or other material. The sample inlet and outlet 48 provide a channel for the flow of liquid. A quartz cover 50 (glass or other light permeable material; quartz is a good material for uv detection) is sealed over the top of the fluid reservoir 46 with silicone or other material. The patch is to facilitate detection of ligands and reaction products inside the device using a photo detection method. If non-optical detection methods are used, the fluid cell top 50 does not need to be made of a light transmissive material.

So far, we have completed the structural description of the single-point gating type micro-electromagnetic unit biochip of the present invention. Partial modifications to the structure and variations in the fabrication process of the individual cores do not depart from the scope of this patent.

FIGS. 13 to 21 show that chemical, biological, pharmaceutical and other types of molecules can be manipulated using the electromagnetic biochip shown in FIG. 4, according to the present invention. These methods include the steps of:

a. a micro-electromagnetic array chip 10 is constructed that can be individually gated as shown in fig. 4.

b. A functional layer 42 is formed on the chip surface. This functional layer serves for the immobilization of ligand molecules.

As described above, the functional layer 42 may be formed by directly chemically modifying the surface of the insulating layer 32, or may be formed by coating a polymer layer, or may be formed by introducing an affinity molecule or an active reactive group. This functional layer may be a hydrophilic or hydrophobic monolayer, a hydrophilic or hydrophobic membrane, a functional hydrophilic or hydrophobic gel, a polymer layer, a dense or non-dense layer or a composite of these materials.

c. The ligand molecules are magnetically modified or supported and immobilized on the functional layer 42.

d. By controlling the current in the individual wires 18 and 30 to generate a magnetic field at the desired micro-electromagnetic unit, the magnetically modified or supported ligand molecules will be drawn and immobilized at the designated locations on the functional layer 42, thereby forming the affinity binding regions on the chip necessary for performing different assays.

By applying a magnetic field, different methods can be used for the directed manipulation and immobilization of ligand molecules. For example, the ligand molecules 44 may be linked to paramagnetic beads 56 by a cleavable linker arm 54. Thus, the ligand molecules can be directionally transported, directionally manipulated and released at fixed points through the magnetic field generated by the electromagnetic biochip. The paramagnetic microbeads 56 may range in size from less than 100 nanometers to greater than 100 microns. They can be manufactured using existing technology and are also commercially available from companies such as Dynal and Seradyn. Cleavable linker 54 may be light-cut, heat-cut, enzymatic or special chemical reaction cleavage. The cleavable linker arm 54 and paramagnetic beads 56 may be covalently linked or linked by affinity binding between the terminal functional group 52 of the cleavable linker arm 54 and the receptor functional group of the beads 56.

The following are examples of complete assembly:

ligand (44) -cleavable linker arm (54) -biotin (52) -streptococcal protein (58) -paramagnetic microbeads (56)

Here, the linkage between the cleavable linker and the paramagnetic microbeads is achieved by the binding between biotin and streptococcal proteins. Such molecular assembly can be used as a routine method for paramagnetic microspherical modification of any ligand molecule using the following steps: first, streptococcal proteins are bound to the surface of paramagnetic microbeads (typically, the surface of paramagnetic microbeads has a layer of carboxyl or amino groups) using methods well established in the art. An alternative is to purchase paramagnetic microbeads coated with streptococcal proteins directly from the manufacturer. Next, a "cleavable linker-biotin" molecular complex is prepared. These two steps can be used for magnetic modification of any type of ligand molecule. The specific ligand molecule is then bound to the cleavable linking arm, such as by means of a covalent bond. And finally, mixing and culturing the paramagnetic magnetic beads covered with the streptococcal protein and the ligand-cleavable connecting arm-biotin molecular complex to promote the generation of biotin-streptococcal protein combination reaction. The entire intermolecular assembly is thus completed.

In ligand molecule immobilization, the magnetic field generated by the energized magnetic unit exerts a magnetic force on paramagnetic microbeads 56, which will bring the entire molecular assembly into contact with the biochip surface above the energized magnetic unit. The cleavable linker arms are then cleaved so that the micro-beads 56 can be removed after the magnetic unit is de-energized. The removal process may be accomplished by fluid flushing or externally applied magnetic field forces, which ultimately leave the ligand molecules immobilized on the functional layer 42.

Another method for magnetically supporting ligands is: paramagnetic microbeads and a solution containing the ligand are mixed and rapidly cooled to form solid particles 60 (typically less than 1 mm in diameter) containing the ligand and paramagnetic microbeads. Solid particles prepared with different samples can be stored in a freezer for later use. Such solid micro-beads can be transported directly to a designated location on the chip using a three-dimensional high precision robot equipped with a specially designed magnetic particle dispensing head 62. After the solid micro-beads are transported to a predetermined position above the designated area of the chip, the particles can be released and fixed in position by controlling the current of the designated micro-electromagnetic unit so that the magnetic field at the designated area on the chip is stronger than the magnetic field at the dispensing head (fig. 14). Thus, the operation of distributing the solid microbeads 60 to the designated regions of the functional layer of the chip 10 is completed (fig. 15). After the solid microbeads 60 are melted, the ligand molecules are then immobilized on the designated chip areas (FIG. 16). The above steps have the following additional advantages: the use of the magnetic dispensing head 62 minimizes cross-contamination between ligand molecules, thus eliminating the need to clean the dispensing head after each transport. After immobilization of the ligand molecules on the chip surface is complete, the microbeads 56 can be removed from the chip by applying a magnetic force or liquid wash over the chip (FIG. 17). The characteristic dimension of the affinity binding region on each of the micro-electromagnetic units on the chip is between 0.1 μm and 5mm (rectangle is length and width; circle is diameter). The size of the bonding area is determined by the size of each core 26 and whether there are multiple core passes and the number of cores that pass. The exact size of the affinity binding domains may also be varied by controlling the shape of the functional layer 42-for example, the functional layer 42 may be formed with lithographic controls (instead, the entire chip is covered).

e. The target-like molecules 62 are labeled (e.g., with fluorochromes 64) and attached to the microbeads 56.

In order to manipulate the target molecules 62 using the single-point gated micro-electromagnetic chip described in the present invention, these molecules need to be first magnetically modified.

There are a variety of methods for magnetically modifying target-like molecules. For example, target-like molecules 62 can be attached to paramagnetic beads 56 by cleavable linker arms 54, such that the target-like molecules can be manipulated and transported to a target area by applying a magnetic field. The linkage of the cleavable linker arm 54 and the paramagnetic microbeads 56 may be achieved by covalent or affinity binding of the terminal functional group 52 of the cleavable linker arm to a functional group or receptor of the paramagnetic microbeads 56. For example, the connection may be the following structure (fig. 18):

an assembly of target-like molecule-cleavable linker arm-biotin-streptococcal protein-paramagnetic beads can be formed by a similar procedure as described above for the "ligand-paramagnetic microbeads" assembly.

f. Target-like molecules 62 attached to paramagnetic beads 56 are placed in the fluid reservoir 46 and brought into contact with ligand molecules 44 immobilized on the biochip surface by a controlled magnetic field.

g. In the row/column cell array, energizing selected electromagnetic cells in the current flow pattern shown in fig. 19 and 20 causes the magnetic field generated by the micro-electromagnetic cells to be alternately turned on and off. In fig. 19 13 of the 25 cells are activated and in fig. 20 the remaining 12 cells are activated. Thus, the magnetic field generated at each of the micro-electromagnetic units attracts the target-like molecules 62 and moves them toward the designated ligand affinity binding region. By alternating the gating of the magnetic field, each electromagnetic unit can attract the target-like molecules in the vicinity of the electromagnetic unit from the solution to achieve the purpose of enrichment. Thus, binding and reaction between the target molecules 62 and the ligand molecules 44 can be carried out (FIG. 21).

When the magnetically modified target-like molecules 62 are introduced onto an electromagnetic biochip for analysis, the movement of the target-like molecules 62 begins to diffuse randomly (FIG. 18). The subsequent manipulation of the directional movement of the target-like molecules towards the micro-electromagnetic units is achieved by alternately switching the magnetic field force generated by the magnetic field of each unit on and off as shown in fig. 19 and 20. The targeted molecules 62 can also be directionally moved toward one or more gated micro-electromagnetic units by selectively gating these units, depending on the particular analytical requirements. Under the influence of the magnetic field generated by the gated micro-electromagnetic units, the magnetically modified target-like molecules 62 rapidly migrate toward the biochip surface and undergo affinity reactions (or other reactions) with the ligand target-like molecules 44 immobilized on the designated unit areas (FIG. 21).

h. In the final step, the target-like molecules 62 (or their reaction products) are separated from the microbeads 56 and then removed. Separation of target-like molecules 62 from the micro-magnetic beads 56 can be achieved by cleaving the cleavable linker arms 54 between the target-like molecules 62 and the micro-magnetic beads 56 by photo-cleavage, enzymatic cleavage, chemical cleavage, or the like (fig. 22). Removal of the microbeads 56 is achieved by applying a magnetic field force external to the chip (this method is not applicable where a closed fluidic cell 46 is used) or by flushing liquid through the cell 46.

In the above methods, the ligand and the target-like molecule may be any type of molecule (e.g., biological, pharmaceutical or any other chemical molecule). The methods described in the present invention can be used for binding assays, drug screening, etc., for determining specific sequences of DNA molecules, antibodies and antigen reactions by hybridization (e.g., binding of a drug molecule or potential pharmaceutical compound to a specific receptor). By way of example, a number of alternative pharmaceutical compounds may be used as ligand molecules and immobilized at predetermined locations on the functional layer 42. The biological receptor can be isolated from the cell or produced by genetic engineering and fluorescently labeled. The receptor is then placed at a specific location on the functional layer 42 to allow binding to the candidate compound. After washing, the candidate compounds that emit light on any area are the compounds that are most likely to interact with biological receptors. Therefore, the present invention can be used for biochemical reactions, biochemical detection and diagnostic tests. In addition, special organic chemical reactions for assembling complex macromolecules can be completed.

When the above-described method is used for DNA hybridization, after step h, non-specifically hybridized DNA molecules can be eliminated by strictly controlling the binding conditions (e.g., hybridization buffer, temperature, etc.). Finally, DNA molecules having a high affinity for ligand molecules are left, and they can be detected by fluorescence or the like.

When the above-described method is applied to an antibody-antigen reaction, following step h, after stringent washing, the non-specifically bound antibody or antigen will be removed, while the specifically bound antigen or antibody molecule will remain in the affinity binding region.

When the above-described method is applied to biological analysis, detection and quantitative analysis of the analysis result can be achieved by several detection methods, such as optical signal (e.g., light absorption detection or fluorescence detection), chemiluminescence or electrochemiluminescence detection, electrochemical detection, and radiolabel detection. Optical detection can be achieved by detecting fluorescence generated by the fluorochrome 64 carried by the laser-induced target molecules. Another optical detection method is to use laser to induce fluorescence from a fluorescent substance labeled on the probe or a fluorescent label on a secondary antibody specifically bound to the target molecule. Fluorescence resonance energy transfer can also be used to detect the proximity of the ligand 44 to the target-like molecule 62. A detailed description of fluorescence resonance energy transfer can be found in the article "fluorescence resonance transfer dye-banded dyes for DNA sequencing and analysis" (Proceedings of the National Academy of Sciences, USA, 92: 4347-. The following is a practical example of the use of the method of the invention to control the directional manipulation of DNA molecules.

Firstly, a micro-electromagnetic array chip with a single-point selectable general formula is manufactured according to the method described by the invention. The surface of the chip is covered with a layer of high molecular polymer for fixing DNA probes.

Paramagnetic microbeads were added to the solution containing the DNA probes and the mixture was then rapidly cooled to form frozen solid particles. The microparticles can be transported onto a designated area on the biochip surface by a high precision robot equipped with a magnetic dispensing head (micro-electromagnetic unit). Different probes are immobilized at different regions. Of course, each chip can have the same number of probes as the number of micro-electromagnetic units on the chip. The micro-electromagnetic units on the biochip are gated to generate a magnetic field stronger than that generated by the magnetic dispensing head, so that the particles containing the probes can be released onto the functional layer in the designated area on the biochip. When the solid particles are melted, the DNA probes in the liquid are immobilized on the designated cells (regions) of the biochip. The microbeads can then be removed by an external magnetic field applied to the biochip surface or cleaned by a fluid. This forms affinity binding regions on the biochip surface.

The target-like DNA molecule is labeled (e.g., with fluorescein or a radiation probe) and linked to the end of a photocleavable linker molecule. At the other end of the linker arm is a biotin molecule. Streptococcal protein molecules are immobilized on the surface of magnetic microbeads. Thus, when the solution containing the target-like DNA-linker-biotin complex is mixed with paramagnetic microbeads coated with streptococcal proteins, the target-like DNA molecules are linked to the magnetic microbeads through the biotin-streptococcal protein interaction.

DNA target molecule-photocleavable linking arm-biotin-streptococcal protein-magnetic micro-magnetic bead.

Next, a solution containing magnetically modified target-like DNA molecules is introduced into a fluidic cell on the biochip. The alternating gating of the micro-electromagnetic elements causes each element on the chip to alternately generate a magnetic field. The target-like DNA molecules modified with paramagnetic microbeads are then moved toward the probe DNA molecules immobilized on the chip surface. Since all electromagnetic units are gated, the target-like DNA molecules are directed to contact all DNA probes. Thus, the target-like DNA molecule can be subjected to hybridization reactions with the probe molecule at the affinity binding region under preselected hybridization conditions.

Any probe that hybridizes to a target-like DNA molecule can be detected by fluorescence, luminescence, or radiation, depending on the label used on the target-like molecule. The method can quickly screen the given DNA target sample molecules aiming at a plurality of DNA probes, and meets the requirements of high analysis speed and high automation degree. If the microbeads interfere with the detection, they can be removed from the target-like DNA molecules, for example by irradiating the arms with light of 250nm to 750nm, as is the case with photo-cleavable linkers. The free magnetic beads can then be removed from the reaction areas on the chip by an applied magnetic force or by a washing solution. In addition, the chip can be subjected to a "hot melt dissociation" temperature to remove the hybridized target-like DNA and reuse it.

The above examples are believed by the inventors to illustrate the preferred methods of applying the invention. However, the parameters mentioned, such as dimensions, materials, geometries, methods, protocols, temperatures, concentrations and times, should not be considered as limiting the invention. Obvious substitutions now or later known to one with ordinary skill in the art, in addition to those who have already claimed, are intended to be encompassed by this patent. The claims are thus to be understood to include what is specifically illustrated above and what may be obviously substituted. The specific embodiments described are for illustrative purposes only and the invention should not be construed as limited thereto. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims (67)

1. An electromagnetic chip with single-point-strobable micro-electromagnetic units (11), comprising:

a substrate (16);

a plurality of micro-electromagnetic elements (11) on a substrate (16), each element being operable to generate a magnetic field by application of an electric current;

means for gating any one of the plurality of cells to generate a magnetic field therein.

2. The electromagnetic chip of claim 1, wherein each of the micro-electromagnetic units comprises:

a magnetic core (26) disposed on the substrate (16);

means for conducting current around the magnetic core (26).

3. The electromagnetic chip of claim 2, wherein the means for conducting current around the magnetic core comprises:

a single or multiple turns of electrical conductor wire surrounding the magnetic core;

a current source for applying a current to the wires.

4. The electromagnetic chip of claim 3, wherein the shape of the electrical conductor surrounding the magnetic core is selected from the group consisting of circular, square, oval, triangular, spiral, and right-angled spiral.

5. The electromagnetic chip of claim 3, wherein the magnitude and direction of the current delivered from the current source is variable.

6. The electromagnetic chip of claim 2, wherein the magnetic core is formed by depositing a ferromagnetic material or a ferrite material on the surface of the substrate.

7. The electromagnetic chip of claim 1, wherein the micro-electromagnetic elements are arranged in a regular, ordered, repeating pattern on the surface of the substrate, with equal spacing between adjacent elements.

8. The electromagnetic chip of claim 1, wherein the micro-electromagnetic elements have a size between 0.1 microns and 1 cm.

9. The electromagnetic chip of claim 1, wherein the means for gating any one of the micro-electromagnetic units comprises:

a switch connected between the current source and each of the micro-electromagnetic units, the switch being operable to control the on/off state, and thereby the gating of the micro-electromagnetic units, either mechanically or electrically.

10. An electromagnetic biochip having a micro-electromagnetic unit (11) that can be gated at a single point, comprising:

a substrate (16);

a plurality of micro-electromagnetic elements (11) on a substrate (16), each element being operable to generate a magnetic field by application of an electric current;

means for gating any one of the plurality of cells to generate a magnetic field therein;

and the functional layer is coated on the surface of the chip and is used for fixing ligand molecules.

11. The electromagnetic biochip of claim 10, wherein the functional layer for immobilizing ligand molecules may be a hydrophilic monolayer, a hydrophilic monolayer with functional groups, a hydrophobic monolayer with functional groups, a hydrophilic film with functional groups, a hydrophobic film with functional groups, a hydrophilic gel layer with functional groups, a hydrophobic gel layer with functional groups, a non-dense material with functional groups, a dense material, and a dense material with functional groups.

12. The electromagnetic biochip of claim 11, wherein the functional group is an aldehyde group, a carbodiimide, a succinimide ester, an antibody, a receptor, or a lectin.

13. An electromagnetic biochip having a micro-electromagnetic unit capable of single-point gating, comprising:

a substrate (16);

a plurality of micro-electromagnetic elements (11) on a substrate (16), each element being operable to generate a magnetic field by application of an electric current;

means for gating any one of the plurality of cells to generate a magnetic field therein;

a functional layer covering the surface of the chip;

ligand molecules fixed on the functional layer.

14. The electromagnetic biochip of claim 13, wherein the ligand molecule is an oligonucleotide, a DNA, an RNA, or a protein.

15. The electromagnetic biochip of claim 13, wherein the ligand molecule is an antibody, a receptor, or a cell.

16. An electromagnetic chip containing a single-point gating type micro electromagnetic unit array comprises:

a substrate (16);

an array of pits (22) arranged in rows and columns in a substrate (16), each pit comprising a pit

An iron core (26);

first layer conductors (18) respectively located between the iron cores of each row;

a second layer of wires (30) insulated from the first layer of wires (18), the second layer of wires (30) being positioned between the rows of cores and perpendicular to the first layer of wires;

a second layer of insulating material is deposited over the surface of the second layer of conductors and the surface of the core.

17. The electromagnetic chip of claim 16, wherein the first layer of insulating material separates the first layer of wires from the second layer of wires.

18. The electromagnetic chip of claim 17, wherein the material of the first insulating layer is any one of silicon oxide, silicon nitride, plastic, glass, ceramic, photoresist, and rubber.

19. The electromagnetic chip of claim 16, wherein the material of the second insulating layer is any one of silicon oxide, silicon nitride, plastic, glass, ceramic, photoresist, and rubber.

20. The electromagnetic chip of claim 16, further comprising another layer of wire positioned between the columns of cores and insulated from other layers of wire.

21. The electromagnetic chip of claim 16, further comprising another layer of wire positioned between the rows of cores and insulated from the other layers of wire.

22. The electromagnetic chip of claim 16, wherein the substrate is made of any one of silicon, glass, ceramic, silicon oxide, or plastic.

23. The electromagnetic chip of claim 16, wherein the material of the wire is any one of aluminum, gold, silver, tin, copper, platinum, palladium, carbon, or a semiconductor.

24. The electromagnetic chip of claim 16, further comprising a functional layer for immobilizing ligand molecules.

25. The electromagnetic chip of claim 24, wherein said functional layer for immobilizing ligand molecules is selected from the group consisting of a hydrophilic monolayer, a hydrophilic monolayer with functional groups, a hydrophobic monolayer with functional groups, a hydrophilic film with functional groups, a hydrophobic film with functional groups, a hydrophilic gel layer with functional groups, a hydrophobic gel layer with functional groups, a non-dense material with functional groups, a dense material, and a dense material with functional groups.

26. The electromagnetic chip of claim 25, wherein the functional group is an aldehyde, a carbodiimide, a succinimidyl ester, an antibody, a receptor or a lectin.

27. The electromagnetic chip of claim 16, further comprising a fluid reservoir for contacting a liquid with the array.

28. An electromagnetic chip containing a single-point gating type micro electromagnetic unit array comprises:

a substrate (16);

an array of pits (22) arranged in rows and columns in a substrate (16), each pit containing a core (26); and a first layer of wires (30), each of the first layer of wires extending at least 90 ° around one of the cores.

29. The electromagnetic chip of claim 28, further comprising another layer of wires, each wire of said set of wires extending at least 90 ° around a core and being insulated from said first set of wires by an insulating layer, said first layer of wires and said another layer of wires being connected by a wire that penetrates said insulating layer in a vertical direction.

30. The electromagnetic chip of claim 29, wherein the first layer of insulating material insulates the first layer of wire from another layer of wire.

31. The electromagnetic chip of claim 30, wherein the material of the first insulating layer is any one of silicon oxide, silicon nitride, plastic, glass, ceramic, photoresist, and rubber.

32. The electromagnetic chip of claim 28, wherein a second layer of insulating material is deposited over the surface of the array.

33. The electromagnetic chip of claim 32, wherein the material of the second insulating layer is any one of silicon oxide, silicon nitride, plastic, glass, ceramic, photoresist, and rubber.

34. The electromagnetic chip of claim 28, further comprising a functional layer for immobilizing ligand molecules.

35. The electromagnetic chip of claim 34, wherein said functional layer for immobilizing ligand molecules is selected from the group consisting of a hydrophilic monolayer, a hydrophilic monolayer with functional groups, a hydrophobic monolayer with functional groups, a hydrophilic film with functional groups, a hydrophobic film with functional groups, a hydrophilic gel layer with functional groups, a hydrophobic gel layer with functional groups, a non-dense material with functional groups, a dense material, and a dense material with functional groups.

36. The electromagnetic chip of claim 35, wherein the functional group is an aldehyde, a carbodiimide, a succinimidyl ester, an antibody, a receptor or a lectin.

37. The electromagnetic chip of claim 28, wherein the substrate is made of any one of silicon, glass, ceramic, silicon oxide, or plastic.

38. The electromagnetic chip of claim 28, wherein the wire material is any one of aluminum, gold, silver, tin, copper, platinum, palladium, carbon, or a semiconductor.

39. The electromagnetic chip of claim 28, further comprising a fluid reservoir for contacting a liquid with the array.

40. A method of manipulating a magnetic bead, comprising:

providing an electromagnetic chip, wherein the chip comprises a plurality of micro-electromagnetic units which can be gated by a single point;

placing magnetic beads on the surface of the chip;

the current applied to each of the micro-electromagnetic units is varied to change the magnetic field distribution on the chip surface, thereby changing the magnetic force applied to the beads and affecting the position, velocity and other kinematic properties of the beads.

41. The method of claim 40, wherein the introduced magnetic beads are suspended in air or a liquid.

42. A method of manipulating magnetically modified biomolecules/microbeads comprising:

providing an electromagnetic biochip, wherein the biochip comprises a plurality of single-point gating type micro electromagnetic units and a functional layer on the surface of the biochip;

placing the magnetically modified biomolecules/microbeads on the surface of the chip;

the current applied to each of the micro-electromagnetic units is varied to vary the magnetic field distribution on the chip surface, thereby varying the magnetic force applied to the magnetically modified biomolecules/microbeads to manipulate and control the local enrichment of the biomolecules/microbeads.

43. The method of claim 42, wherein the magnetically modified biomolecules/microbeads comprise biomolecules/microbeads attached to a magnetic substance.

44. The method according to claim 43, wherein the attachment of the biomolecules/microbeads to the magnetic substance is accomplished by a linker molecule, covalent bond or biological affinity.

45. The method according to claim 42, wherein the biomolecules/microbeads are DNA molecules, cDNA fragments, protein molecules or cellular microparticles.

46. A method of directing a reaction between a ligand and a target-like molecule comprising the steps of:

providing a unit comprising a plurality of single-point-selectable micro-magnetic cores;

forming a functional layer for fixing ligand molecules on the magnetic core;

modifying the ligand molecule such that the ligand molecule can be positioned by a magnetic field;

depositing a solution containing modified ligand molecules on the functional layer;

gating the magnetic core to form a magnetic field, positioning the ligand molecules on a predetermined position by the magnetic field, and fixing the positioned ligand molecules on the functional layer so as to generate a pattern of the immobilized ligand molecules;

modifying the target-like molecules such that the target-like molecules can be localized by a magnetic field;

depositing a solution comprising modified target-like molecules on the pattern of immobilized ligand molecules;

the gate core forms a magnetic field that positions the modified target-like molecule adjacent to the predetermined immobilized ligand molecule to cause the predetermined target-like molecule and the predetermined ligand molecule to react.

47. The method of claim 46, further comprising the step of detecting a predetermined reaction between the target-like molecule and the ligand molecule.

48. The method of claim 47, wherein the step of detecting the reaction comprises optical detection.

49. The method according to claim 46, wherein the functional layer for immobilizing ligand molecules is selected from the group consisting of a hydrophilic monolayer, a hydrophilic monolayer having functional groups, a hydrophobic monolayer having functional groups, a hydrophilic film having functional groups, a hydrophobic film having functional groups, a hydrophilic gel layer having functional groups, a hydrophobic gel layer having functional groups, a non-dense material having functional groups, a dense material and a dense material having functional groups.

50. The method of claim 49, wherein the functional group is an aldehyde, a carbodiimide, a succinimide ester, an antibody, a receptor, or a lectin.

51. A method according to claim 46, wherein the step of modifying the ligand molecule comprises attaching the ligand molecule to a magnetic substance.

52. A method according to claim 51, wherein the ligand molecule and the magnetic substance are linked by a cleavable linker arm.

53. The method of claim 52, wherein said cleavable arm is a photocleavable linker, a hot-cleavable linker, an enzymatically cleavable linker or a linker cleavable with other chemical reagents.

54. A method according to claim 51, wherein the ligand molecule and the magnetic substance are linked by covalent bonding or bioaffinity.

55. The method according to claim 54, wherein said bioaffinity is an antibody-antigen affinity, a lectin-anticoagulant affinity or a receptor-ligand affinity.

56. The method of claim 46, wherein the magnetic modification of the target-like molecule comprises attaching the target-like molecule to a magnetic substance.

57. The method of claim 56, wherein the target-like molecule and the magnetic substance are linked by a cleavable linker arm.

58. The method of claim 57, wherein said cleavable arm is a photocleavable linker, a thermal cleavable linker, an enzymatic cleavable linker or a linker cleavable with another chemical agent.

59. The method according to claim 56, wherein the target-like molecule and the magnetic substance are linked by covalent bonding or bioaffinity.

60. The method according to claim 59, wherein said bioaffinity is an antibody-antigen affinity, a lectin-anticoagulant affinity or a receptor-ligand affinity.

61. A method according to claim 46, wherein the magnetic species associated with the target-like molecule or ligand molecule is isolated by cleavage of the cleavable arm.

62. The method of claim 61, wherein the cleaved magnetic species are removed by applying a magnetic field to the chip or by rinsing with a fluid.

63. A method according to claim 46, wherein the ligand molecules are modified by mixing together a solution of the ligand molecules and a magnetic substance and forming solid magnetic microparticles of the mixed magnetic substance and ligand molecules by rapid freezing.

64. The method of claim 63, further comprising the step of dispensing small solid magnetic particles to each cell with a magnetic dispensing head.

65. A method according to claim 46, wherein said ligand and target-like molecule are biomolecules, chemical molecules or drug molecules.

66. A method according to claim 46, wherein said ligand and target-like molecule are nucleic acid molecules.

67. A method according to claim 46 wherein the ligand and the target-like molecule are antibodies and antigens.