JP7401871B2 - Biological particle collection device - Google Patents

Description

The present invention relates to a biological particle collection device.

There are various types of cells, which are one type of biological particles, and there are multiple evaluation and analysis methods for each type. Especially for rare cells such as circulating cancer cells that exist in the blood, evaluation and analysis performed at the single cell level have been attracting attention in recent years. Diagnosis of various types of cancer cells is important for preliminary diagnosis and postoperative diagnosis.

Conventionally, in order to identify cancer cells, immunostaining methods such as enzyme-linked immunosorbent assay (ELIZA method) have been widely used. However, in the immunostaining method, when increasing the number of times the reagent is changed during staining, the cells are often separated using a centrifuge each time, and rare cells are often lost. Therefore, for example, an evaluation analysis method performed at a single cell level using an apparatus as disclosed in Non-Patent Document 1 has become widespread.

Here, a conventional example of an apparatus for performing the above-mentioned evaluation analysis method, particularly immunostaining, is shown in FIG. The diagrams indicated by reference numerals 601 to 603 in FIG. 10 are schematic diagrams showing how cells in a solvent are once held in a depression and then separated from the depression in the apparatus disclosed in Non-Patent Document 1. The figure indicated by reference numeral 604 is a top view of the device disclosed in Non-Patent Document 1.

The apparatus 200 shown in FIG. 10 is an apparatus for efficiently immunostaining cells. As shown in the diagram indicated by reference numeral 601 in FIG. 10, in the device 200, an electrode 101 is arranged on a glass substrate 100, and a layer 104 formed by coating the glass substrate with resin or the like is arranged on the electrode 101.

The electrode 101 is a transparent electrode represented by, for example, ITO made of a mixture of indium oxide and tin oxide. A recess 102 is formed in the layer 104 and is large enough to hold cells 106 (see the figures 602 and 603 in FIG. 10) to be evaluated and analyzed. Further, in the downstream region in the flow direction in the peripheral region of the depression 102 of the layer 104, a microwall 103 is formed to physically stop the cells 106 that are separated from the depression 102.

Further, in the device 200, a microfluidic channel 110 made of dimethylpolysiloxane resin (hereinafter referred to as PDMS) is adhered to the substrate 100. As a result, a space is formed between the substrate 100 and the microfluidic channel 110, and it becomes possible to flow a liquid such as the solvent 105 (see the figures indicated by reference numerals 602 and 603 in FIG. 10) into the space.

Next, a series of processes from collecting biological particles to replacing the solvent will be explained. As shown in the diagram 602 in FIG. 10, first, a single cell 106 is flowed into the space formed in the device 200 together with a solvent 105 having low conductivity, and the dielectrophoretic force F _DEP generated at the electrode 101 is The cells 106 are collected in the depression 102 by acting on the cells 106.

Thereafter, as shown in the diagram 603 in FIG. 10, a reagent 107 used for immunostaining is caused to flow into the space formed in the apparatus 200, thereby replacing the solvent 105 with the reagent 107. Many of the reagents 107 used for immunostaining have high electrical conductivity. Here, if the solvent 105 is replaced with the reagent 107, the dielectrophoretic force F _DEP acting on the cells 106 will be weakened, and the cells 106 will move away from the depressions 102. However, since the cells 106 are physically stopped by the microwall 103 formed on the downstream side of the depression 102, it is possible to prevent the cells 106 from leaving the depression 102.

M. Takeuchi, et.al., “Highly efficient trapping and analysis of rare cells using an electroactive microwell array with barriers”, Proceeding of 21st International conference on Miniaturized Systems for Chemistry and Life Sciences (microTAS)2017, pp880-881.

However, in the device 200 disclosed in Non-Patent Document 1, the distance between the side surface of the depression 102 and the wall surface of the microwall 103 on the upstream side of the flow of cells 106 is large. Therefore, when the cells 106 held in the depressions 102 come into contact with the solvent 107 flowing from upstream and leave the depressions 102, there is a problem that it becomes difficult for the cells 106 to return to the depressions 102. Specifically, there was a problem in that after the cells 106 separated from the depressions 102 were stopped by the microwalls 103, they remained in the space formed between the depressions 102 and the microwalls 103.

Further, in the device 200 disclosed in Non-Patent Document 1, although the electrode 101 is formed on the glass substrate 100, the electrode 101 cannot have a laminated wiring structure. Therefore, the device 200 also has a problem in that the plurality of electrodes 101 cannot be arranged in an array on the glass substrate 100. Furthermore, the device 200 also has a problem in that the operation of the electrode 101 cannot be controlled for each of the plurality of depressions 102.

One aspect of the present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a biological body that can stably hold biological particles in a cavity and make it easier to observe biological particles. An object of the present invention is to provide a particle collection device.

In order to solve the above problems, a biological particle collection device according to one aspect of the present invention applies a dielectrophoretic force to biological particles in a solvent embedded in a semiconductor substrate and flowing onto the semiconductor substrate. and a first protective film formed on the semiconductor substrate to prevent the semiconductor substrate from coming into contact with the solvent, and the first protective film includes: , a recess for holding the biological particles to which the dielectrophoretic force has been applied is formed at a location corresponding to the arrangement position of the electrode on the first protective film; A first wall is formed for holding the bioparticles to which a dielectrophoretic force is applied in the recess, and a side surface of the recess is in contact with a wall surface portion of the first wall on the recess side, The first wall is composed of a first wiring layer formed on the first protective film, and the first wiring layer and the second wiring layer including the electrode are each made of copper or aluminum, A second protective film is formed on the bottom surface of the depression and on the surface of the first wall to prevent the first wiring layer and the second wiring layer from coming into contact with the solvent.

According to one aspect of the present invention, it is possible to realize a bioparticle collection device that can stably hold bioparticles in recesses and easily observe bioparticles.

A diagram designated by reference numeral 511 is a top view of the biological particle collection device according to Embodiment 1 of the present invention, showing an example of the arrangement of electrodes, depressions, and first walls formed on a semiconductor substrate. The figure indicated by the reference numeral 512 is a sectional view taken along the line A-A' in the figure indicated by the reference numeral 511. The figures indicated by reference numerals 513 and 514 are top views of the biological particle collection device according to Embodiment 1 of the present invention, showing other examples of the arrangement of electrodes, depressions, and first walls formed on a semiconductor substrate. It is. The figure designated by reference numeral 521 is a top view of the biological particle collection device according to Embodiment 1 of the present invention, showing another example of the arrangement of electrodes, depressions, and first walls formed on a semiconductor substrate. The figure indicated by the reference numeral 522 is a sectional view taken along the line A-A' in the figure indicated by the reference numeral 521. The figures indicated by reference numerals 531 to 536 are schematic views showing an example of the formation process of each layer formed above the semiconductor substrate. Diagrams 541 to 545 are schematic diagrams showing another example of the formation process of each layer formed above the semiconductor substrate. The figures indicated by reference numerals 551 and 552 are top views of the biological particle collection device according to Embodiment 1 of the present invention. The diagrams indicated by reference numerals 561 and 602 are schematic diagrams showing the flow up to replacing the solvent with the reagent used for immunostaining. FIG. 2 is a cross-sectional view showing the configuration of a biological particle collection device according to Embodiment 2 of the present invention. The figure designated by reference numeral 581 is a top view of the biological particle collection device according to Embodiment 3 of the present invention. The figure indicated by reference numeral 582 is a sectional view taken along the line B-B' in the figure indicated by reference numeral 581. The figure indicated by reference numeral 583 is a top view of the biological particle collection device according to Embodiment 3 of the present invention. The figure indicated by reference numeral 584 is a vertical cross-sectional view taken along the line CC' in the figure indicated by reference numeral 583. Diagrams 591 to 593 are schematic diagrams showing the flow from replacing the solvent with the reagent used for immunostaining to confining cells. The figures indicated by reference numerals 601 to 603 are schematic diagrams showing how cells in a solvent are once held in a depression and then separated from the depression in the apparatus disclosed in Non-Patent Document 1. The diagram indicated by 604 is a top view of the device disclosed in Non-Patent Document 1.

[Embodiment 1]
The biological particle collection device 1a according to this embodiment will be described below based on FIGS. 1 to 6. In this specification, a "biological particle collection device" is a device that collects cells 15 in the solvent 34 flowing onto the semiconductor substrate 10.

First, the configuration of a biological particle collection device 1a according to this embodiment will be explained using FIG. 1. A diagram indicated by reference numeral 511 in FIG. 1 is a top view of the biological particle collection device 1a, showing an example of the arrangement of the electrode 11, the recess 12, and the first wall 13 formed on the semiconductor substrate 10. The diagram indicated by reference numeral 512 in FIG. 1 is a longitudinal sectional view taken along the line A-A' in the diagram indicated by reference numeral 511 in FIG. The figures indicated by reference numerals 513 and 514 in FIG. 1 are top views of the biological particle collection device 1a, showing other examples of the arrangement of the electrodes 11, the recesses 12, and the first walls 13 formed on the semiconductor substrate 10. It is.

As shown in the diagram 512 in FIG. 1, the biological particle collection device 1a includes an electrode 11, a semiconductor substrate 10, a first protective film 16, a second protective film 18, a first wall 13, a recess 12, and a microfluidic channel. It is equipped with 30. The electrode 11 outputs a signal for causing a dielectrophoretic force F _DEP to act on cells (biological particles) 15 (details will be described later; see FIG. 6, etc.) in the solvent flowing onto the semiconductor substrate 10. The electrode 11 can be formed of aluminum, copper, or the like, which is a material for forming a wiring layer in the semiconductor substrate 10 . The electrode 11 may have a plurality of electrodes 11 formed therein, as shown in the diagram 511 in FIG. 1, or a single electrode 11, as shown in the diagram 514 in FIG. It's okay.

The first protective film 16 prevents the semiconductor substrate 10 from coming into contact with the solvent. The second protective film 18 prevents the electrode 11 from coming into contact with the solvent 34 . This is because wiring layers such as the electrodes 11 are formed of copper, aluminum, or the like, so that if a liquid such as the solvent 34 comes into direct contact with the electrodes 11, corrosion or the like will occur. The first protective film 16 is made of silicon dioxide (SiO ₂ ), silicon nitride (Si ₃ N ₄ ), or the like. The first protective film 16 is preferably formed of silicon dioxide because it is advantageous for bonding with the microfluidic channel 30 formed of dimethylpolysiloxane (PDMS) or the like.

The thickness of the second protective film 18 is preferably thinner than the thickness of the first protective film 16. This is because the thinner the second protective film 18 is, the greater the dielectrophoretic force F _DEP applied from the electrode 11 to the cell 15 (details will be described later; see the diagram indicated by reference numeral 561 in FIG. 6). The thickness of the second protective film 18 is more preferably, for example, 1 μm or less.

If the first protective film 16 is formed as is, the dielectrophoretic force F _DEP applied to the cells 15 is reduced. On the other hand, by forming the second protective film 18, the dielectrophoretic force F _DEP applied to the cells 15 becomes smaller than when the second protective film 18 is not formed, but it does not prevent electric leakage or corrosion of the electrode 11. I can do it. Furthermore, by forming the second protective film 18 as thin as the above-mentioned thickness, it is possible to prevent the dielectrophoretic force F _DEP acting on the cells 15 from becoming small as much as possible.

The first wall 13 allows the cell 15 on which the dielectrophoretic force F _DEP is applied to be held in the depression 12 . The shape of the first wall 13 is not particularly limited, and for example, the first wall 13 is formed in a shape (e.g., an arch shape) along the outer peripheral wall of the depression 12, as shown in the diagram indicated by the reference numeral 511 in FIG. be done. Furthermore, the first wall 13 may be formed in a polygonal shape, as shown in the diagram indicated by reference numeral 513 in FIG. In this case, when the shape of the depression 12 is formed into a circular shape, the outer shape of the depression 12 and a part of the first wall 13 are formed on a straight line.

The first wall 13 is preferably formed of a photosensitive resin material. This is because when vertical etching is performed on a photosensitive resin material, it is easy to etch the photosensitive resin material in a substantially straight line.

The depression 12 holds the cell 15 on which the dielectrophoretic force F _DEP has been applied. The depression 12 is formed by etching the surface of the semiconductor substrate 10, for example. The shape of the depression 12 may be square, but since many biological particles such as cells 15 are spherical, it is desirable that the shape of the depression 12 is circular. Further, the depressions 12 have a particle size that is approximately the same as the maximum diameter length of the cells 15 .

In addition, as shown in the diagrams indicated by reference numerals 511, 513, and 514 in FIG. It is formed.

Next, the configuration of a biological particle collection device 1b having another configuration according to this embodiment will be explained using FIG. 2. The diagram indicated by reference numeral 521 in FIG. 2 illustrates a case where the electrode 11 formed on the semiconductor substrate 10 is formed of a wiring layer formed at the lower layer among the wiring layers formed on the semiconductor substrate 10. , is a top view of the biological particle collection device 1b. Specifically, an example of the arrangement of the depression 12 and the first wall 131 is shown. The diagram indicated by reference numeral 522 in FIG. 2 is a longitudinal sectional view taken along the line A-A' in the diagram indicated by reference numeral 521 in FIG.

As shown by reference numeral 522 in FIG. 2, the biological particle collection device 1b includes an electrode 11, a semiconductor substrate 10, a first protective film 16, a second protective film 18, a first wall 131, a depression 12, and a microfluidic channel 30. It is equipped with The first wall 131 has the same shape as the first wall 13 of the biological particle collection device 1a. Note that the shape of the first wall 131 may be the same as the shape of the first wall 13 in the drawing indicated by the reference numeral 513 in FIG. 1, or the same shape as the first wall 13 in the drawing indicated by the reference numeral 514 in FIG. The first wall 131 is formed of a top wiring layer 132 that is thicker than other wiring layers (including the wiring layer in which the electrodes 11 are formed) among the wiring layers formed on the semiconductor substrate 10. There is. In other words, the uppermost wiring layer 132 refers to a wiring layer formed on the opposite side of the bottom surface of the semiconductor substrate 10 from the arrangement position of the electrode 11. The electrode 11 is formed of aluminum, copper, or the like, and is formed of a wiring layer that is not the wiring layer closest to the microfluidic channel 30 and is not the uppermost wiring layer 132.

The first protective film 16 is formed of an interlayer insulating film 161 that insulates between wiring layers on the semiconductor substrate 10. The interlayer insulating film 161 prevents the semiconductor substrate 10 from coming into contact with the solvent 34 . The second protective film 18 covers the surfaces of each of the electrode 11 and the semiconductor substrate 10 that face the microfluidic channel 30, and the surfaces of the first wall 131 other than the surface that faces the semiconductor substrate 10. The second protective film 18 prevents the electrode 11 and the uppermost wiring layer 132 from coming into contact with the solvent 34 . Since the wiring layer such as the electrode 11 is formed of copper or aluminum, if a liquid such as the solvent 34 comes into direct contact with the electrode 11, corrosion or the like will occur. Therefore, it is preferable to cover the surfaces of the electrodes 11 and the like with the second protective film 18.

Note that the surfaces of the first wall 131 other than the surface facing the semiconductor substrate 10 do not need to be covered with the second protective film 18. The surface may be covered with at least a film made of the same material as the material forming the second protective film 18.

Examples of the material for forming the first protective film 16 include silicon dioxide (SiO ₂ ) and silicon nitride (Si ₃ N ₄ ). Among these materials, the first protective film 16 is preferably formed of silicon dioxide because it has good bonding with the microfluidic channel 30 formed of dimethylpolysiloxane (PDMS) or the like.

Examples of the material for forming the second protective film 18 include silicon dioxide. The material for forming the second protective film 18 is also preferably silicon dioxide for the same reason as described above.

The thickness of the second protective film 18 is preferably thinner than the thickness of the first protective film 16 formed of the interlayer insulating film 161. The thinner the second protective film 18 is than the first protective film 16, the more dielectrophoretic force F _DEP is applied from the electrode 11 to the cell 15 (details will be described later: see the diagram indicated by reference numeral 551 in FIG. 5). ) becomes large. The thickness of the second protective film 18 is more preferably, for example, 1 μm or less.

In order to maintain the dielectrophoretic force F _DEP acting on the cells 15 , a depression 12 is formed in the interlayer insulating film 161 directly above the surface of the electrode 11 facing the microfluidic channel 30 . Further, the thickness of the portion of the second protective film 18 that covers the bottom surface of the recess 12 is thinner than the thickness of other portions. By forming the second protective film 18 to such a thickness, it is possible to prevent the dielectrophoretic force F _DEP acting on the cells 15 from becoming small as much as possible.

The first wall 131 allows the cell 15 on which the dielectrophoretic force F _DEP is applied to be held in the depression 12 . The first wall 131 is formed of the uppermost wiring layer 132 as described above. This is because if the first wall 131 is formed of the uppermost wiring layer 132, it is easier to etch the uppermost wiring layer 132 in a substantially straight line when etching the uppermost wiring layer 132 in the vertical direction (up and down direction as viewed from the drawing).

The depression 12 holds the cell 15 on which the dielectrophoretic force F _DEP has been applied. The depression 12 is formed by etching the surface of the semiconductor substrate 10, for example. Although the shape of the depression 12 may be square, it is preferable that the depression 12 be circular, since many biological particles such as cells 15 are spherical. Further, the depressions 12 have a particle size that is approximately the same as the maximum diameter length of the cells 15 .

Further, as shown in the diagram indicated by reference numeral 521 in FIG. 2, a space 17 is formed in the first wall 131 through which cells 15 cannot pass, but through which other biological particles having a particle size smaller than that of the cells 15 can pass. .

Next, using FIG. 3, an example of the formation process of each layer formed above the semiconductor substrate 10 having the configuration shown in FIG. 1 will be described. FIG. 3 is a schematic diagram showing an example of the formation process of each layer formed above the semiconductor substrate 10. As shown in FIG.

First, in a normal method for forming a semiconductor integrated circuit, an electrode 11 formed of a metal layer (for example, the uppermost wiring layer 132: not shown in FIG. 3) is buried in the semiconductor substrate 10 (indicated by reference numeral 531 in FIG. 3). figure). After that, the first protective film 16 is formed on the semiconductor substrate 10 and the electrode 11 (as shown by reference numeral 532 in FIG. 3). Thereafter, etching is performed on the first protective film 16 at a location corresponding to the arrangement position of the electrode 11 on the first protective film 16 . Thereby, the electrode 11 is placed in the semiconductor substrate 10 at a location directly below the bottom surface of the recess 12 (as shown by reference numeral 533 in FIG. 3).

After forming the recess 12 by etching, the second protective film 18 is formed on the semiconductor substrate 10, the electrode 11, and the first protective film 16 to have a desired thickness (for example, 1 μm or less) (see FIG. 3). 534). After that, a photosensitive resin 19 is coated on the second protective film 18 (as shown by reference numeral 535 in FIG. 3), and then the photosensitive resin 19 is irradiated with light and the photosensitive resin 19 is etched. Forming the wall 13 (illustrated by reference numeral 536 in FIG. 3).

In the step illustrated by reference numeral 536 in FIG. 3, the first wall 13 is formed so that the distance in the horizontal direction between the side surface 12a of the recess 12 and the wall surface 13a on the recess 12 side of the first wall 13 is zero. .

Note that the diagram indicated by reference numeral 536 in FIG. 3 is an example in which the distance is zero, but the length of the distance is such that the cell 15 is continued to be retained. If so, there are no particular limitations. In addition, in this specification, "the cells 15 continue to be held" means that the cells 15 in the solvent 34 are attracted to the electrode 11 by the dielectrophoretic force F _DEP and after the cells 15 are settled in the recesses 12. This refers to maintaining the state of being contained in the depression 12 and not moving away from the depression 12.

Furthermore, the concept of "the cells 15 continue to be retained" includes that even if the cells 15 that have settled in the recesses 12 are once separated from the recesses 12 by the flow of the solvent 34, the cells 15 that have left the recesses 12 are attached to the first wall 13. This also includes being physically stopped by and returned to the recess 12.

Thereafter, by forming a microfluidic channel 30 (not shown in FIG. 3) made of PDMS or the like above the first wall 13, it is possible to flow the solvent 34 (not shown in FIG. 3) in which the cells 15 etc. are mixed. The biological particle collection device 1a can be obtained. Note that in order to bond PDMS forming the microfluidic channel 30 to the side wall (not shown) of the biological particle collection device 1a, the bonding surface is preferably silicon dioxide.

Next, using FIG. 4, an example of the formation process of each layer formed above the semiconductor substrate 10 having the configuration shown in FIG. 2 will be described. FIG. 4 is a schematic diagram showing another example of the formation process of each layer formed above the semiconductor substrate 10.

First, in a normal method for forming a semiconductor integrated circuit, an electrode 11 formed by an intermediate metal layer (for example, a wiring layer one to several layers below the topmost wiring layer) is buried in the semiconductor substrate 10 (reference numerals in FIG. 4). 541). Thereafter, an interlayer insulating film 161 is formed on the semiconductor substrate 10 and the electrodes 11 (as shown by reference numeral 542 in FIG. 4). After that, etching is performed on the interlayer insulating film 161 at a location corresponding to the arrangement position of the electrode 11 in the interlayer insulating film 161. Thereby, the electrode 11 is placed in the semiconductor substrate 10 at a location directly below the bottom surface of the recess 12 (as shown by reference numeral 543 in FIG. 4).

After forming the recess 12 by etching, the uppermost wiring layer 132 is formed on the interlayer insulating film 161 (as shown by reference numeral 544 in FIG. 4). In this step, the uppermost wiring layer 132 is formed so that the distance in the horizontal direction between the side surface 12a of the recess 12 and the wall surface 132a of the uppermost wiring layer 132 on the recess 12 side is zero. Thereafter, the second protective film 18 is formed on the electrode 11, the interlayer insulating film 161, and the uppermost wiring layer 132 so that the second protective film 18 has a desired thickness (for example, 1 μm or less). A first wall 131 is formed (as shown by reference numeral 545 in FIG. 4).

Note that the shape illustrated in the figure indicated by reference numeral 545 in FIG. 4 is an example in which the distance is zero, but the length of the distance is such that the cell 15 is separated to the extent that the depression 12 continues to hold the cell 15. If there is, there are no particular limitations.

The figures indicated by reference numerals 551 and 552 in FIG. 5 are top views of the biological particle collection device 1a. It is assumed that inside the biological particle collection device 1a shown in FIG. 5, a solvent 34 (not shown) (cells 15 in the solvent 34) flows from direction B to direction C. The configuration of the biological particle collection device 1b when viewed from above is also the same as the configuration of the biological particle collection device 1a shown in FIG. 5 when viewed from above.

As shown in the drawings indicated by reference numerals 551 and 552 in FIG. 5, one unit 21 is formed by the electrode 11, the depression 12, and the first wall 13. Further, the plurality of units 21 are arranged in an array on the semiconductor substrate 10 like units 21a to 21c.

Preferably, the plurality of units 21 are arranged as follows. In other words, if two units 21 are adjacent to each other in the direction in which the cells 15 and the like flow, when the two units 21 are viewed from the direction in which the cells 15 and the like flow, the other unit 21 located behind the one unit 21 that appears in the foreground is Preferably, at least a portion of the unit 21 is visible. Taking the unit 21a and the unit 21b as an example, when the unit 21a and the unit 21b are viewed from the direction in which the cells 15 and the like flow, it is preferable that at least a part of the unit 21b placed after the unit 21a that is visible in the front can be seen. .

Here, the plurality of units 21 arranged in an array constitute a plurality of unit groups, as shown in the diagram indicated by reference numerals 551 and 552 in FIG. Specifically, one unit group is composed of a plurality of units 21 arranged in a line in a direction intersecting the direction in which cells 15 flow. The arrangement of the plurality of units 21 described above using this unit group will be explained as follows. In other words, when looking at the two unit groups from the direction in which the cells 15 flow, the placement position of one unit group that appears in front and the placement position of the other unit group placed after the one unit group are as follows: They differ in the direction in which the cells 15 flow and the direction that intersects.

Further, as shown by an arrow X in the figure indicated by reference numeral 552 in FIG. 5, the cells 15 flowing onto the semiconductor substrate 10 are collected in the depression 12 included in the unit 21a. However, if the position of the cells 15 is shifted from the depression 12 of the unit 21a, as shown by the arrow Y in the above figure, the cells cannot be collected in the depression 12. However, by arranging the unit 21b at a position slightly shifted from the position of the unit 21a, it becomes easier to collect the cells 15 that have not been collected into the unit 21a into the depression 12. Therefore, the collection rate of cells 15 can be increased.

In the biological particle collection device 1a, a control circuit 22 is formed on a semiconductor substrate 10. The control circuit 22 is connected to the electrode 11 of each unit 21 via the connection member 2. The control circuit 22 individually controls the output of the signal by the electrodes 11 for each electrode 11 included in each of the plurality of units 21 .

In the device 200 disclosed in the above-mentioned Non-Patent Document 1, the electrode 101 is made of a transparent electrode made of ITO on the glass substrate 100. Therefore, when a plurality of electrodes 101 are arranged in an array on the glass substrate 100, the electrodes 101 in the depressions 102 are formed with the same wiring. As a result, even if the electrodes 101 are arranged in a plurality of arrays, the same signal is applied to all the electrodes 101, making it impossible to collect the cells 106 only in the desired depressions 102. On the other hand, since the biological particle collection device 1a can give the signal only to the specific controlled unit 21, it is possible to collect the cells 15 only at a desired position.

Next, a series of steps from collecting cells 15 as single cells using the biological particle collecting device 1a to performing immunostaining on the cells 15 will be described. Note that the same applies to the configuration of the biological particle collection device 1b. The diagrams indicated by reference numerals 561 and 562 in FIG. 6 are schematic diagrams showing the flow up to replacing the solvent 34 with the reagent 35 used for immunostaining. Note that the structure of the biological particle collection device 1b is similar to that shown in the diagrams indicated by reference numerals 551 and 552 in FIG. 5.

As shown in the diagram 561 in FIG. 6, in order to collect the cells 15 on the electrode 11 using the dielectrophoretic force F _DEP , it is necessary to It is necessary to select an appropriate solvent 34.

Here, the dielectrophoretic force F _DEP that the electrode 11 exerts on the cell 15 is expressed by the following equation 1.

d is the diameter of the cell 15, ε _p ^* and ε _m ^* are the complex dielectric constants of the cell 15 and the solvent 34, respectively, and E is the electric field provided by the electrode 11.

Depending on the sign of the real number component of the CM factor shown in Equation 1 above and Equation 2 below, the cells 15 are either attracted to the electrode 11 by the dielectrophoretic force F _DEP or are repelled and moved away. can be calculated.

When the value of Re [(ε _p ^* - ε _m ^* / (ε _p ^* + 2ε _m ^* )] in the above equation 1 is positive, the force directed toward the electric field given by the electrode 11 on the cell 15, i.e. , a positive dielectrophoretic force F _DEP acts.On the other hand, if the value of Re [(ε _p ^* - ε _m ^* )/(ε _p ^* + 2ε _m ^* )] in the above equation 1 is negative, the cell 15 On the other hand, a force that repels and separates the electric field applied by the electrode 11, that is, a negative dielectrophoretic force F _DEP acts.

In the case of the configuration of the biological particle collection device 1a, by applying a voltage signal that generates a positive dielectrophoretic force F _DEP to the electrode 11 from a voltage source (not shown), cells existing in the vicinity of the electrode 11 are collected. 15 can be attracted to the electrode 11 and collected in the depression 12.

On the other hand, cells 15 existing near the electrode 11 can be moved away from the electrode 11 by applying a voltage signal that generates a negative dielectrophoretic force F _DEP to the electrode 11 from a signal source (not shown). It is possible.

First, the cells 15 and the solvent 34 are flowed into the biological particle collection device 1a. The electrode 11 applies dielectrophoretic force F _DEP to attract the cells 15 to the electrode 11 and collect the cells 15 . Next, as shown by reference numeral 562 in FIG. 6, the solvent 34 is replaced with the reagent 35 by flowing the reagent 35 for immunostaining into the biological particle collection device 1a. Many of the reagents 35 used for immunostaining have high electrical conductivity. Therefore, when the solution is replaced with the reagent 35, the dielectrophoretic force F _DEP acting on the cells 15 is weakened, and the cells 15 are separated from the electrode 11. However, the cells 15 are held in the depression 12 by the first wall 13 formed near the depression 12 in which the cells 15 are collected. Furthermore, by individually controlling each electrode 11 with the control circuit 22, cells 15 can be collected only at desired locations among the plurality of units 21 arranged in an array.

[Embodiment 2]
The biological particle collection device 1c according to this embodiment will be described below with reference to FIG. FIG. 7 is a sectional view showing the configuration of the biological particle collection device 1c according to this embodiment. As shown in FIG. 7, in the biological particle collection device 1c according to this embodiment, a photodiode 40 that receives light emitted from the cells 15 is embedded directly below the electrode 11. According to the configuration, the characteristics of the cells 15 can be analyzed by evaluating the light received by the photodiode 40 from the cells 15.

As an example of the light, for example, when immunostaining is performed on the cells 15, the photodiode 40 receives fluorescence. By evaluating the characteristics of the fluorescence (eg, fluorescence wavelength, fluorescence lifetime, fluorescence intensity, etc.), the characteristics of cells that emit the fluorescence can be analyzed. Note that the type of photodiode 40 is not particularly limited, and is appropriately selected depending on the type of biological particles used.

[Embodiment 3]
The biological particle collection device 1d according to this embodiment will be described below based on FIGS. 8 and 9. First, the configuration of the biological particle collection device 1d according to this embodiment will be explained using FIG. 8. The figure indicated by the reference numeral 581 in FIG. 8 is a top view of the biological particle collection device 1d. The diagram indicated by reference numeral 582 in FIG. 8 is a BB' cross-sectional view in the figure indicated by reference numeral 581. The diagram indicated by reference numeral 583 in FIG. 8 is a top view of the biological particle collection device 1d. The diagram indicated by reference numeral 584 in FIG. 8 is a longitudinal sectional view taken along line CC' in the diagram indicated by reference numeral 583 in FIG.

As shown in the drawings indicated by reference numerals 581 to 583 in FIG. 8, the biological particle collection device 1d includes, in addition to the unit 21 described above, a second wall 50 formed around the unit 21. The second wall 50 allows the cell 15 (not shown in FIG. 8) on which the dielectrophoretic force F _DEP is applied to be held in the recess 12 . Further, a region 51 is formed by the second wall 50 so as to surround the unit 21 . The first wall 13 may be the same as the biological particle collection device 1a, or may be formed of the uppermost wiring layer 132 described in FIG. 4.

In the example of the biological particle collection device 1d shown by reference numerals 581 and 582 in FIG. 8, the first wall 13 and the second wall 50 are made of different resin materials. On the other hand, in the example of the biological particle collection device 1d shown by reference numerals 583 and 584 in FIG. 8, the first wall 13 and the second wall 50 are integrally formed of the same resin material. Note that from the viewpoint of simplifying the manufacturing process and reducing manufacturing costs, it is preferable that the first wall 13 and the second wall 50 are integrally formed of the same resin material.

The height H' of the side wall of the second wall 50 is the same as the height H of the first wall 13, or is higher than the height H, since the cells 15 are later confined in the unit 21 for analysis. It is preferable to form it high. Moreover, the shape of the side wall of the second wall 50 is not particularly limited.

Next, the flow from collecting the cells 15 as single cells using the biological particle collection device 1d to replacing the solvent 34 with the reagent 35 used for immunostaining will be explained using FIG. 9. . The diagrams indicated by reference numerals 591 to 593 in FIG. 9 are schematic diagrams showing the flow up to replacing the solvent 34 with the reagent 35 used for immunostaining.

As shown in the diagram indicated by reference numeral 591 in FIG. 9, first, the cells 15 are flowed into the biological particle collection device 1d together with the solvent 34, the cells 15 are drawn toward the electrode 11, and the cells 15 are collected. Next, as shown in the diagram indicated by reference numeral 592 in FIG. 9, a reagent 35 containing an enzyme-labeled antibody containing a biomarker and the like and a fluorogenic substrate is flowed into the biological particle collection device 1d, thereby removing the solvent. 34 is replaced with reagent 35. At this time, the cells 15 are held in the depressions 12 and the regions 51 by the first wall 13 and the second wall 50 .

Next, as shown in the diagram indicated by reference numeral 593 in FIG. The cells 15 are stopped within the region 51 to prevent the cells 15 from moving to other regions 51. Thereafter, the cells 15 are destroyed and the destroyed cells 15 are irradiated with excitation light (not shown). As a result, the characteristics of RNA and DNA present inside the cells 15 can be evaluated by detecting the light generated from the cells 15 by a detectable detection means.

By the method described above, an ELISA method using a minute amount of cells 15 can be realized. This is because by housing the biological sample within one area 51, detection sensitivity can be further increased. Note that the ELISA method using a minute amount of cells 15 in this embodiment is realized by pressing the microfluidic channel 30 arranged above the region 51. However, the method is not limited to the above method as long as the region 51 can be separated by covering the upper side of the region 51 by some method.

[Additional notes]
The present invention is not limited to the embodiments described above, and various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. are also included within the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment.

〔summary〕
A biological particle collection device (1a, 1b, 1c, 1d) according to aspect 1 of the present invention includes biological particles ( an electrode (11) that outputs a signal for applying a dielectrophoretic force (F _DEP ) to cells 15); and an electrode (11) formed on the semiconductor substrate for preventing the semiconductor substrate from coming into contact with the solvent. 1 protective film (16), the first protective film has a recess (12) for holding the biological particles on which the dielectrophoretic force has acted, and the electrode in the first protective film A first wall (13) is formed on the first protective film to hold the biological particles on which the dielectrophoretic force has acted in the recess. and the distance in the horizontal direction between the side surface (12a) of the recess and the wall surface (13a) on the recess side of the first wall is zero or is far enough away that the biological particles are continued to be retained by the recess. .

According to the above configuration, since the horizontal distance between the side surface of the depression and the wall surface on the depression side of the first wall is zero or extremely close, even if the biological particles leave the depression due to being replaced by the solvent, the first 1 Wall makes it easy for biological particles to accumulate in the depression. Therefore, even if the bioparticles are replaced by a solvent, the biological particles contained in the solvent housed in the depressions can be stably held in the depressions. Furthermore, since the biological particles can be stably held in the recesses, the biological particles can be easily observed.

The biological particle collection device (1a, 1b, 1c, 1d) according to the second aspect of the present invention is the one unit (21, 21a, 21b, 21c) is formed, a plurality of the units are arranged in an array on the semiconductor substrate, and the output of the signal by the electrode is controlled for each of the electrodes included in each of the plurality of units. The apparatus further includes a control circuit (22) to perform the following operations.

According to the configuration, only the electrodes of a specific unit desired by the user can output signals under control by the control circuit. Therefore, biological particles can be held only in specific depressions desired by the user.

In the biological particle collection device (1a, 1b, 1c, 1d) according to aspect 3 of the present invention, in the aspect 2, the two units adjacent to each other in the direction in which the biological particles flow are When the two units are viewed from above, at least a portion of the other unit placed behind the one unit visible in front can be seen.

According to the above configuration, even if the direction of movement of biological particles due to dielectrophoretic force deviates from the desired direction and does not fit into the recess of a specific unit, the direction of movement of biological particles due to dielectrophoretic force deviates from the desired direction and does not fit into the recess of a specific unit. It is more likely that it will fit into the cavity. Therefore, it is possible to improve the collection rate of bioparticles into the depressions.

In the biological particle collection device (1a, 1b, 1c, 1d) according to aspect 4 of the present invention, in the aspect 2, the plurality of units arranged in an array are constituted by a plurality of unit groups, The unit group is composed of a plurality of units arranged in a line in a direction intersecting the direction in which the biological particles flow, and two adjacent unit groups are separated from the direction in which the biological particles flow. When looking at the two unit groups, the arrangement position of one of the unit groups visible in front and the arrangement position of the other unit group arranged after the one unit group are such that the biological particles flow They differ in the direction and in the intersecting direction.

According to the above configuration, a biological particle collection device in which a plurality of units arranged in an array form a plurality of unit groups can achieve the same effect as the biological particle collection device according to aspect 3 of the present invention. play.

In the biological particle collection device (1d) according to aspect 5 of the present invention, in any one of aspects 2 to 4, the biological particles on which the dielectrophoretic force has acted are placed around the one unit in the recess. A second wall (50) for holding is formed.

According to the above configuration, an ELISA method using a minute amount or a single cell can be realized. By performing the ELISA method using a minute amount or a single cell, detection sensitivity can be further increased by containing the biological sample within one region 51.

In the biological particle collection device (1d) according to aspect 6 of the present invention, in aspect 5, the first wall and the second wall are integrally formed of the same resin material. According to the configuration, the first wall and the second wall can be formed simultaneously by etching or the like using one mask, for example. Therefore, it is possible to simplify the manufacturing process and reduce manufacturing costs.

In the biological particle collection device (1d) according to aspect 7 of the present invention, in any one of aspects 1 to 4, the first wall is located downstream of the depression when viewed from the direction in which the biological particles flow. A space (17) is formed in the first wall through which other biological particles having a particle size smaller than the biological particles can pass.

According to the configuration, biological particles having a particle size larger than the particle size of the biological particles to be collected are physically stopped by the first wall. Furthermore, if the particle size of the biological particles physically stopped by the first wall is larger than the particle size of the biological particles to be collected, there is a high possibility that the biological particles will not fit into the depression. Therefore, the probability that the bioparticles returned to the depression by the first wall become the bioparticles to be collected is increased, and the bioparticles to be collected that have moved away from the depression can be more reliably retained in the depression.

In the biological particle collection device (1a, 1b, 1c, 1d) according to aspect 8 of the present invention, in any one of aspects 1 to 5, the electrode is located at a location directly below the bottom surface of the recess in the semiconductor substrate. A second protective film (18) having a thickness of 1 μm or less is formed on the bottom surface of the recess to prevent the electrode from coming into contact with the solvent.

According to the configuration, the second protective film can prevent the electrode from coming into contact with the solvent. Further, since the thickness of the second protective film is 1 μm or less, the dielectrophoretic force generated from the electrode can be applied to the biological particles to the extent that the biological particles are not hindered in being accommodated in the recesses.

In the biological particle collection device (1a, 1b, 1c, 1d) according to aspect 9 of the present invention, in aspect 8, the first wall is located on the opposite side of the bottom surface side of the semiconductor substrate from the arrangement position of the electrode. The wiring layer (132) is formed on a wiring layer (132), and the surface other than the surface facing the semiconductor substrate is covered with a film made of the same material as the material forming the second protective film. .

According to the above configuration, when etching the wiring layer formed on the opposite side of the bottom surface of the semiconductor substrate from the electrode arrangement position in the vertical direction (in the vertical direction as viewed from the drawing), it becomes easier to etch the wiring layer in a substantially straight line.

In the biological particle collection device (1b) according to aspect 10 of the present invention, in aspect 8 or 9, the second protective film is formed of silicon dioxide. According to the above configuration, the bonding with the microfluidic channel formed of dimethylpolysiloxane (PDMS) or the like is improved.

In the biological particle collection device (1a, 1b, 1c, 1d) according to aspect 11 of the present invention, in any one of aspects 1 to 6, the first wall is formed of a photosensitive resin material. Photosensitive resin materials can be easily etched in the vertical direction. In this respect, according to the configuration, the side surface of the first wall on the side of the recess and the side surface of the recess can be easily formed into a substantially linear shape.

In the biological particle collection device (1a, 1b, 1c, 1d) according to aspect 12 of the present invention, in any one of aspects 1 to 11, the biological particle collection device (1a, 1b, 1c, 1d) is provided in which the biological particle is A photodiode (40) is buried therein to receive light emitted from the photodiode (40). According to the configuration, the characteristics of the biological particles can be analyzed by evaluating the characteristics of the light received by the photodiode from the biological particles.

The present invention can be mainly used for research in biology and medicine, clinical tests, and the like.

1a, 1b, 1c, 1d biological particle collection device 10 semiconductor substrate 11 electrode 12 depression 12a side surface 13, 131 first wall 13a, 132a wall surface 15 cell (biological particle)
16 First protective film 18 Second protective film 21, 21a, 21b, 21c Unit 34 Solvent 132 Top wiring layer (wiring layer formed on the opposite side of the bottom surface of the substrate from the electrode arrangement position)
F _DEP dielectrophoretic force

Claims (10)

an electrode embedded in a semiconductor substrate that outputs a signal for applying a dielectrophoretic force to biological particles in a solvent flowing onto the semiconductor substrate;
a first protective film formed on the semiconductor substrate for preventing the semiconductor substrate from coming into contact with the solvent;
A depression for holding the biological particles to which the dielectrophoretic force has been applied is formed in the first protective film at a location corresponding to a position of the electrode in the first protective film,
A first wall is formed on the first protective film for holding the biological particles on which the dielectrophoretic force has acted in the recess;
A side surface of the recess is in contact with a wall surface portion of the first wall on the recess side,
The first wall is composed of a first wiring layer formed on the first protective film,
The first wiring layer and the second wiring layer including the electrode are each made of copper or aluminum,
A biological particle collection device, wherein a second protective film that prevents the first wiring layer and the second wiring layer from coming into contact with the solvent is formed on the bottom surface of the recess and on the surface of the first wall. one unit is formed by the electrode, the recess and the first wall,
A plurality of the units are arranged in an array on the semiconductor substrate,
The biological particle collection device according to claim 1, further comprising a control circuit that controls output of the signal by the electrode for each of the electrodes included in each of the plurality of units. The two units adjacent to each other in the direction in which the bioparticles flow are such that the other unit is located behind one of the units that appears in front when the two units are viewed from the direction in which the bioparticles flow. The biological particle collection device according to claim 2, wherein at least a portion of the other unit is visible. The plurality of units arranged in an array are composed of a plurality of unit groups,
The unit group is composed of a plurality of units arranged in a line in a direction intersecting the direction in which the biological particles flow,
The two unit groups adjacent to each other are
When the two unit groups are viewed from the direction in which the biological particles flow, the arrangement position of one of the unit groups that appears in front, and the arrangement of the other unit group that is placed after the one unit group. The biological particle collection device according to claim 2, wherein the positions are different in a direction intersecting the direction in which the biological particles flow. The living body according to any one of claims 2 to 4, wherein a second wall is formed around the one unit for holding the biological particles to which the dielectrophoretic force has acted in the recess. Particle collector. The first wall is formed on the downstream side of the depression when viewed from the direction in which the biological particles flow,
The biological particle collection device according to any one of claims 1 to 4, wherein a space is formed in the first wall through which other biological particles having a particle size smaller than that of the biological particles can pass. The second wiring layer is located directly below the bottom surface of the recess in the semiconductor substrate,
The biological particle collection device according to any one of claims 1 to 5, wherein the second protective film has a thickness of 1 μm or less. The biological particle collection device according to any one of claims 1 to 5, wherein the first wiring layer is a wiring layer formed on the opposite side of the bottom surface side of the semiconductor substrate from the arrangement position of the electrode. . The biological particle collection device according to any one of claims 1 to 8, wherein the second protective film is made of silicon dioxide. The biological particle collector according to any one of claims 1 to 9, wherein a photodiode that receives light emitted from the biological particles is embedded in a location directly under the electrode in the semiconductor substrate. Device.

Images