JP2006184250A - Biosensor, method for measuring object, cartridge for biosensor, and nonwoven fabric - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor capable of simply and quickly carrying out measurements. <P>SOLUTION: A sensor chip 1 is disposed on the inner base of a cartridge 5, which is made up such that it can be inserted into and pulled out of an analyzer body 6, and a nonwoven fabric 46, impregnated with magnetic substance particles Mg, is arranged at a position opposite to the sensor chip 1, and a sample solution is dropped from an aperture of a lid 45. The magnetic substance particles Mg dissolved in the sample solution, drop on the surface of the sensor chip 1 and react therewith. After this reaction, magnetic field is applied to the sensor chip; and while keeping non-coupled magnetic substance particles away from the sensor chip 1, the strength of the magnetic field is detected, whereby an object to be measured can be analyzed. Accordingly, analysis can be carried out, simply and quickly without the labor of preparing the magnetic substance particles as another sample. <P>COPYRIGHT: (C)2006,JPO&amp;NCIPI

Description

  The present invention relates to a biosensor, an object measurement method, a biosensor cartridge, and a nonwoven fabric, and more particularly, to a biosensor, an object measurement method, a biosensor cartridge, and a nonwoven fabric that can be measured easily and quickly.

In recent years, in clinical diagnosis / detection and gene analysis, in order to detect antigens, antibodies, DNA (Deoxyribonucleic Acid), RNA (Ribonucleic Acid), etc., specific molecules such as the binding of an antigen to an antibody against it can be detected. An immunological technique using specific binding is used.
One of these analysis methods, solid phase binding analysis, includes a method using magnetic particles. FIG. 13 shows a schematic diagram of solid phase analysis using magnetic particles.

As shown in FIG. 13, this analysis is performed using a solid phase 91, a second molecular receptor 95, magnetic particles Mg, and a secondary antibody as the first molecular receptor 93. The measurement object 94 is analyzed.
The solid phase 91 has a solid surface in contact with the sample solution, and the second molecular receptor 95 is fixed to the solid surface. For the solid phase 91, polystyrene beads, reaction vessel walls, substrates and the like are used.
The second molecular receptor 95 is a substance that selectively holds the measurement target 94 such as an antigen, antibody, DNA, RNA, etc. present in the sample solution on the solid phase 91. As the second molecular receptor 95, a molecule that specifically binds to the measurement target 94 is used, and an antigen, an antibody, DNA, RNA, or the like is used.

  The magnetic particles Mg are magnetic particles and are used as a labeling substance. That is, by detecting the magnetic field formed by the magnetic particles Mg, the amount of the magnetic particles Mg is specified, and the presence or concentration or the concentration of the measurement object 94 in the sample solution is recognized. In addition to the magnetic particles Mg, those that emit detectable signals such as radioactive substances, phosphors, chemiluminescent substances, and enzymes are used as labels. Test methods using these labels include enzyme immunoassay using antigen-antibody reaction (EIA method), chemiluminescent method (CLIA) in the narrow sense of labeling with chemiluminescent compounds as immunoassay label compounds, and chemiluminescence. A chemiluminescent method (CL method) such as a chemiluminescent enzyme method (CLEIA), which uses a chemical compound in a detection system to detect enzyme activity with high sensitivity, is known.

The first molecular receptor 93 specifically binds to the measurement object 94 and is fixed to the magnetic particles Mg in advance.
In the analysis shown in FIG. 13, first, a sample solution containing the measurement object 94 is introduced into the solid phase 91 on which the second molecular receptor 95 is fixed in advance. As a result, the measurement target 94 is bound to the solid phase 91 via the second molecular receptor 95. Other substances contained in the sample solution float in the sample solution without binding to the solid phase 91. Next, the magnetic particles Mg to which the first molecular receptor 93 is fixed are put into the sample solution. At this time, the first molecular receptor 93 specifically binds to the measurement target 94, whereby the magnetic particles Mg are bound to the solid phase 91 via the second molecular receptor 95. Next, the magnetism of the magnetic particles Mg is detected, and the amount of the magnetic particles Mg bound to the solid phase 91 is specified. Thereby, the density | concentration or position of the measuring object 94 couple | bonded with the solid phase 91 can be specified. Patent Document 1 discloses a method of detecting the magnetism formed by the magnetic particles by using a magnetoresistive element arranged in an array.

Further, Patent Document 2 and Patent Document 3 disclose a method of arranging Hall elements in an array and detecting magnetic particles.
US Pat. No. 5,981,297 WO03 / 67258 pamphlet International Publication No. 03/102546 Pamphlet

In these analysis techniques, techniques relating to sensors that detect magnetic particles are disclosed, but techniques relating to the entire system that actually analyzes the measurement object are not disclosed.
Furthermore, in order to perform quick and simple analysis, a technique that requires a small number of steps and does not require labor is desired.
The present invention has been made in view of the above-described problems. A biosensor, a target measurement method, a biosensor cartridge, and a non-woven fabric capable of quickly and easily analyzing a measurement target such as an antigen, an antibody, DNA, or RNA. It is to provide.

In order to solve the above-described problem, a biosensor according to claim 1 of the present invention is a biosensor that analyzes an object to be measured by measuring the amount of magnetic particles. A magnetic particle having a first molecular receptor that specifically binds to a substance on the surface, a reaction vessel for holding a solution containing the measurement object, a reaction vessel installed in the reaction vessel, A sensor chip for measuring a quantity, wherein the sensor chip includes a magnetic sensor in which a plurality of magnetic field detection elements that output an output value corresponding to the detected magnetic field strength are two-dimensionally arranged, and the magnetic field detection A selection circuit that selects each of the elements, an amplification circuit that amplifies the output signal of the magnetic sensor, a second molecular receptor formed on the surface of the magnetic sensor that specifically binds to the measurement object, To have And butterflies.
In this way, by arranging magnetic particles provided with the first molecular receptor in advance in the reaction tank together with the sensor chip, at the time of measurement, just dropping the solution containing the measurement object into the reaction tank, Magnetic particles can be measured quickly and easily without the need for preparing magnetic particles separately as a reagent.

The biosensor according to claim 2 of the present invention is characterized in that, in claim 1, a magnetic field generator is provided at a position facing the sensor chip.
When a solution containing the measurement object is dropped and a predetermined time has elapsed, some of the magnetic particles are specifically bound to the surface of the sensor chip via the measurement object, and the remaining magnetic particles are not bonded. Without floating in the solution or settling on the surface of the sensor chip. Therefore, by providing a magnetic field generator at a position facing the sensor chip and generating a magnetic field, magnetic particles that are not specifically bonded can be moved away from the sensor chip surface. Further, while maintaining this state, that is, magnetic particles that are not specifically bound to the sensor chip surface are kept away from the sensor chip surface and a magnetic field is applied to the sensor chip. Can be detected by the sensor chip.

The biosensor according to claim 3 of the present invention is characterized in that, in claim 1 or 2, the periphery of the magnetic particles is covered with a dried saccharide and a buffer.
By placing the magnetic particles covered with dry saccharides and a buffer, it becomes possible to stably store the first molecular receptor, and it was exposed to a solution containing the measurement object at the time of measurement. At this time, the magnetic particles are easily dissolved. In addition, it is easy to dispose the magnetic particles in the reaction vessel.

A biosensor according to a fourth aspect of the present invention is characterized in that, in any one of the first to third aspects, the magnetic particles are arranged in the reaction tank in a state of being dispersed and fixed to a nonwoven fabric.
By impregnating and drying the magnetic particles in the nonwoven fabric, handling of the dried magnetic particles is facilitated and fixed to the nonwoven fabric in a more dispersed state, so that it is magnetic when exposed to a solution containing the measurement object. Body particles are easier to melt.

The biosensor according to claim 5 of the present invention is characterized in that, in claim 4, the nonwoven fabric is disposed at a position facing the magnetic sensor.
By disposing the nonwoven fabric impregnated with magnetic particles at a position facing the magnetic sensor, when the solution containing the measurement object is dropped, the magnetic particles that dissolve from the nonwoven fabric immediately reach the surface of the magnetic sensor, and the reaction time Can be shortened. Moreover, the solution containing the measurement object passes through the non-woven fabric, and impurities contained in the solution that inhibit the binding of the magnetic particles to the surface of the sensor chip can be removed.

A biosensor according to a sixth aspect of the present invention is characterized in that, in any one of the first to fifth aspects, a vibration generator for vibrating the reaction vessel is provided.
By providing the vibration generator, the solution in the reaction vessel can be stirred, and the reaction between the measurement object and the first and second molecular receptors can be promoted. Moreover, dissolution of the dried magnetic particles can be promoted.
A biosensor according to a seventh aspect of the present invention is the biosensor according to any one of the first to sixth aspects, wherein an electrode for supplying power to the sensor chip, an electrode for sending a signal to the selection circuit, and the amplification circuit The electrode for taking out the output signal from is formed on the bottom or side of the reaction vessel.
By arranging the electrodes on the back surface of the housing, that is, the bottom surface or the side surface of the reaction vessel, the housing can be reduced in size.

The biosensor according to claim 8 of the present invention is characterized in that, in claim 7, the electrode formed on the bottom surface of the reaction vessel is connectable with a smart card connector.
By using the smart card connector, the reliability of the connection between the electrode on the back side of the reaction tank and the connector can be improved.
According to a ninth aspect of the present invention, in any one of the first to eighth aspects, the sensor chip includes a memory element that stores in advance information on the measurement object.
By storing the information of the measurement object in advance, it is possible to automatically set different measurement conditions for each measurement object without the operator setting.

A biosensor according to a tenth aspect of the present invention is characterized in that, in any one of the first to ninth aspects, the reaction tank includes a plurality of the magnetic sensors.
By providing a plurality of magnetic sensors, a plurality of types of measurement objects can be analyzed simultaneously. At this time, a plurality of magnetic sensors may be arranged on the same sensor substrate, or a plurality of sensor substrates may be arranged.
A biosensor according to an eleventh aspect of the present invention is characterized in that, in the tenth aspect, different second molecular receptors are formed on the surfaces of the plurality of magnetic sensors.

According to a twelfth aspect of the present invention, in any one of the first to eleventh aspects, the magnetic field detecting element is a semiconductor Hall element, and the sensor substrate constituting the sensor chip is a silicon substrate. .
By using silicon as a substrate, a plurality of semiconductor Hall elements and electronic circuits having uniform and uniform performance can be manufactured on the same substrate easily and at low cost.

A biosensor according to a thirteenth aspect of the present invention is the biosensor according to any one of the first to twelfth aspects, wherein two or more insulating films made of different materials are formed on a surface of a sensor substrate constituting the sensor chip, and the insulation is performed. At least one insulating film of the film is characterized in that a portion at a position facing the magnetic sensor provided on the sensor substrate in the stacking direction is removed.
Here, the surface of the magnetic sensor refers to a region including at least the surface of each magnetic field detection element arranged in the magnetic sensor.
By removing a part of the insulating film only on the surface of the magnetic sensor, the distance between the magnetic sensitive surface and the surface of the magnetic sensor can be shortened, and the sensitivity to magnetic particles can be improved. Further, by using different materials for the insulating film to be removed and the insulating film to be left, it is possible to accurately control the thickness of the film to be removed by using the difference in the removal speed depending on the material in the removing process.

A biosensor according to a fourteenth aspect of the present invention is the magnetic sensor according to the thirteenth aspect, comprising a silicon oxide film and a silicon nitride film formed on the silicon oxide film as the two or more insulating films. The silicon nitride film portion at a position facing the substrate in the stacking direction is removed.
In general, under the dry etching conditions for a nitride film, a silicon nitride film has a higher etching rate than a silicon oxide film. Therefore, this speed difference makes it possible to leave the silicon oxide film to the extent that the metal wiring is not exposed while completely removing the silicon nitride film.

A biosensor according to a fifteenth aspect of the present invention is the biosensor according to any one of the first to fourteenth aspects, wherein the first insulating film having a high removal rate sandwiches a predetermined layer on the surface of the sensor substrate constituting the sensor chip. The first insulating film and the predetermined layer are stacked on a second insulating film having a slower removal rate through a predetermined layer, and the first insulating film and the predetermined layer are located at positions facing the magnetic sensor provided on the sensor substrate in the stacking direction. Is removed.
Here, the predetermined layer is an arbitrary layer formed between the first insulating film and the second insulating film, for example, a metal wiring layer.
As described above, when the insulating films having different removal rates are continuously laminated, due to the difference in the removal rates, the metal wiring is exposed to the second insulating film while completely removing the first insulating film. It is possible to leave

A biosensor according to a sixteenth aspect of the present invention is the biosensor according to any one of the first to fifteenth aspects, wherein a plurality of metal wiring layers are formed on a surface of a sensor substrate that constitutes the sensor chip, and among the metal wiring layers, The metal wiring layer formed in the uppermost layer in the stacking direction is characterized in that a portion at a position facing the magnetic sensor installed on the sensor substrate in the stacking direction is removed.
By not arranging the uppermost metal wiring on the part facing the surface of the magnetic sensor, when the insulating film on the uppermost metal wiring is a single layer, even if this insulating film in the magnetic sensor area is removed, the metal wiring Can be prevented.

A biosensor according to a seventeenth aspect of the present invention is the biosensor according to any one of the first to sixteenth aspects, wherein the sensor chip is provided with electrodes for electrical connection to the outside in a predetermined region. A wire separating the magnetic sensor is provided on the surface.
By installing a wire separating the electrode region and the magnetic sensor region on the surface, it is possible to prevent the resin from flowing into the magnetic sensor region when the electrode region is sealed with resin.

  An object measuring method according to claim 18 of the present invention is a measuring object measuring method using the biosensor according to any one of claims 2 to 17, wherein the magnetic particles are used as the sensor chip. A step of dissolving in a solution containing the measurement object introduced above, a step of bonding the magnetic particles to the surface of the magnetic sensor via the measurement object, and the magnetic substance floating in the solution without being bonded. The body particles and the magnetic particles bonded to the surface of the magnetic sensor without passing through the measurement object are separated from the surface of the magnetic sensor by the magnetic field generator, and then bonded to the surface of the magnetic sensor through the measurement object A step of specifying the amount of the magnetic particles, and a step of specifying the amount of the measurement object based on the amount of the magnetic particles, and these steps are continuously set in advance. Characterized in that the automatically performed on the basis of the ram.

A biosensor cartridge according to a nineteenth aspect of the present invention is a cartridge used for the biosensor according to any one of the first to seventeenth aspects, wherein the reaction vessel including the magnetic particles and the sensor chip is provided as an apparatus. It is formed in a housing that can be inserted into and removed from the main body.
A non-woven fabric according to claim 20 of the present invention is a non-woven fabric used in the biosensor according to any one of claims 1 to 17, wherein magnetic particles having the first molecular receptor on the surface thereof are dispersed and fixed. It is characterized by that.

  According to the present invention, measurement can be performed easily and quickly.

Next, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows a schematic configuration of the biosensor according to the present embodiment. The biosensor according to the present invention binds to a sensor chip that is a solid phase via the first molecular receptor, the measurement object, and the second magnetic molecule as described in the section of the prior art. The measurement object is analyzed by measuring the amount of the magnetic particles.

As shown in the figure, the biosensor of this embodiment includes an analyzer body 6 and a cartridge 5 configured to be detachable from the analyzer body 6.
As will be described in detail later, the cartridge 5 is a casing that functions as a reaction tank, in which a sensor chip 1 and magnetic particles Mg are arranged. The upper part is covered with a lid 45 having an opening, and the solution containing the measurement object is introduced into the sensor chip 1 provided in the cartridge 5 by dropping the solution of the measurement object from above the opening. It is possible. The sensor chip 1 is controlled by a signal from the analyzer body 6 and sends a measurement result to the analyzer body 6.

The analyzer main body 6 includes a coil 61 and a magnetic core 62 as a magnetic field generation device, a display device 63 that displays an analysis result, a control device 64 that controls the magnetic field generation device, the display device 63, and the sensor chip 1, and a cartridge. 5 and a connector 65 connected to the electrode on the back surface.
The magnetic core 62 is disposed at a position facing the sensor chip 1, and forms a magnetic field that attracts the magnetic particles Mg present on the sensor chip upward by energizing the coil 61 wound around the magnetic core 62.

The display device 63 is controlled by the control device 64 and displays the progress of measurement and the measurement result to the operator.
The control device 64 controls the magnetic field applied to the sensor chip 1 by passing a current through the coil 61, and controls the sensor chip 1 and processes signals from the sensor chip 1. Further, the display device 63 is controlled to display the analysis result. In the present embodiment, the measurement is configured by including various control circuits for controlling the magnetic field generation device, the display device 63, and the sensor chip 1, an arithmetic processing device, and storage devices such as a RAM and a ROM, and is programmed. Measurement is automatically executed by sending commands to various control circuits based on the procedure.

  The connector 65 connects the electrode of the cartridge 5 and the analyzer main body 6 so that power can be supplied to the cartridge 5 by bidirectionally transmitting and receiving signals between the control device 64 and the cartridge 5. The interface is not particularly limited as long as such connection is made, and a USB connector, a smart card connector, or the like can be used. The smart card connector is suitable as a connector used for the analyzer main body 6 for inserting and removing the cartridge 5 because the guaranteed number of insertions and removals is large.

Further, although not shown, a vibration generator for agitating the sample solution introduced into the cartridge 5 by vibrating the cartridge 5 may be provided. As the vibration generator, for example, a vibration motor, an ultrasonic vibrator, or the like is used.
Further, although not shown, an actuator may be provided that retracts the coil 61 and the magnetic core 62 before measurement and moves them to a position facing the sensor chip 1 during measurement. Thereby, the sample solution can be easily dropped onto the sensor chip 1.

(Basic cartridge configuration)
Next, the configuration of the cartridge 5 of the present embodiment will be described with reference to FIG. 2A is a cross-sectional view of the cartridge 5, FIG. 2B is a plan view of the cartridge 5, and FIG. 2C is a view showing the bottom surface of the cartridge 5.
The lid 45 of the cartridge 5 is provided with an opening at a position facing a Hall element array 9 area (described later) that is a magnetic field detection area of the sensor chip 1, and a fixture (see FIG. Not shown). A nonwoven fabric 46 impregnated with magnetic particles Mg is attached so as to close the opening. When analyzing the measurement object, the sample solution is dropped onto the region of the nonwoven fabric 46. Thereby, the magnetic particles in the nonwoven fabric 46 are dissolved in the sample solution and dispersed on the surface of the sensor chip 1. The details of the nonwoven fabric 46 impregnated with the magnetic particles Mg will be described later.

  A second molecular receptor that specifically binds to the measurement object is fixed on the surface of the sensor chip 1 via a fixed layer. This immobilization layer is a layer for immobilizing the second molecular receptor on the surface of the sensor chip. For example, when the second molecular receptor is an antibody, it is preferably formed by a silane coupling agent. By using a silane coupling agent, the surface of the fixing layer can be made hydrophobic, and the antibody can be fixed to the surface of the sensor chip 1 by hydrophobic bonding. The fixed layer may be formed by applying a spin coater in a wafer state before being divided into chips when the sensor chip 1 is manufactured, or the sensor chip 1 is set on the cartridge 5 and the electrode thereof is resin 44 as described later. You may apply | coat with a dispenser after sealing with.

The second molecular receptor is fixed by sealing the sensor chip 1 with the resin 44 and then applying a solution containing the second molecular receptor onto the sensor chip 1 in a state where the fixing layer is formed. . The second molecular receptor may be fixed only to the Hall element array 9 region on the sensor chip 1 or may be fixed to the entire region where the surface of the sensor chip 1 is exposed.
Further, electrodes (electrodes for supplying power to the sensor chip 1, electrodes for taking out signals from the sensor chip 1, and electrodes for sending signals to the sensor chip 1) are provided on the surface of the sensor chip 1. In addition, the metal wiring 43 is connected to an electrode provided on the inner bottom surface of the cartridge 5. Further, the electrode is connected to the electrode 42 on the bottom surface of the cartridge 5 (see FIG. 2C). The electrodes on the metal wiring 43 and the sensor chip 1 and the electrode inside the cartridge 5 are sealed with a resin 44 so as to prevent leakage even if a sample solution is introduced into the recess. The sealing of the resin 44 will be described later.

  The electrode 42 on the bottom surface of the cartridge 5 is connected to the electrode of the connector 65 of the analyzer body 6 so that power is supplied from the analyzer body 6 to the sensor chip 1 and between the sensor chip 1 and the analyzer body 6. Send and receive signals between them. Thus, by arranging the electrode 42 on the bottom surface of the cartridge 5, the cartridge 5 can be downsized as compared with the case where the electrode 42 is installed on the top surface while avoiding the space as a reaction tank.

Next, FIG. 3 shows (a) a cross-sectional view, (b) a plan view, and (c) a bottom view of the cartridge when no nonwoven fabric is used.
On the surface of the sensor chip 1, magnetic particles Mg are arranged in a lump state. The magnetic particles Mg are those obtained by immobilizing the second molecular receptor and then dipping it in a saccharide and a buffer solution onto the sensor chip 1 and freeze-drying it. When analyzing the measurement object, the sample solution is dropped into the opening of the lid 45, so that the mass of the magnetic particles Mg is melted and the magnetic particles Mg are dispersed on the sensor chip 1.
(Cartridge for measuring multiple types of measurement objects)

Next, another configuration example of the cartridge 5 will be described with reference to FIG.
In the cartridge 5 shown in FIG. 4A, two sensor chips 1 are arranged on the inner bottom surface, and one Hall element array 9 is arranged on each sensor chip. The electrodes on the sensor chip 1, the electrodes in the cartridge 5, and the wiring connecting them are sealed with a resin 44. Different two types of second molecular receptors are fixed to the two sensor chips 1, whereby two types of measurement objects can be measured simultaneously. When measuring two or more types of measurement objects, two or more sensor chips 1 are installed in accordance with the measurement object.

Further, in the cartridge 5 shown in FIG. 4B, Hall element arrays 9 are arranged at both ends of one sensor chip 1. The center electrode on the sensor chip 1, the electrode in the cartridge 5, and the wiring connecting them are sealed with resin 44. Two kinds of molecular receptors are fixed to the Hall element arrays 9 and 9 at both ends, respectively. When measuring two or more kinds of measurement objects, two or more Hall element arrays 9 are arranged on one sensor chip 1 in accordance therewith.
Further, a plurality of cartridges 5 as shown in FIG. 2 or FIG. 3 may be formed in one casing, and a plurality of types of measurement objects may be measured. In this case, it is necessary to dispense a sample solution containing a plurality of types of measurement objects into the respective reaction vessels.

(About magnetic particles)
Next, the magnetic particles Mg arranged in a state impregnated in a nonwoven fabric or freeze-dried on the surface of the sensor chip 1 will be described.
The magnetic particles Mg have a first molecular receptor on the surface. The magnetic particles Mg themselves may be those obtained by making the magnetic material itself into particles, or those obtained by impregnating a polymer such as polystyrene with the magnetic material.
When fixing the magnetic particles Mg to the nonwoven fabric 46, the nonwoven fabric 46 is impregnated with a solution obtained by immersing the magnetic particles Mg in a saccharide and a buffer solution and freeze-dried. The material of the nonwoven fabric 46 is not particularly limited, and examples thereof include cellulose, polyester, and glass.

When a non-woven fabric is not used as shown in FIG. 3, the magnetic particles fixed with molecular receptors are dripped onto the sensor chip 1 after being immersed in saccharides and a buffer solution.
As described above, when a plurality of types of measurement objects are measured, the first molecule acceptor corresponding to the measurement object is provided in the sensor chip 1 or Hall element array 9 region for each measurement object. Drop magnetic particles with the body fixed. In this case, a plurality of types of first molecular receptors may be fixed to one magnetic particle, or may be fixed to different magnetic particles for each type of first molecular receptor.

(Resin sealing)
FIG. 5 is a view for explaining sealing of the metal wiring 43 in the cartridge 5 of the present embodiment with resin, and shows a part of the inner bottom surface of the cartridge 5. FIG. 5 illustrates an example in which the Hall element array 9 is arranged on both sides of the sensor chip 1.
First, the metal wiring 43 connects the electrodes on the sensor chip 1 and the electrodes provided on the inner bottom surface of the cartridge 5. Next, wires 48 are provided on both sides of the region where the wiring 43 is provided. The wire 48 is provided so as to contact the surface of the sensor chip 1 from the inner bottom surface of the cartridge 5 to the inner bottom surface on the opposite side across the sensor chip 1 so as to cover the entire area where the wiring is provided. The metal wiring 43 and the wire 48 may be the same wire material such as gold or aluminum. Next, resin 44 is applied to seal the electrodes and metal wiring 43. When the resin 44 is applied, it flows on the surface of the sensor chip 1, but is blocked by the wire 48.
Thus, by providing the wire 48, the spread of the resin 44 can be prevented, and the resin 44 can be prevented from flowing on the Hall element array 9. There is no need to limit the material of the resin 44 as long as the insulation of the metal wiring 43 can be maintained, and an epoxy resin, a silicone resin, or the like is used.

(About sensor chip configuration)
Next, the configuration of the sensor chip 1 will be described. FIG. 6 is a schematic view showing a part of the sensor chip 1 according to this embodiment, and FIG. 7 is a block diagram of the sensor chip 1.
The sensor chip 1 includes a Hall element array 9 in which Hall elements 2 that are magnetic field detection elements are arranged in a plurality of arrays, a memory circuit 82 that stores information on an object to be measured in advance, an arbitrary Hall element 2 and A selection circuit 71 that selects a memory element constituting the memory circuit 82 and an amplification circuit 81 that amplifies a signal from the Hall element 2 are provided.
As will be described in detail later, the Hall element 2 outputs a voltage proportional to the magnetic flux density when a magnetic flux perpendicular to the magnetosensitive surface (described later) is applied from the outside.

The selection circuit 71 selects a designated Hall element based on an address signal for selecting a specific Hall element 2 from the control device 64 as described later. Then, the output voltage of the selected Hall element is output to the amplifier circuit 81. A memory element can also be selected based on an address signal from the control device 64.
The amplifier circuit 81 amplifies the detection signal of the Hall element 2 and outputs it to the control device 64. For example, a differential amplifier circuit is provided.

The memory circuit 82 holds information on the measurement object in a non-volatile manner, and outputs information held inside in response to a request from the control device 64. This measurement object information is the ID of the measurement object in this embodiment.
Such a sensor chip 1 is formed on the silicon substrate 11 by a CMOS (complementary mental-oxide semiconductor device) manufacturing process which is a well-known technique. Hall elements are formed below the recesses 13 on the surface of the sensor chip 1, and input and output of the individual hall elements are performed via the gate electrode 30 and the metal wiring 37. The outermost surface is covered with a silicon nitride film or a silicon oxide film formed by plasma CVD (chemical vapor deposition).

(About Hall element)
Next, the Hall element 2 of the present embodiment formed as described above will be described with reference to FIG. 8A is a top view of the Hall element 2, FIG. 8B is a cross-sectional view taken along the alternate long and short dash line a, and FIG. 8C is a cross section taken along the alternate long and short dash line b.
The Hall element 2 includes a gate electrode 30, a source electrode 31, a drain electrode 32, output electrodes 33 and 34, and an insulating layer 35, and is formed in a P well region 36. Except for the output electrodes 33 and 34, the configuration is the same as that of the n-type MOSFET, and the metal wiring to each electrode is omitted in the drawing. The output electrodes 33 and 34 are formed such that current flows perpendicularly to the magnetic flux formed substantially perpendicular to the surface of the sensor chip 1 and the current flowing between the source and drain electrodes.

  Next, the operation of the Hall element 2 will be described. A bias is applied to the gate electrode 30, the source electrode 31, and the drain electrode 32 to set the operation state similar to that of the MOSFET. It is desirable that the operation state at this time be in a linear region. In this state, when there is no magnetic flux applied from the outside, the two output electrodes 33 and 34 are at the same potential. On the other hand, when a magnetic flux perpendicular to the Hall element surface is applied from the outside, a voltage proportional to the magnetic flux density appears as a differential voltage between the output electrodes 33 and 34, and this differential voltage is amplified by an amplifier circuit as will be described later. 81.

  Although the illustration of the metal wiring layer is omitted in FIG. 8, FIG. 9 shows the metal wiring layer 37 and the insulating film 12 with respect to the same cross section as in FIG. 8C. That is, in the sensor chip 1 of FIG. 9, two metal wiring layers 37a and 37b are formed. Among these, a silicon oxide film 12b as an insulating film formed on the first metal wiring layer 37b is formed. Compared to other regions, the region is thinner at the portion facing the gate electrode 30 in the stacking direction. This can be formed as follows. First, the first metal wiring layer 37b, the silicon oxide film 12b, and the second metal wiring layer 37a are formed in this order, and then the portion of the metal wiring layer 37a facing the gate electrode 30 in the stacking direction is removed. Next, a silicon nitride film 12a is formed as an insulating film of the second metal wiring layer 37a. As a result, different types of insulating films 12, that is, the silicon oxide film 12b and the silicon nitride film 12a are continuously stacked on the portion facing the gate electrode 30. After the formation of the silicon nitride film 12a, a portion of the silicon nitride film 12a that faces the gate electrode 30 in the stacking direction is formed with a bonding pad forming portion for electrically connecting the sensor chip 1 and the outside. At the same time, it is removed by dry etching. At this time, the silicon nitride film 12a is removed at the bonding pad forming portion to expose the metal wiring layer 37a, but the silicon oxide film 12b is slightly removed at the portion facing the gate electrode 30. By adjusting the formation conditions of the insulating film and setting the conditions such that the etching rate of the silicon nitride film 12a is higher, it is possible to reliably etch the silicon oxide film and expose the underlying metal wiring layer. Can be prevented.

Thus, by reducing the distance between the magnetosensitive surface of the Hall element (the interface between the gate electrode 30 and the P well region 36) and the surface of the sensor chip 1, the sensitivity of the Hall element becomes excellent.
In this embodiment, the first and second metal wiring layers 37a and 37b are not provided in the portion facing the gate electrode 30, but the first metal wiring layer 37b may be provided. However, if the uppermost metal wiring layer (in this embodiment, the second metal wiring layer 37a) is provided, the distance between the magnetic sensing surface of the Hall element and the surface of the sensor chip 1 becomes longer, and the sensitivity deteriorates. It is not preferable.

  Although not shown in the figure, it is desirable that the uppermost metal wiring layer is not provided for the wiring in the Hall element array region (a region in which the Hall element regions and the regions between the Hall elements are combined). Thereby, the silicon nitride film 12a in the entire Hall element array region can be removed. If the uppermost metal wiring is present in the Hall element array region, only the silicon nitride film on each Hall element must be removed, and etching with higher accuracy must be performed.

(Hall element array arrangement, selection method of each Hall element by selection circuit)
Next, a method of selecting each Hall element arranged in an array by using a selection circuit and taking out an output will be described with reference to FIG.
A source electrode, a drain electrode, and a pair of output electrodes of each Hall element (E (0,0), E (0,1),...) Are connected to V via a switch (R0, R1,...). _L , V _H , OUT1, and OUT2 are connected to the same column in the column direction Y in common. The gate electrodes in the same row in the row direction X are also common, and are connected to common gate electrode lines C0, C1,. V _L and V _H are wirings for supplying a bias to the Hall element side, and OUT 1 and OUT 2 are wirings for sending an output from the Hall element to the amplifier circuit 81.

  A case where the Hall element E (0, 0) is selected will be described. Only the switch R0 is turned on, and the switches R1, R2,. Further, only the gate electrode line C0 is set to a voltage at which the Hall element is activated, and the gate electrode lines C1, C2,... -Set so that no current flows between the drains.

At this time, V _L and V _H are applied to the Hall element E (0, 0) and the source electrode and drain electrode of the Hall element in the same column, but only the Hall element E (0, 0) flows. . A voltage corresponding to the magnetic flux density appears at the output electrode of the Hall element E (0, 0). Since the output electrodes of the Hall elements arranged in the row direction are not in an operating state, the output voltage of the Hall element E (0, 0) is output as it is to OUT1 and OUT2. In this configuration, even if the number of arrays increases, the number of wires in the array is the same and only switches are added to the ends, so the area of the sensor chip is almost proportional to the number of arrays and easily has a large number of Hall elements A sensor chip can be configured.

(About measurement program)
Next, the operation of the biosensor according to the program according to the present embodiment will be described.
First, the controller 64 detects that the cartridge 5 has been inserted into the connector 65. At this time, the sample solution containing the measurement object is introduced into the cartridge 5 by the operator.
Next, when it is detected that the cartridge 5 has been inserted into the analyzer body 6, the operation of the sensor chip is checked. At this time, if it does not operate normally, an error message is output to the display device 63. In this case, the operator again drops the sample containing the measurement object into the opening of another cartridge 5 and inserts it into the analyzer body 6.

In the case of normal operation, the process proceeds to the next step, the ID information of the measurement object is acquired from the memory circuit built in the sensor chip 1, and the process proceeds to the next step.
Next, based on the acquired ID information, the inspection conditions held in the storage device of the analyzer body 6 are extracted in advance, and the inspection conditions are set. From the next step onward, measurement is performed according to the inspection conditions. Here, examples of the inspection condition include a reaction time, a magnetic field strength in magnetic cleaning described later, a cleaning time, a measured value, and a calibration curve of the measurement object. As described above, the information to be mounted on the cartridge 5 is only the ID information, so that the cartridge can be configured at low cost. Note that the ID information of the measurement object may be input to the analyzer body 6 by the operator before measurement.

Next, a predetermined reaction time is waited based on the set inspection conditions. At this time, the magnetic particles held in the freeze-dried state are melted and dispersed and settled on the sensor chip, and a part of the particles is bonded to the surface of the sensor chip via the measurement object. After the reaction time has passed, the process proceeds to the next step.
Next, by sending a command to the magnetic field generator, a weak magnetic field that is insufficient to pull up the magnetic particles that are present on the sensor chip and are not bonded via the measurement object is applied, The dispersion state of the magnetic particles on the surface of the sensor chip 1 is measured by acquiring the output signal from the sensor chip 1 at this time.

Next, a predetermined magnetic field for pulling up the magnetic particles that are not bonded via the measurement object is applied for a predetermined time. At this time, the magnetic particles bonded through the measurement object remain on the sensor chip surface as they are, and the magnetic particles that are not bonded to the sensor chip surface without passing through the measurement object. Body particles are pulled upwards.
Next, measurement is performed by applying a predetermined magnetic field for measurement so as not to drop the pulled magnetic particles and obtaining each Hall element output at this time, and the result is stored in a storage device inside the control device 64. Save to. After the measurement is completed, signal processing is performed, the analysis result is output to the display device 63, and the process ends.

The present invention will be described based on examples.
[Example 1]
(Sensor chip)
The sensor chip 1 was produced by a 0.6 μm CMOS process. Hall element 2 having a magnetosensitive surface size of about 5 μm square is arranged as an array of 16 × 16 at a pitch of about 15 μm, and a selection circuit 71 for Hall element 2 and an amplifier circuit 81 for amplifying a signal from Hall element 2 are provided. Arranged. The number of layers of the metal wiring (Al wiring) is two, and the area of the Hall element array 9 is not the upper Al wiring but only the lower Al wiring. Further, the insulating film on the upper Al wiring was a silicon nitride film, and this silicon nitride film was removed from the region of the Hall element array 9.

(Preparation of antibody)
Two monoclonal antibodies against ribosomal protein L7 / L12 derived from Haemophilus influenzae were prepared according to the method described in WO00 / 06603. Of these, one was fixed to the sensor chip 1 (hereinafter referred to as primary antibody) and the other was fixed to the magnetic particles (hereinafter referred to as secondary antibody).

(Immobilization of primary antibody to the surface of sensor chip 1)
The surface of the sensor chip 1 is coated with aminoalkyl functional T-structure polydimethylsiloxane (manufactured by United Chemical Technologies) as a hydrophobic coating agent, and 0.1 M MES (2- (N-morpholine) on the surface of the sensor chip 1. The primary antibody prepared at a concentration of 10 μg / ml at room temperature was allowed to stand under humid conditions at room temperature. After 5 hours, the surface was rinsed with PBS-T (Phosphate Buffered Saline, pH 7.4, with 0.05% Tween 20 (Tween is a registered trademark, polyoxyethylene sorbitan monochlorate)), and then the storage solution 1 (5% sucrose, 0%). 1% Alkali-treated casein (Clinica Chimica Acta, 1982, 123, 193-198), 0.1% polyvinylcohol, 0.1M MOSPO (3- (N-morpholino) -2-hydroxypronesulfuric acid), pH 7 After covering and allowing to stand at room temperature for 5 minutes, water on the surface was removed with water-absorbing paper and dried.

(Immobilization of secondary antibody to magnetic particles)
A secondary antibody purified by an antibody purification kit using Protein G (MabTrap Kit, manufactured by Amersham Biosciences) was prepared to 9.3 mg / ml with PBS (Phosphate Buffered Saline, pH 7.4). To 108 μl of this secondary antibody solution, 3.3 μl of 20 mM Biotin-PEG-NHS reagent aqueous solution (Nektar Therapeutics, MW 3400) was added and reacted at room temperature for 2 hours. After the reaction, the mixture was concentrated with a centrifugal ultrafiltration membrane filter unit (Ultrafree-CL, manufactured by Millipore Corporation) having a molecular weight cut off of 30,000, and washed by repeating dilution and concentration three times with PBS. When the biotin labeling rate of the obtained Biotin-PEG labeled secondary antibody was quantified with a biotin quantitative reagent in EZ-Link Sulfo-NHS-Biotinylation Kit (PIERCE), the number of biotin labels per molecule of the antibody was 2.8 molecules. Met.

  Next, 33 μl of 3 mg / ml Biotin-PEG-labeled secondary antibody solution was added to 100 μl of Dynabeads MyOne Streptavidin (Dynabeads is a registered trademark, manufactured by Dynal Biotech) to 1% (w / v) with PBS. Furthermore, after making the final volume 500 μl with 367 μl of PBS, the reaction was carried out with stirring for 1 hour at room temperature. After the reaction, only the secondary antibody immobilized with magnetic particles was collected using a magnet (Dynamic Magnetic Particle Concentrators), and the supernatant was removed. 1 ml of PBS is added to the recovered secondary antibody immobilized with magnetic particles, and washing and recovery are performed in the same manner, and finally, storage solution 2 (10% sucrose, 0.25% Alkaline-treated casein (Clinica) Chimica Acta, 1982, 123, 193-198), 0.5% polyvinyl alcohol, 50 mM MES (2- (N-morpholino) ethanolsulfonic acid), pH 6.0 to 2% (w / v) concentration.

(Impregnation of non-woven fabric with secondary particles fixed with magnetic particles)
A magnetic particle-immobilized secondary antibody prepared at a concentration of 2% (w / v) is 0.5% in 10 mM MES (2- (N-morpholino) etheresulphonic acid, pH 6.0) containing 3% trehalose. The concentration was adjusted to w / v), and 1.5 μl was impregnated into a non-woven fabric (BEMCOT, manufactured by Asahi Kasei Co., Ltd.) attached to the opening of the cartridge lid, and then freeze-dried. For freeze-drying, the nonwoven fabric is put into a polypropylene container, the whole container is cooled with hexane-dry ice to freeze the solution, and then dried for 16 hours with a freeze-dryer (FD-1000, manufactured by Tokyo Rika Kikai Co., Ltd.). I let you.

(About production of cartridge)
The sensor chip 1 was fixed to the PCB substrate, and the electrode on the sensor chip 1 and the electrode on the upper surface of the PCB substrate were connected by an Al wire. The electrode and the Al wire were sealed with an epoxy resin. This is inserted into a case formed of ABS resin, and the above-mentioned primary antibody is fixed to the surface of the sensor chip 1, and then the nonwoven fabric 46 impregnated with magnetic particles Mg as shown in FIG. It installed in the position which opposes.

(About preparation of measurement sample)
Haemophilus influenzae ATCC10211 was cultured at 37 ° C. for 24 hours using chocolate agar medium (Poremedia, manufactured by Eiken Chemical Co., Ltd.). After culturing, the cells were collected with physiological saline and stored at 4 ° C., a part of the collected solution was diluted, and the number of viable bacteria was counted by the same culturing. To 10 μl of each sample diluted to an appropriate viable cell count with physiological saline, an extract (1% Triton X-100 (Triton is a registered trademark, t-octylphenoxypolyethylethanol), 20 mM dithiothreitol, 0.1 M MES (2- (N-morpholino) ethansulfonic acid), pH 6.0) was added, and then diluted solution (30% Fetal Calf Serum, 6% Alkaline-treated casein (Clinica Chimica Acta, 1982, 123, 1983)). Diluted 2-fold with 2M MOPSO (3- (N-morpholino) -2-hydroxysulfonic acid), pH 7.5) Things were a measurement sample. The viable count display for each measurement sample represents the final value after dilution.

(About measurement and measurement results)
A cartridge lid with a non-woven fabric dried by freeze-drying was set on the cartridge body on which the primary antibody was fixed and dried to complete the measurement cartridge. A measurement sample diluted to the above viable count was dropped onto the completed cartridge and inserted into the analyzer body for measurement. A measurement sequence is shown in FIG. By allowing to stand for 2 minutes after insertion, the magnetic particles were eluted from the nonwoven fabric, and the reaction between the measurement object, the primary antibody, and the secondary antibody was performed (step S2). After standing, unbound magnetic particles were pulled up by an AC magnetic field (alternating magnetic field) of 12 mT for 5 seconds and a frequency of 625 Hz (step S3). Thereafter, AC signals of 16 × 16 Hall elements were measured in an AC magnetic field of 12 mT and a frequency of 625 Hz, that is, a weak magnetic field (step S4). Next, a DC magnetic field (DC magnetic field) of 50 mT, that is, a strong magnetic field that saturates the magnetization of the magnetic particles was applied to an AC magnetic field of 12 mT and a frequency of 625 Hz, and the AC signal was measured (step S5). As the signal processing in step S6, the deviation value (average deviation) of 16 × 16 AC signals in the AC magnetic field (ratio of the change amount of the AC component of the output of each Hall element to the minute change of the AC magnetic field component) and The difference between the deviation values of 16 × 16 AC signals in the AC + DC magnetic field was calculated, and the result was output in step S7. After standing for 2 minutes (step S2), the time until the measurement result was output was about 1 minute. The measurement results are shown in FIG. The negative object, that is, the sample that does not contain viable bacteria, showed a difference in deviation that was nearly zero. The difference in deviation increased as the concentration of viable bacteria increased. From this result, it was confirmed that it was possible to carry out in 3 minutes from dropping of the sample to output of the measurement result, and it was possible to quantify a very small amount of viable bacteria of 1.7 × 10 ⁵ CFU / ml.

It is a figure which shows schematic structure of the biosensor which concerns on this embodiment. It is a figure explaining the structure of the cartridge of this embodiment (when a nonwoven fabric is used). It is a figure explaining the structure of the cartridge of this embodiment (when a nonwoven fabric is not used). The configuration of the cartridge according to this embodiment is not described (when a plurality of magnetic sensors are provided). It is a figure explaining sealing with resin of metal wiring. It is the schematic which shows a part of sensor chip concerning this embodiment. It is a block diagram of a sensor chip. It is a figure explaining the structure of a Hall element. It is a figure explaining the formation state of a metal wiring layer and an insulating film. It is a figure explaining the method of selecting each Hall element and taking out an output. It is a figure which shows the measurement sequence of an Example. It is a graph which shows the result of an Example. It is a schematic diagram of a solid phase analysis using magnetic particles.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sensor chip 2 Hall element 9 Hall element array 11 Silicon substrate 12 Insulating film 12a Silicon nitride film 12b Silicon oxide film 13 Recess 30 Gate electrode 31 Source electrode 32 Drain electrode 33, 34 Output electrode 35 Insulating layer 36 Well region 37 Metal wiring layer 37a, 37b Metal wiring layer 38 Metal plug 42 Electrode 43 Metal wiring 44 Resin 45 Lid 46 Nonwoven fabric 48 Wire 5 Cartridge 6 Analyzer body 61 Coil 62 Magnetic core 63 Display device 64 Controller 65 Connector 91 Solid phase 93 First molecule Receptor 94 Object to be measured

Claims (20)

In a biosensor that analyzes a measurement object by measuring the amount of magnetic particles,
Magnetic particles disposed on the reaction vessel and having a first molecular receptor on the surface thereof that specifically binds to the measurement object;
A reaction vessel for holding a solution containing the measurement object;
A sensor chip installed in the reaction vessel and measuring the amount of the magnetic particles;
With
The sensor chip includes a magnetic sensor in which a plurality of magnetic field detection elements that output an output value corresponding to the detected magnetic field strength are two-dimensionally arranged, a selection circuit that selects each of the magnetic field detection elements, A biosensor comprising: an amplification circuit that amplifies an output signal of the magnetic sensor; and a second molecular receptor formed on a surface of the magnetic sensor that specifically binds to the measurement object.   The biosensor according to claim 1, further comprising a magnetic field generator at a position facing the sensor chip.   The biosensor according to claim 1 or 2, wherein the magnetic particles are covered with a dried saccharide and a buffer.   The biosensor according to any one of claims 1 to 3, wherein the magnetic particles are arranged in the reaction tank in a state of being dispersed and fixed on a nonwoven fabric.   The biosensor according to claim 4, wherein the nonwoven fabric is disposed at a position facing the magnetic sensor.   The biosensor according to any one of claims 1 to 5, further comprising a vibration generator that vibrates the reaction vessel.   An electrode for supplying power to the sensor chip, an electrode for sending a signal to the selection circuit, and an electrode for taking out an output signal from the amplification circuit are formed on the bottom surface or side surface of the reaction tank. The biosensor according to claim 1, wherein the biosensor is characterized in that:   The biosensor according to claim 7, wherein the electrode formed on the bottom surface of the reaction vessel is connectable with a smart card connector.   The biosensor according to any one of claims 1 to 8, wherein the sensor chip includes a memory element in which information on the measurement object is stored in advance.   The biosensor according to any one of claims 1 to 9, wherein the reaction tank includes a plurality of the magnetic sensors.   The biosensor according to claim 10, wherein different second molecular receptors are formed on the surfaces of the plurality of magnetic sensors.   The biosensor according to any one of claims 1 to 11, wherein the magnetic field detection element is a semiconductor Hall element, and a sensor substrate constituting the sensor chip is a silicon substrate.   Two or more insulating films made of different materials are formed on the surface of the sensor substrate constituting the sensor chip, and at least one insulating film of the insulating films is stacked with the magnetic sensor provided on the sensor substrate. The biosensor according to any one of claims 1 to 12, wherein a portion at a position facing each other is removed.   A silicon oxide film and a silicon nitride film formed on the silicon oxide film are provided as the two or more insulating films, and the silicon nitride film portion at a position facing the magnetic sensor in the stacking direction is removed. The biosensor according to claim 13, wherein the biosensor is used.   A first insulating film having a high removal rate is stacked on a surface of a sensor substrate constituting the sensor chip with a predetermined layer sandwiched between a second insulating film having a slower removal rate and the first layer. 15. The insulating film and the predetermined layer are removed from the magnetic sensor provided on the sensor substrate at a position facing the magnetic sensor in the stacking direction. Biosensor.   A plurality of metal wiring layers are formed on the surface of the sensor substrate constituting the sensor chip, and the metal wiring layer formed in the uppermost layer in the stacking direction of the metal wiring layers includes the magnetic sensor installed on the sensor substrate. The biosensor according to any one of claims 1 to 15, wherein portions at positions facing each other in the stacking direction are removed.   17. The sensor chip according to claim 1, wherein an electrode for electrical connection with the outside is provided in a predetermined area, and a wire separating the area and the magnetic sensor is provided on the surface. The biosensor described. A measurement method of an object to be measured using the biosensor according to any one of claims 2 to 17,
Dissolving the magnetic particles in a solution containing the measurement object introduced onto the sensor chip;
Binding the magnetic particles to the magnetic sensor surface via the measurement object;
The magnetic particles floating in the solution without being bound and the magnetic particles bound to the surface of the magnetic sensor without passing through the measurement object are moved away from the surface of the magnetic sensor by the magnetic field generator, and then measured. Identifying the amount of magnetic particles bound to the surface of the magnetic sensor through the object;
Identifying the amount of the measurement object based on the amount of the magnetic particles,
An object measuring method characterized in that these steps are automatically performed continuously based on a preset program. A cartridge used for the biosensor according to any one of claims 1 to 17,
A biosensor cartridge, wherein a reaction vessel including the magnetic particles and the sensor chip is formed in a housing that can be inserted into and removed from an apparatus main body. A nonwoven fabric used for the biosensor according to any one of claims 1 to 17,
A non-woven fabric, wherein magnetic particles having the first molecular receptor on the surface are dispersed and fixed.

Images