JP2013029440A - Biomolecule target detector, biomolecule target detection system, and biomolecule target detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biomolecule target detector and its detection method that actualize detection of a very small quantity of biomolecule targets.SOLUTION: There is provided the biomolecule target detector which detects a biomolecule target by detecting a leakage magnetic field of a magnetic bead coupled to the biomolecule target, the biomolecule target detector includes: a main flow passage in which a specimen liquid including the biomolecule target and the magnetic bead flows; a sub flow passage which is connected to the main flow passage and in which a specimen liquid including the biomolecule target and the magnetic bead flows in a different direction from the main flow passage; a circuit which has a power source and an electrode wire connected to the power source and generates an induction magnetic field in the direction where the specimen liquid of the sub flow passage flows to the main flow passage and an AC magnetic field to the sub flow passage by applying a current to the electrode wire; a ligand which is provided to the sub flow passage and can be coupled to the biomolecule target; and a magnetic sensor which detects the magnetic field of the sub flow passage.

Description

  The present invention relates to a technique for detecting a leakage magnetic field of a magnetic bead bound to a biomolecule target with high sensitivity to detect a biomolecule target, and relates to a biomolecule target detector, a biomolecule target detection system, and a detection method thereof.

In the present specification, a minute substance having a size of several tens of nm or less containing virus, DNA (Deoxyribonucleic Acid), or RNA (Ribonucleic Acid) is referred to as a biomolecular target. Biomolecular targets are
Even very small or low concentrations of biomolecular targets can have a negative impact on the environment. Therefore, there is an increasing need to detect the biomolecular target with high sensitivity and prevent the expansion of the influence of the biomolecular target at an early stage. For early detection, the detection system is required to be highly sensitive and capable of high-speed detection, and to be portable and inexpensive.

  Patent Document 1 shows an example in which a magnetoresistive element that detects a leakage magnetic field of a magnetic bead by a magnetic resistance change is used as a magnetic sensor. Patent Document 2 shows an example in which a Hall element that detects a leakage magnetic field of a magnetic bead by a change in Hall voltage is used as a magnetic sensor. Patent Document 3 shows an example in which a magnetoresistive element that detects a change in the magnetic permeability μ of a biomolecule target molecule is used as a magnetic sensor. Patent Document 4 describes an example in which a magnetic field that attracts magnetic beads to a magnetic bead fixing site is generated.

Special table 2007-514932 gazette Japanese Patent No. 4240303 JP 2004-205495 A JP 2003-279570 A

In order to realize detection of a trace amount of biomolecule target, it is necessary to induce the biomolecule target in a detectable region and detect the biomolecule target with high sensitivity. For example, in the case of about 600 molecules (1 fM level) in the channel volume μL (width 100 μm × height 100 μm × length 100 mm), the existence probability of molecules in a sensor region of 10 μm × 10 μm is about 6 × 10 ⁻³ . Therefore, high-sensitivity detection cannot be expected without inducing a biomolecular target in the detection region. Patent Documents 1 to 4 disclose that a highly sensitive tunneling magnetoresistive element (MTJ) or semiconductor Hall element is used as a sensor. Specifically, a small amount of biomolecular target is induced and detected. There is no description of the method.

  Moreover, in the prior art represented by patent documents 1-4, not only a target but the non-target containing a contaminant etc. flows through a main flow path similarly. Therefore, in the sensor region in which the ligand is arranged, mutual aggregation and unnecessary adsorption are caused, and it is difficult to detect the biomolecular target with high sensitivity. A configuration that selectively guides the target to the sensor region and enables high-sensitivity detection is required. An object of the present invention is to solve the above-described conventional problems and to provide a method for realizing highly sensitive detection of a biomolecular target.

  In order to solve the above problems, a biomolecule target detector of the present invention is a biomolecule target detector that detects the biomolecule target by detecting a leakage magnetic field of a magnetic bead bound to the biomolecule target. A main flow path through which the sample liquid containing the biomolecule target and the magnetic beads flows, and a sub flow through which the sample liquid containing the biomolecule target and the magnetic beads flows in a direction different from the main flow path. An induction magnetic field having a flow path, a power supply, and an electrode wiring connected to the power supply, and applying a current to the electrode wiring to flow the sample liquid in the sub-flow path with respect to the main flow path And a circuit that generates an alternating magnetic field in the sub-channel, a ligand that can bind to the biomolecule target provided in the sub-channel, and a magnetic sensor that detects the magnetic field of the sub-channel Obtain.

  According to the present invention, highly sensitive biomolecular target detection can be realized.

The structure schematic perspective view of an example of the biomolecule target detector of this invention. The structure schematic sectional drawing of an example of the biomolecule target detector of this invention. The principle schematic diagram of the biomolecule target detection of this invention. FIG. 6 is a schematic diagram showing the relationship between biomolecular target detection in the prior art. The structure schematic sectional drawing of another example of the biomolecule target detector of this invention. The structure schematic of the magnetoresistive element used for the biomolecule target detector of this invention. The structure schematic of the magnetoresistive element used for the biomolecule target detector of this invention. The structure schematic perspective view of another example of the biomolecule target detector of this invention. The structure schematic sectional drawing of another example of the biomolecule target detector of this invention. The structure schematic of the electrode wiring for magnetic field application of the biomolecule target detector of this invention. The structure schematic of the electrode wiring for magnetic field application of the biomolecule target detector of this invention. The circuit schematic diagram of the magnetic sensor used for the biomolecule target detector of this invention. The circuit schematic diagram of the magnetic sensor used for the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows an example of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows the manufacturing method of the biomolecule target detector of this invention. The schematic sectional drawing which shows an example of the biomolecule target detector of this invention. The schematic sectional drawing which shows an example of the biomolecule target detector of this invention. Process drawing which shows the detection method of the biomolecule target detector of this invention. The figure which shows an example of the data which shows the detection method of the biomolecule target detector of this invention. The figure which shows an example of the data which shows the detection method of the biomolecule target detector of this invention. Process drawing which shows the detection method of the biomolecule target detector of this invention. The principle schematic diagram of the biomolecule target detection of this invention. The figure which shows an example of the data of the biomolecule target detection of this invention. The figure which shows an example of the base sequence of complementary strand DNA used as DNA of the biomolecule target 1 used in Example 1, and a ligand.

(Embodiment 1)
Embodiments of the present invention will be described below. In addition, this invention is not limited to description of the following embodiment and an Example. In the following description, specific numerical values and specific materials may be exemplified, but other numerical values and other materials may be applied as long as the effect of the present invention is obtained.

  FIG. 1 shows a perspective view of a biomolecule target detector 10 of the present embodiment. FIG. 2 shows a cutaway view of the biomolecule target detector 10 shown in FIG.

  The biomolecule target detector 10 includes a main flow path 11 through which a specimen liquid including the biomolecule target 1 and the magnetic beads 2 flows, and a side flow having a groove-like structure connected to the main flow path 11 and running in parallel with the main flow path 11. A path 12, an electrode wiring 5 and an electrode wiring 6 for generating an induced magnetic field 4 for guiding the magnetic beads 2 from the main flow path 11 to the sub flow path 12, and a biomolecule target 1 provided in the sub flow path 12. A ligand 7 capable of binding and a magnetic sensor 8 formed of a magnetoresistive element capable of detecting a leakage magnetic field provided around the side surface of the sub-flow channel 12 are provided.

  FIG. 1 shows the X axis, the Y axis, and the Z axis. The X axis is the direction in which the sample liquid flows through the main channel 11. The Y axis is a direction orthogonal to the X axis on the surface of the main channel 11 through which the sample liquid flows. The Z-axis is a direction perpendicular to the surface through which the sample liquid flows through the main channel 11.

<Biomolecular target 1 and magnetic beads 2>
The biomolecule target 1 refers to a minute substance having a size of several tens of nm or less including virus, DNA (Deoxyribonucleic Acid), or RNA (Ribonucleic Acid). The biomolecule target detector 10 detects the biomolecule target 1. As shown in FIG. 2, the magnetic beads 2 are provided with a ligand 9 that binds complementarily to the biomolecule target 1. The magnetic bead 2 binds to the biomolecular target 1 via the ligand 9.

<Main flow path 11 and sub flow path 12>
The main flow path 11 and the sub flow path 12 are formed of an insulator 13.

  For example, the main path 11 has a pair of side walls and a lower surface that connects between the pair of side walls. A concave groove is formed by the pair of side walls and the lower surface. The sample liquid containing the biomolecule target 1 and the magnetic beads 2 flows through the groove.

  The sub-channel 12 is a channel through which the sample liquid flows in a direction different from the direction in which the sample liquid flows in the main channel 11. The sub-channel 12 preferably has a groove extending in the Z-axis direction perpendicular to the direction in which the sample liquid flows in the main channel 11.

  For example, a sub-flow channel 12 having a groove shape is formed on the lower surface of the main path 11. The groove that is the sub-flow channel 12 extends through the main path 11 in a direction different from the direction in which the sample liquid flows. As a result, the biomolecule target 1 bound to the magnetic beads 2 in the sample liquid can be selectively guided to the sub-channel 12. A specific guidance method will be described later.

<Electrode wiring 5 and 6>
The electrode wiring 5 and the electrode wiring 6 are connected to a power source (not shown). For example, a circuit is formed by electrically connecting both ends of the electrode wiring 5 and both ends of the electrode wiring 6. A power supply is inserted into this circuit. For example, as shown in FIG. 1, the electrode wiring 5 and the electrode wiring 6 are formed inside an insulator 13 that forms the main flow path 11 and the sub flow path 12.

  By applying currents in opposite directions to the electrode wiring 5 and the electrode wiring 6, a magnetic field in the vertical direction (Z-axis direction) is generated. Here, the “longitudinal direction” is a direction different from the direction in which the sample liquid flows through the main path 11 and the direction in which the groove of the sub-channel 12 extends.

  By applying currents in opposite directions in the X-axis direction in FIG. 1, a vertical magnetic field is generated. As shown in FIG. 2, in the electrode wiring 5, a current flows from the front surface to the back surface. In the electrode wiring 6, a current flows from the back surface to the front surface of the paper. As a result, an induced magnetic field is generated in the direction of the arrow. The induced magnetic field 4 can guide the magnetic beads 2 from the main channel 11 to the sub-channel 12.

  The longitudinal magnetic field is an example of the induced magnetic field 4. The induced magnetic field 4 may be a magnetic field in the direction in which the sample liquid flows through the sub-channel 12 with respect to the sample liquid flowing through the main channel 11.

  The biomolecule target 1 that is not bound to the magnetic bead 2 or the foreign matter that is the non-target molecule 3 does not have magnetism, and therefore is not induced in the sub-flow channel 12 by the vertical magnetic field. Hereinafter, contaminants that are the biomolecule target 1 and the non-target molecule 3 that are not bound to the magnetic beads 2 are referred to as “non-target substances”. When simply referred to as “biomolecule target 1”, it may include a biomolecule target 1 that is bound to magnetic beads 2 and a biomolecule target 1 that is not bound to magnetic beads 2.

  The biomolecule target 1 bound to the magnetic bead 2 can be selectively guided to the secondary flow path 12. As a result, the density of the biomolecule target 1 contained in the sample liquid in the sub-channel 12 can be improved, and the density of the non-target substance can be reduced. Therefore, the detection probability of the biomolecule target 1 can be improved.

<Magnetic sensor 8>
The biomolecule target 1 guided to the sub-channel 12 approaches the sub-channel side surface 15 of the sub-channel 12 by being moved by the influence of thermal fluctuation or the like, or by the adsorption force on the side surface of the sub-channel. The biomolecule target 1 then binds to the ligand 7 disposed on the side surface 15 of the subchannel 12. Hereinafter, the sub-channel side surface 15 of the sub-channel 12 is also referred to as “detection region”.

  The magnetic sensor 8 detects a leakage magnetic field generated from the magnetic beads 2 bound to the biomolecule target 1. Specifically, the magnetic sensor 8 detects a magnetic field in the sub flow channel 12. The magnetic sensor 8 determines that the biomolecule target 1 is bound to the ligand 7 when the leakage magnetic field is equal to or greater than the threshold value. The magnetic sensor 8 determines that the biomolecule target 1 is not bound to the ligand 7 when the leakage magnetic field is smaller than the threshold value. The magnetic sensor 8 is preferably composed of a magnetoresistive element. A specific configuration will be described later.

  For example, as shown in FIG. 1, the magnetic sensor 8 is formed inside an insulator 13 that forms a main flow path 11 and a sub flow path 12. The magnetic sensor 8 should just be arrange | positioned in the position which can detect the magnetic field in the subchannel 12.

<Detection of biomolecular target 1>
FIG. 3 shows how the biomolecular target 1 is detected using a magnetic method. The biomolecule target 1 binds complementarily to the ligand 9 arranged on the magnetic bead 2. The biomolecule target 1 also binds complementarily to the ligand 7 disposed on the side surface 15 of the subchannel 12. This state of the biomolecule target 1 is referred to as a double hybridized state 30.

  The magnetic sensor 8 detects the leakage magnetic field 31 from the magnetic beads 2. When the magnetic sensor 8 detects the leakage magnetic field 31 having a predetermined threshold value or more, it is determined that the biomolecule target 1 is present. When the magnetic sensor 8 detects a leakage magnetic field 31 smaller than a predetermined threshold, it is determined that there is no biomolecule target 1.

  Ligand 7 and ligand 9 shown in FIG. 2 assume that biomolecule target 1 is DNA or RNA. However, as shown in FIG. 19, even when the biomolecule target 135 is an antigen, the ligand 136 is a first antibody molecule, and the ligand 137 is a second antibody molecule, the magnetic sensor 8 can be similarly identified.

  The step of binding the ligand 9 of the magnetic bead 2 and the biomolecule target 1 may be performed in the main channel 11. Alternatively, a sample liquid containing the biomolecule target 1 bound to the ligand 9 of the magnetic bead 2 may be introduced into the main path 11.

<Comparison with conventional technology>
FIG. 4 shows how a biomolecular target is detected in the prior art. The case where a trace amount biomolecule target 41 is induced | guided | derived to the detection area | region is shown. The induced biomolecule target 41 approaches a detection region where the magnetic sensor 8 is present due to thermal fluctuation or adsorption force and binds to the ligand 47.

  However, the non-target 43 is also adsorbed by approaching the detection region due to thermal fluctuation and adsorption force. Therefore, in the detection region, the non-target 43 including the non-target molecule 3 and the like is adsorbed, and the density of the biomolecule target 41 is lowered. Therefore, the detection sensitivity of the biomolecule target 41 is lowered. In the induced magnetic field 46 induced by current application to the electrode wiring 45 of the prior art, a driving force component exists in the normal direction with respect to the detection region, so that it can be expected to promote adsorption to the detection region, but non-target adsorption We cannot expect suppression of this.

  On the other hand, the biomolecule target detector 10 shown in FIG. 2 selectively guides the biomolecule target 1 to the sub-channel 12 via the magnetic beads 2. As a result, improvement in target detection probability and suppression of non-target adsorption can be expected.

<Modification of electrode wiring>
FIG. 5 shows an example of the biomolecule target detector 50. The biomolecule target detector 50 has electrode wirings 51, 52, 53 and 54. The electrode wiring 51 and the electrode wiring 52, and the electrode wiring 53 and the electrode wiring 54 are set as one set. An induced magnetic field 55 is generated by applying a current in the reverse direction to the electrode wiring 51 and the electrode wiring 52. An induced magnetic field 55 is generated by applying a current in the opposite direction to the electrode wiring 53 and the electrode wiring 54.

  Driving force with respect to the normal direction of the side surface 15 of the sub-channel 12 that is the detection region is canceled by the induction magnetic field 55 caused by the electrode wiring 51 and the electrode wiring 52 and the induction magnetic field 55 caused by the electrode wiring 53 and the electrode wiring 54. Can be reduced. As a result, it is preferable because the detection signal can be easily separated from the induction magnetic field 55 and the leakage magnetic field from the magnetic beads 2.

<Magnetic resistance element>
6A and 6B show a magnetoresistive element which is a preferred example of the magnetic sensor 8. The magnetoresistive element is composed of a magnetic layer (61, 65) / nonmagnetic layer (62, 66) / magnetic layer (63, 67).

  The magnetoresistive element shown in FIG. 6A is an element using GMR (Giant Magnetoresistance). In the magnetoresistive element shown in FIG. 6A, the material of the metal nonmagnetic layer 62 is a metal such as Cu, and a magnetic resistance is detected by applying a current 64 in parallel to each layer.

  The magnetoresistive element shown in FIG. 6B is an element using TMR (Tunnel Magnetoresistance). In the magnetoresistive element shown in FIG. 6B, the insulating nonmagnetic layer 66 is an insulator tunnel barrier, and a current 68 is applied perpendicularly to each layer to detect the magnetoresistance.

  Further, a magnetoresistive element using AMR (Anisotropic Magnetoresistance) for detecting the anisotropic magnetoresistance of a magnetic material, or a magnetoresistive element using CMR (Colossal Magnetoresistance) using a strongly correlated electron material can be used.

In the present embodiment, the magnetic sensor 8 is preferably a magnetoresistive element using TMR. By using MgO (magnesium oxide) or MgAl ₂ O ₄ (magnesium aluminate) for the nonmagnetic layer 66, an MR ratio (magnetoresistivity change rate) of several hundred percent can be realized.

  The detected magnetic field sensitivity of the magnetoresistive element is inversely proportional to the magnitude of the MR ratio. Therefore, it can be expected that a biomolecule target is detected with high sensitivity when the magnetoresistive element has a high MR ratio. In order to detect a small amount of biomolecule target, it is preferable to use a magnetoresistive element that can induce the biomolecule target in the detection region and detect the biomolecule target with high sensitivity.

  In order to detect the leakage magnetic field from the magnetic beads 2, the magnetization directions of the two magnetic layers (magnetic layer 61 and magnetic layer 65 and magnetic layer 63 and magnetic layer 67) constituting the magnetoresistive element are orthogonal to each other. It is preferable to arrange in such a manner.

  For example, the magnetic layer is formed in a magnetic field, or post-annealing is performed in the magnetic field after forming the magnetic layer. Thereby, a magnetoresistive element having two magnetic layers (the magnetic layer 61 and the magnetic layer 65, and the magnetic layer 63 and the magnetic layer 67) whose magnetization directions are orthogonal to each other can be manufactured.

<Modification of biomolecular target detector>
FIG. 7 shows a perspective view of the biomolecule target detector 70. FIG. 8 shows a cross-sectional view of the biomolecule target detector 70 along the line bb shown in FIG. The biomolecule target detector 70 shown in FIG. 7 has the same configuration as that of the biomolecule target detector 10 shown in FIG.

  The biomolecule target detector 70 has a main channel 71 through which a sample liquid containing the biomolecule target 1 and the magnetic beads 2 flows, and a sub-structure having a via-like structure connected to the main channel 71 and connected in parallel to the main channel 71. A flow path 72, an electrode wiring 75 and an electrode wiring 76 arranged in the opening of the sub flow path 72 that generate an induced magnetic field 74 for guiding the magnetic beads 2 from the main flow path 71 to the sub flow path 72, A ligand 7 capable of binding to the biomolecule target 1 provided in the path 72 and a magnetic sensor 78 formed of a magnetoresistive element capable of detecting a leakage magnetic field provided around the side surface of the sub-channel 72 are provided.

  By applying currents in opposite directions to the electrode wiring 75 and the electrode wiring 76, an induced magnetic field 74 in the vertical direction is generated. The induced magnetic field 74 guides the magnetic beads 2 to the sub-channel 72. The biomolecule target 1 approaches the sub-channel side surface 73 of the sub-channel 72 due to thermal fluctuation or adsorption force. The biomolecule target 1 that has approached the sub-channel 72 is bound to the ligand 7 disposed on the side surface 73 of the sub-channel. Whether or not the biomolecule target 1 is bound is detected by detecting the leakage magnetic field of the similarly bound magnetic beads 2 with a magnetic sensor 78 disposed around the side surface 73 of the sub-channel.

  The electrode wiring 75 and the electrode wiring 76 can be connected and arranged as shown in FIG. 9A to form a single turn coil. Further, a desired operation can be realized even if a large number of turn coils are arranged as shown in FIG. 9B. Furthermore, even if the coil is arranged as a solenoid coil, a desired operation can be realized.

<Configuration example of magnetic sensor 8>
In order to determine the presence or absence of the biomolecule target 1 with high sensitivity, the magnetic sensor 8 preferably uses differential detection. FIG. 10A shows a configuration example of the magnetic sensor 8.

  Using the magnetoresistive element 101 and the reference resistor 102, the displacement voltage Vsig 105 and the reference voltage Vref 106 are detected through the negative feedback amplifier 103 and the previous value negative feedback amplifier 104 using a negative feedback circuit. The amplifier 107 differentially amplifies the displacement voltage Vsig 105 and the reference voltage Vref 106 to obtain a detection voltage Vout108.

  The detection voltage Vout108 can also be obtained using the bridge circuit shown in FIG. 10B. The bridge circuit illustrated in FIG. 10B includes a reference resistor 111, a reference resistor 112, and a reference resistor 113. The detection output voltage Vout 114 can be obtained as a resistance change of the magnetoresistive element 110 by the bridge circuit. The reference resistor 102, the reference resistor 111, the reference resistor 112, and the reference resistor 113 are preferably magnetoresistive elements having resistances equivalent to those of the magnetoresistive element 101 and the magnetoresistive element 110.

<Manufacturing method>
11A to 11J show an example of a method for manufacturing the biomolecule target detector of the present embodiment. 11A to 11J show cross-sectional views of the biomolecule target detector.

<Step S1>
As shown in FIG. 11A, the insulator layer 120, the magnetic layer 121, the tunnel insulating layer 122, and the conductor layer 123 are deposited in layers to form a film. Hereinafter, a film including the insulator layer 120, the magnetic layer 121, the tunnel insulating layer 122, and the conductor layer 123 is referred to as a “multilayer film 1200”.

  This will be specifically described below. A plurality of magnetic layers 121 and a tunnel insulating layer 122 are formed on the insulator layer 120. The tunnel insulating layer 122 is sandwiched between the magnetic layers 121. An insulating layer 120 is formed on the plurality of magnetic layers 121 and the tunnel insulating layer 122. A conductor layer 123 is formed over the insulator layer 120. An insulator layer 120 is formed on the conductor layer 123. If the surface in contact with the insulator layer 120 is the magnetic layer 121, a plurality of tunnel insulating layers 122 may be formed.

The material of the insulator layer 120 may be an insulator. For example, the material of the insulator layer 120 is a SiO ₂ film using TEOS (Tetra ethyl orthosilicate) as a raw material, thermally oxidized Si (SiO ₂ ), SiOC, SiN (Si ₃ N ₄ ), or an organic system having a low dielectric constant. Material.

  Suitable materials for the magnetic layer 121 are Fe, Co, and Ni. A suitable material for the magnetic layer 121 is an alloy containing any of Fe, Co, and Ni, and an amorphous magnetic alloy containing B (boron). The magnetic layer 121 may be a magnetic layer having perpendicular magnetization such as FePt.

A suitable material for the tunnel insulating layer 122 is Al ₂ O ₃ , TiO ₂ , MgO, or MgAl ₂ O ₄ . Since these materials have a high MR ratio, they are preferably used.

  The material of the conductor layer 123 only needs to have conductivity with a resistivity of 100 mΩcm or less. For example, the material of the conductor layer 123 is copper (Cu), aluminum (Al), platinum (Pt), tantalum (Ta), tungsten (W), tantalum nitride (Ta—N), titanium nitride (Ti—N). Or aluminum titanium nitride (Ti-Al-N).

  For example, a silicon substrate or the like can be used as a substrate (not shown) that forms the biomolecule target detector of the present embodiment.

  The multilayer film 1200 illustrated in FIG. 11A may be formed over a substrate on which a semiconductor circuit is mounted. When the multilayer film 1200 shown in FIG. 11A is formed over a substrate on which a transistor, a contact plug, or the like is formed in advance, it is preferable for signal measurement.

<Step S2>
As shown in FIG. 11B, both ends of the multilayer film 1200 shown in FIG. 11A are removed using an etching technique. The space removed by using the etching technique is referred to as a removal space 124 (removal space). As the etching technique, dry etching or wet etching can be used.

  In order to make the multilayer film 1200 after etching have a steep shape, RIE (Reactive Ion Etching) is suitable. The steep shape means that the angle (θ) in the cross-sectional shape of the multilayer film 1200 is an acute angle, as shown in FIG. 11B.

  Argon ion milling is suitable for processing the magnetic metal contained in the material of the multilayer film 1200.

<Step S3>
As illustrated in FIG. 11C, the insulator layer 125 is deposited so as to cover the multilayer film 1200. An insulator layer 125 is formed over the removal space 124 and the multilayer film 1200. It is preferable to planarize the upper surface portion of the insulating layer 125 using CMP (Chemical Mechanical Polishing) or the like. The material of the insulator layer 125 is similar to that of the insulator layer 120.

<Step S4>
As shown in FIG. 11D, a part of the insulating layer 125 formed on the multilayer film is removed using an etching technique. The removed portion 126 becomes the main flow path 11.

<Step S5>
As shown in FIG. 11E, a pattern mask material 140 is applied on the removed portion 126 and the insulator layer 125. The pattern mask material is a resist material or a photosensitive polymer. The pattern mask material 140 becomes a photosensitive pattern for forming the portion 127 to be a sub flow path.

<Step S6>
As shown in FIG. 11F, a part of the multilayer film 1200 is removed using an etching technique. The removed portion 127 corresponds to the sub flow path. The removed portion 127 has a groove shape of the sub-channel 12 shown in FIG. 1 or a via-shape of the sub-channel 72 shown in FIG. As shown in FIG. 11F, the removed portion 127 becomes a space that penetrates the multilayer film 1200.

<Step S7>
As shown in FIG. 11G, the protective layer 128 is formed so as to cover the multilayer film exposed to the external space by forming the removed portion 127. The protective layer 128 is also formed so as to cover the pattern mask material 140.

The material of the protective layer 128 is an insulator and a material that is resistant to an aqueous solution. A suitable material for the protective layer 128 is SiO ₂ or SiN.

<Step S8>
As shown in FIG. 11H, surface treatment is performed on the protective layer 128 in contact with the removed portion (sub-channel) 127. A ligand 129 is disposed on the side surface of the sub-flow channel 127 that has been subjected to a surface treatment that imparts a carboxyl group (—COOH), a thiol group (—SH), an amino group (—NH 2), or the like.

<Step S9>
As shown in FIG. 11I, the biomask detector of the present embodiment is formed by removing the pattern mask material 140 and the protective film 128 formed on the pattern mask material 140.

  As shown in FIG. 11J, the conductor layer 123 corresponds to the electrode wiring 5 and the electrode wiring 6. The plurality of magnetic layers 121 and the tunnel layer 122 correspond to the magnetic sensor 8. A current is applied to the electrode wiring 5 and the electrode wiring 6 to generate the induced magnetic field 4. Due to the induced magnetic field 4, the magnetic beads 2 are guided to the auxiliary flow path 127. The biomolecule target 1 that binds to the magnetic beads 2 is detected by the magnetic sensor 8.

  In the manufacturing process shown in FIGS. 11A to 11J, the photosensitive pattern mask material 140 is shown in FIGS. 11E to 11H. In the present embodiment, photolithography using the same photosensitive pattern material is applied to the processing of the main path 126 and the sub-flow path 127.

  When the sub-channel 127 penetrates the multilayer film, the lower main path 131 is formed in the lower part of the sub-channel 127 as shown in FIG. 12B. The sample liquid that has flowed through the sub flow path 127 then flows through the lower path 131.

  As shown in FIG. 12A, when the lower main path 131 is provided, a sacrificial layer 130 is disposed in advance under the multilayer film. In step S <b> 6, the lower main channel 131 can be formed by removing the sacrificial layer 130 when forming the sub-channel 127. The sample liquid passes from the upper main flow path 126 via the sub flow path 127. It flows into the lower main channel 131. As a result, the non-target substance does not remain in the sub-flow channel 126, which is preferable.

  The sacrificial layer 130 uses glass or SiN used in MEMS processing, or an organic material typified by a resist.

  When there are a plurality of electrode wiring sets as shown in FIG. 5, FIG. 9A, or FIG. 9B, a plurality of conductor layers 123 are formed in step S1 of FIG. 11A. For example, the conductor layer 123 is formed below the plurality of magnetic layers 121 and the tunnel insulating layer 122 with the insulating layer 120 interposed therebetween.

  A biomolecule target system in which the biomolecule target detectors of this embodiment are arranged in a plurality of arrays can also be formed. The biomolecule target system can detect a variety of biomolecule targets according to the ligand by arranging various ligands on the biomolecule target detector arranged in an array.

<Other manufacturing methods>
FIG. 13A to FIG. 13J show an example of another method for manufacturing a biomolecule target detector. 13A-13J show cross-sectional views of the biomolecule target detector. The manufacturing method described below is generally the same except that the process of FIG. 13G is different from the process of FIG. 11G of the manufacturing method described above.

<Step S11>
As shown in FIG. 13A, an insulator layer 120, a magnetic layer 121, and a conductor layer 123 are deposited to form a multilayer film. A film including the insulator layer 120, the magnetic layer 121, and the conductor layer 123 is denoted as “multilayer film 1300”.

  Specifically, the magnetic layer 121 is formed on the insulator layer 120. An insulating layer 120 is formed on the magnetic layer 121. A conductor layer 123 is formed on the insulator layer 120. There is an insulator layer 120 between the magnetic layer 121 and the conductor layer 123.

<Step S12>
As shown in FIG. 13B, both ends of the multilayer film 1300 shown in FIG. 13A are removed using an etching technique. The space removed by using the etching technique is referred to as a removal space 124 (removal space).

<Step S13>
As illustrated in FIG. 13C, the insulator layer 125 is deposited so as to cover the multilayer film 1300. At this time, the upper surface portion is preferably planarized by using a method such as CMP (Chemical Mechanical Polishing).

<Step S14>
As shown in FIG. 13D, a part of the insulator layer 125 formed on the multilayer film is removed using an etching technique. The removed portion 126 becomes the main flow path 11.

<Step S15>
As shown in FIG. 13E, a pattern mask material 140 is applied on the removed portion 126 and the insulator layer 125. The pattern mask material is a resist material or a photosensitive polymer. The pattern mask material 140 becomes a photosensitive pattern for forming the portion 127 to be a sub flow path.

<Step S16>
As shown in FIG. 13F, a part of the multilayer film 1300 is removed using an etching technique. A part of the removed portion 127 corresponds to a sub flow path.

<Step S17>
As shown in FIG. 13G, a tunnel insulating layer 132, a magnetic layer 133, and a protective layer 128 are deposited on the removed portion of the multilayer film 1300. The material of the tunnel insulating layer 132 is preferably Al ₂ O ₃ or TiO ₂ . This is because the multilayer film 1300 can be easily formed on the side surface.

  In particular, a method of natural oxidation in an oxygen atmosphere after depositing Al or Ti in advance is preferable. In addition, an ultrathin high-quality tunnel insulating layer can be formed by a multi-step process of deposition and oxidation.

Suitable materials for the magnetic layer 121 are Fe, Co, and Ni. A suitable material for the magnetic layer 121 is an alloy containing any of Fe, Co, and Ni, and an amorphous magnetic alloy containing B (boron). The magnetic layer 121 may be a magnetic layer having perpendicular magnetization such as FePt. The material of the protective layer 128 is a substance having resistance to an aqueous solution. A suitable material for the protective layer 128 is SiO ₂ or SiN.

<Step S18>
As shown in FIG. 13H, surface treatment is performed on the protective layer 128 in contact with the removed portion (sub-channel) 127. A ligand 134 is arranged on the side surface of the sub-flow channel 127 that has been subjected to a surface treatment that imparts a carboxyl group (—COOH), a thiol group (—SH), an amino group (—NH 2), or the like.

<Step S19>
As shown in FIG. 13I, by removing the pattern mask material 140 and the protective film 128 formed on the pattern mask material 140, the biomolecule detector of the present embodiment is formed.

  The biomolecule target detector shown in FIG. 13J induces the magnetic beads 2 by applying a current to the electrode wiring 5 and the electrode wiring 6. The same structure as shown in FIG. 2 can be realized in which the induced magnetic beads 2 are detected by the magnetic sensor 8 made of a magnetoresistive element. The magnetoresistive element shown here can be formed in a vertical array as shown in FIG. 14, and the tunnel insulating layer 132 can be formed in a single process, so that the effect of suppressing variations in element characteristics can be expected. .

  Each of the steps shown in FIGS. 11A to 11J, 12A to 12B, and 13A to 13J is a known technique, for example, a technique used in a semiconductor element manufacturing process, a thin film forming process, or a microfabrication process. Can be applied.

  The formation of each layer includes, for example, atomic layer deposition (ALD), pulse laser deposition (PLD), ion beam deposition (IBD), cluster ion beam, and RF, DC, electron cycloton resonance (ECR), helicon. Various sputtering methods such as inductively coupled plasma (ICP) and a counter target, molecular beam epitaxial method (MBE), ion plating method, and the like can be applied. In addition to the PVD (Physical Vapor Deposition) method, a CVD (Chemical Vapor Deposition) method, a MOCVD (Metalorganic Chemical Vapor Deposition) method, a plating method, a MOD (Metalorganic Decomposition) method, or a sol-gel method may be used.

  For microfabrication of each layer, for example, a method used in a manufacturing process of a semiconductor element or a manufacturing process of a magnetic device (such as a magnetoresistive element such as GMR or TMR) can be applied. For example, physical or chemical etching methods such as ion milling, RIE (Reactive Ion Etching), and FIB (Focused Ion Beam) may be used. Further, a stepper for forming a fine pattern, a lithography technique using an EB (Electron Beam) method, or the like may be used in combination.

  The planarization of the interlayer insulating layer and the surface of the conductor deposited in the contact hole can be performed by, for example, CMP or cluster ion beam etching.

  The oxidation treatment or reduction treatment at the time of manufacturing the tunnel insulating layer is performed in an appropriate atmosphere containing oxygen atoms, molecules, ions, radicals, or the like. The oxidation treatment may change the atmosphere, temperature, time, and reactivity. As means for generating plasma and radicals, for example, known means such as ECR discharge, glow discharge, RF discharge, helicon or ICP can be applied. Nitriding using nitrogen can be performed by the same method.

<Biomolecular target detection method>
A method for detecting the biomolecule target 1 by the biomolecule target detector of the present embodiment will be described. FIG. 15 shows a detection process of the biomolecule target 1. As an example, the case where the biomolecule target detector 70 shown in FIG. 8 detects the biomolecule target 1 will be described.

<Step S151>
By applying a current to the electrode wiring 75 and the electrode wiring 76, a vertical magnetic field is induced in the main channel 71. For example, the electrode wiring 75 and the electrode wiring 76 are formed by the turn coils shown in FIG. 9A. A static magnetic field 74 is generated for guiding the magnetic beads 2 bound to the biomolecule target 1 flowing in the main flow path 71 to the sub flow path 72.

  The static magnetic field represents a DC magnetic field induced with a constant current. However, the static magnetic field may be a low-frequency magnetic field having a vibration period sufficiently longer than the movement of the magnetic beads. For example, a magnetic field having a low frequency of 0.01 Hz to 100 Hz may be used.

<Step S152>
By applying an alternating current to the electrode wiring 75 and the electrode wiring 76, an alternating magnetic field 74 is generated with respect to the sub flow path 72. As a result, the magnetic beads 2 bound to the biomolecule target 1 are vibrated in the sub-channel 72 to promote the binding with the ligand 7. For example, an alternating magnetic field is generated after a predetermined time after the vertical magnetic field is generated in step S151.

  The biomolecule target 1, which is a minute substance having a size of several tens of nm or less, is very small with respect to the width of the sub flow path 72. Therefore, there is a possibility that the secondary flow path 72 flows without being combined with the ligand 7 arranged in the secondary flow path 72. The probability of binding to the ligand 7 can be improved by vibrating the magnetic beads 2 bound to the biomolecule target 1 in the sub-channel 72 by the alternating magnetic field 74.

  The AC magnetic field 74 is preferable when the direction in which the sample liquid flows through the sub-channel 72 is the direction of gravity. The AC magnetic field 74 has a suitable vibration cycle that varies depending on the length of the sub flow path. For example, a frequency in the range of 1 Hz to 10 kHz is preferable.

<Step S153>
The magnetic sensor 78 detects the magnetic field in the sub flow path 72 (step S153). Specifically, the magnetic sensor 78 detects a leakage magnetic field from the magnetic beads 2 bound to the ligand 7.

<Step S154>
When the magnetic sensor 8 detects the leakage magnetic field 31 above a predetermined threshold, it determines that the biomolecule target 1 is present. The magnetic sensor 8 determines that there is no biomolecule target 1 when it detects a leakage magnetic field 31 smaller than a predetermined threshold. The magnetic sensor 8 may detect the leakage magnetic field, transmit leakage magnetic field data to a determination unit (not shown), and the determination unit may determine the presence or absence of the biomolecule target 1. Further, the magnetic sensor 8 determines the presence or absence of the biomolecule target 1. When it is determined that the biomolecule target 1 is present, the number or type of the biomolecule target 1 may be determined.

<Step S155>
If it is determined that the biomolecule target 1 is present, the detected leakage magnetic field 31 data is collected and the detection is completed. For example, it records in the recording part which the biomolecule target detector 78 has.

  You may hold | maintain the relationship matched with the magnitude | size of the leakage magnetic field, and the number or kind of biomolecule target 1 previously. The magnetic sensor 8 or the discriminating unit may specify the number or type of the biomolecule target 1 existing in the sub-channel 72 by referring to the magnitude of the detected leakage magnetic field using this relationship.

  For example, when the magnetic beads 2 present in the sample liquid have the same magnetic force, the number of biomolecular targets 1 increases as the magnitude of the detected leakage magnetic field increases. Therefore, it is only necessary to have a relationship in which the magnitude of the leakage magnetic field is proportional to the number of biomolecule targets 1.

  Moreover, you may change the kind of the ligand 9 couple | bonded for every magnitude | size of the magnetic force of the magnetic bead 2. FIG. For example, when the ligand 9 to be bound differs depending on the type of the biomolecule target 1, there is a relationship in which the magnitude of the magnetic force (including an integer multiple value) is associated with the type of the biomolecule target 1. If you do.

FIG. 18 shows a modification of the detection process of the biomolecule target 1. The detection step shown in FIG. 18 includes a step (step S181) of removing the extra magnetic beads 2 by applying a static magnetic field between step S152 and step S153 shown in FIG. The other detection steps shown in FIG. 18 are the same as the detection steps shown in FIG.
Furthermore, it is more preferable to repeat step S152 and step 181 a predetermined number of times.

  Here, the extra magnetic beads 2 mean the magnetic beads 2 to which the biomolecule target 1 is not bound. By eliminating the extra magnetic beads 2, it becomes easier to obtain an output signal corresponding to the biomolecule target 1 more accurately.

  FIG. 16 shows an example of the output voltage detected by the magnetic sensor 8 from step S151 to step S153.

  A signal due to the magnetic field generated in step S151 and step S152 also appears in the output voltage. In addition, although modulation appears in the output voltage in the middle of step S152, the output due to the leakage magnetic field of the coupled magnetic beads is detected in a superimposed manner. In step S153, the presence or absence of an output voltage is determined and detected without applying a magnetic field.

  FIG. 17 shows different examples of the output voltage detected by the magnetic sensor 8. Step S151 is the same as the example shown in FIG. By using a constant frequency for the input modulation in step S152 shown in FIG. 17, the frequency component can be removed from the output voltage. Therefore, it is preferable because the biomolecule target 1 can be detected even in the middle of step S152.

  The biomolecule detector of the present embodiment is desirable because an amplification step including a polymerase chain reaction (PCR) is not required. Conventional methods for detecting biomolecular targets have been proposed and partially used. However, the method using a crystal resonator, the method using a field effect, and the optical detection method using a fluorescent marker require many molecules, so the detection sensitivity is low and usually includes an amplification step using PCR. . In addition, the use of a fluorescent marker is not desirable because it includes a step of washing away unbound markers and biomolecule targets.

  According to the biomolecule detector of the present embodiment, the biomolecule target 1 bound to the magnetic beads 2 can be detected by the leakage magnetic field. Therefore, since the magnetic beads themselves can be driven with a magnetic field, unbound markers can be removed with a magnetic field. Since the biomolecule target unbound with the magnetic beads is not detected, the biomolecule target 1 can be detected without performing an extra washing step in principle. Since the biomolecule target 1 is a non-magnetic or low-permeability molecule, there is an advantage that the noise component is small compared to other methods. That is, if the leakage magnetic field of the magnetic beads can be detected with high sensitivity, detection with a high S / N can be expected.

  Hereinafter, the present invention will be described in more detail by way of specific examples.

Example 1
In Example 1, the biomolecule target detector 70 of FIG. 7 was manufactured using the biomolecule target detector manufacturing method shown in FIGS. 11A to 11J. The detection characteristics of the produced biomolecule target detector 70 (Sample 1-1) were evaluated.

A multilayer film shown in FIG. 11A was prepared. Co40Fe40B20 as the magnetic layer 121 and MgO (2 nm) as the nonmagnetic layer 122 were deposited on a Si substrate having a thermal oxide film (SiO ₂ film) of 500 nm formed on the surface of the multilayer film. However, the structure of the magnetic layer (lower part) / non-magnetic layer / magnetic layer (upper part) is specifically, Ta (5) / Cu (200) / Ta (5) / Pt47Mn53 (30) / Co50Fe50 (3) in order from the bottom. /Ru(0.7)/CoFeB(3) as the lower magnetic layer and CoFeB (5) / Ta (5) / Cu (200) / Ta (5) / Pt (20) as the upper magnetic layer in order from the bottom Formed. The numbers in parentheses indicate the film thickness (nm).

  The upper and lower Cu correspond to wiring electrodes for obtaining the output voltage of the magnetoresistive element. Lower PtMn / CoFe / Ru / CoFeB corresponds to the pinned magnetic layer. The upper Pt corresponding to the upper CoFeB and the free magnetic layer corresponds to passivation.

  Each layer was formed by magnetron sputtering. Film formation was performed under standard conditions for forming a magnetoresistive element at room temperature. As specific conditions, CoFeB was formed at a pressure of 0.8 mTorr and an RF output of 50 W. MgO was formed at room temperature with a pressure of 4 mTorr and an RF output of 50 W. Thereafter, a TEOS film 800 nm as the insulator layer 120, a titanium nitride (TiN) film 200 nm as the conductor layer 123, and a TEOS film 800 nm as the insulator layer 120 were deposited thereon. The TiN layer was deposited by magnetron sputtering using a Ti target.

  Sputtering is performed in an atmosphere (pressure: 0.1 Pa) in a mixed gas of nitrogen gas and argon gas (nitrogen gas: argon gas volume ratio is about 4: 1) in a range of 0 to 350 ° C. (mainly 280 ° C.) and the applied power was DC 4 kW. Thereafter, in order to orthogonalize the magnetization direction, a vacuum heat treatment at 280 ° C. was performed in a magnetic field of 5 KOe.

Next, it processed using the standard lithography and the etching method as FIG. 11B. Lithography here was contact exposure and electron beam exposure, and etching was performed using an argon ion milling technique. The wiring width formed by processing was set to 100 μm. Thereafter, a TEOS film having a thickness of 1 μm as shown in FIG. 11C was deposited. Subsequently, a recess serving as the main flow path 126 according to FIG. 11D was formed. The width of the recess was 50 μm and the height was 1-1.2 μm. Next, using the positive resist 140, a via serving as the sub-channel 127 according to FIG. 11F was formed. Five vias were formed in a row at about 100 μm intervals with a diameter of about 10 μmφ. Next, as shown in FIG. 11G, SiO ₂ 100 nm was formed as a protective film. Since the film formation was performed by RF sputtering, the thickness of the side wall portion of the sub-flow channel was ½ or less of the formed film thickness, and was typically about 30 nm.

Next, the surface of the subchannel was treated to modify the carboxyl group (—COOH). In the modification treatment, carbomethoxyethyltrichlorosilane (CMETS) whose terminal group is a methyl ester is used to make a 0.5 mmol / l toluene solution, and the whole secondary flow path is immersed for 1 hour, and then acetone, The surface was subjected to ultrasonic cleaning for 5 minutes in order of methanol and pure water to give CMETTS to the surface. Thereafter, the methyl ester, which is a terminal group of CMETS, was hydrolyzed to form -COOH on the surface. The hydrolysis treatment was performed for 2 hours so that the entire sub-channel was immersed in the HCl solution. After the surface is converted to -COOH, activation is performed with an N-hydroxysuccinimide group, and then streptavidin is added and immobilized. In this example, since the DNA having the wild type (2) sequence using a part of the ALDH2 gene of (1) shown in FIG. 21 as the biomolecule target 1 was used, biotin modification was performed on the 3 ′ end side ( Ligand 7 (and 129) was constructed by providing a streptavidin-biotin conjugate using a complementary (c-) DNA having the sequence of 3). The introduction of each reagent into the main and sub-channels was facilitated by reducing the amount of air trapped by degassing the solution, and the entire channel was slightly decompressed to facilitate introduction. . The magnetic beads were modified with biotin on the 5 ′ end side based on spherical magnetic beads (diameter 2 μm: Micromer-M made by micromod) made of iron oxide (Fe ₃ O ₄ ) with streptavidin attached to the surface. Complementary (c-) DNA having the sequence (4) was used to form a magnetic bead 2 provided with a ligand 9 by providing a streptavidin-biotin conjugate. Evaluation of a biomolecular target detector prepared by introducing 1 nM biomolecular target 1 dispersed in TE buffer composed of trishydroxymethylaminomethane (Tris) and ethylenediaminetetraacetic acid (EDTA) into the main channel together with magnetic beads 2 Carried out. Evaluation was performed by installing a biomolecule target detector in air at 25 ° C.

The arrangement shown in FIG. 21 is as follows.
(1) A part of ALDH2 gene
ttgggcgagt acgggctgca ggcatacact g aagtgaaaac tgtgagtgtg ggacctgctg ggggctcagg
gcctgttggg gcttgagggt ctgctggtgg ctcggagcct gctgggggat tggggtctgt tgggggctcg gggcctgcca
gaggttcagg acctgccggg gactcagggc gactcagggc ctgctggaag ttcaggacct gctggggatc agggcctgcc
(2) Gene sequence 5′-ttgggcgagt acgggctgca ggcatacact g aagtgaaaac tgtgagtgtg ggacctgctg ggggctcagg-3 ′
(3) Gene sequence of complementary DNA 3'-aacccgctca tgcccgacgt ccgtatgt-5 '
(4) Gene sequence of complementary DNA 3'-ctcacac cctggacgac ccccgagtcc-5 '
In accordance with the detection process of FIG. 18, after applying a constant current of 10 mA to the electrode wiring to generate a static magnetic field to promote induction of the magnetic beads 2 to the sub-channel 127, an AC magnetic field is applied by applying 10 mA at a frequency of 1 Hz. Generated. A voltage due to a change in magnetoresistance was measured by a detection method according to FIG. FIG. 20 shows an example of a detection waveform after removing the magnetic field effect for driving the magnetic beads. In the figure, τ and Vs indicate the AC magnetic field application time and the detected output voltage, respectively. The decrease in the output voltage after τ time is a signal when a DC magnetic field is applied to remove the unbound extra magnetic beads, and the detected output voltage Vs is the detected output voltage. The change in the detected output voltage when the AC magnetic field application time is changed is as shown in Table 1. It was shown that the detection probability is improved by applying the AC magnetic field. Furthermore, since the detection sensitivity per magnetic bead calculated from the catalog magnetization amount (3 emu / g) of the magnetic beads used in the biomolecule target detector of this example is about 10 μV / piece, it is at several levels of biomolecule targets. It was shown that the detection of can also be performed at a low concentration of 1 nM.

  In this example, a biomolecule target of DNA consisting of a part of the ALDH2 gene was used, but basically, any DNA can be used with the same high sensitivity detection.

(Example 2)
In Example 2, instead of the target DNA and complementary DNA used in Example 1, a sample 2-1 formed using a target antigen and an antibody was prepared, and leakage of magnetic beads bound by the antigen-antibody reaction was leaked Magnetic field detection characteristics were evaluated.

  The used biomolecule target detector was exactly the same as in Example 1, and the structure according to FIG. 7 was formed by the manufacturing method shown in FIGS. 11A to 11J.

The surface of the subchannel was treated to modify the carboxyl group (—COOH). The method was the same as in Example 1. After the surface was converted to -COOH, activation was performed with an N-hydroxysuccinimide group. In this example, since BSA (bovine serum albumin) antigen was used as the biomolecule target 1, an anti-BSA antibody (polyclonal antibody) was attached and immobilized on the activated surface to obtain ligand 7 (and 129). The introduction of each reagent into the main and sub-channels was facilitated by reducing the amount of air trapped by degassing the solution, and the entire channel was slightly decompressed to facilitate introduction. . Further, as magnetic beads, magnetic beads provided with ligand 9 using spherical magnetic beads (diameter 250 nm: nanomag-D manufactured by micromod) made of iron oxide (Fe ₃ O ₄ ) with a BSA antibody attached to the surface in advance are used. 2 was constructed. The 1 nM biomolecule target 1 dispersed in the phosphate buffer was introduced into the main channel together with the magnetic beads 2, and the prepared biomolecule target detector was evaluated. Evaluation was performed by installing a biomolecule target detector in air at 25 ° C.

  According to the detection process of FIG. 18, a constant current of 10 mA is applied to the electrode wiring to generate a static magnetic field to promote induction of the magnetic beads 2 to the sub-channel 127, and then 10 mA is applied at a frequency of 10 Hz to generate an alternating magnetic field. Generated. A voltage due to a change in magnetoresistance was measured by a detection method according to FIG. As the AC magnetic field application time increases, the detection output voltage increases, indicating that the detection probability is improved by applying the AC magnetic field.

  In this example, a biomolecular target composed of a BSA antigen was used, but basically any antigen can be used for high-sensitivity detection using the same technique.

  The present invention can be applied to biomolecular target detectors and electronic devices including the same. In addition, since the detection method of the biomolecule target of the present invention can improve the detection probability and shorten the detection time, the present invention can be applied as an in-situ diagnostic technique such as a virus or food inspection. .

DESCRIPTION OF SYMBOLS 1,135 Biomolecule target 2 Magnetic bead 3 Non-target molecule 4, 46, 55 Induced magnetic field 5, 6, 45, 51, 52, 53, 54, 75, 76 Electrode wiring 7, 9, 47, 129, 134, 136 137 Ligand 8, 78 Magnetic sensor 10, 50, 70 Biomolecule target detector 11, 71 Main flow path 12, 72 Sub flow path 13 Insulator 15, 73 Sub flow path side 30 Double hybridized state 31 Leakage magnetic field 41 Biomolecule Target 43 Non-target 61, 63, 65 Magnetic layer 62 Metal non-magnetic layer 66 Insulating non-magnetic layer

Claims (9)

A biomolecule target detector for detecting the biomolecule target by detecting a leakage magnetic field of a magnetic bead bound to the biomolecule target,
A main channel through which a specimen liquid containing the biomolecule target and the magnetic beads flows,
A sub-flow path connected to the main flow path and through which a specimen liquid containing the biomolecule target and the magnetic beads flows in a direction different from the main flow path;
A power source and an electrode wiring connected to the power source, and applying an electric current to the electrode wiring to generate an induced magnetic field in a direction in which the specimen liquid in the sub-channel flows in the main channel; and A circuit for generating an alternating magnetic field for the sub-flow path;
A ligand capable of binding to the biomolecule target provided in the subchannel;
A magnetic sensor for detecting the magnetic field of the sub-flow path;
Biomolecular target detector equipped with. The sub-channel is a plurality of via-like structures,
The biomolecule target detector according to claim 1. The ligand is DNA or RNA or an antibody molecule;
The biomolecule target detector according to claim 1. The magnetic sensor is at least two magnetoresistive elements;
The biomolecule target detector according to claim 1. The magnetic sensor includes at least two or more magnetoresistive elements, and includes a differential circuit using at least one of the magnetoresistive elements as a reference element.
The biomolecule target detector according to claim 4.   5. The magnetic sensor is configured to include a bridge circuit using at least two or more magnetoresistive elements, and at least one of the magnetoresistive elements is a reference element. The described biomolecular target detector. A plurality of biomolecule target detectors according to claim 1 are arranged in a matrix.
Biomolecular target detection system. A biomolecule target detection method for detecting the biomolecule target by detecting a leakage magnetic field of magnetic beads bound to the biomolecule target using a biomolecule target detector,
The biomolecule target detector is
A main channel through which a specimen liquid containing the biomolecule target and the magnetic beads flows,
A sub-flow path connected to the main flow path and through which a specimen liquid containing the biomolecule target and the magnetic beads flows in a direction different from the main flow path;
A power source and an electrode wiring connected to the power source, and applying an electric current to the electrode wiring to generate an induced magnetic field in a direction in which the specimen liquid in the sub-channel flows in the main channel; and A circuit for generating an alternating magnetic field for the sub-flow path;
A ligand capable of binding to the biomolecule target provided in the subchannel;
A magnetic sensor for detecting the magnetic field of the sub-flow path,
Generating an induced magnetic field in the main flow path by the circuit;
Generating an alternating magnetic field in the auxiliary flow path by the circuit;
The magnetic sensor detects the magnetic field of the sub-flow channel, and when the detected magnetic field is equal to or greater than a predetermined threshold, it is determined that the biomolecule target is present, and when the detected magnetic field is smaller than the predetermined threshold, Determining that the biomolecular target is absent.
Biomolecular target detection method. After the step of generating the alternating magnetic field, eliminating magnetic beads that are not bound to the biomolecular target;
The biomolecule target detection method according to claim 8.

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