JP5157246B2 - Small-diameter cell body capturing chip and manufacturing method thereof - Google Patents

Description

The present invention relates to a small-sized cell body capturing chip and a method for producing the same, and in particular, in a microinjection system that injects a gene or a drug into a small-sized cell body such as a stem cell having a diameter of about several μm, damages the small-sized cell body The present invention relates to a small-diameter cell body capturing chip and a method for producing the same, which is characterized by a configuration for surely arranging and immobilizing without any problems.

In recent years, modification of cell properties by injecting genes or the like into cells has attracted attention as a method for treating diseases caused by genetic causes, and research is progressing.
The results of this research include elucidation of the role of genes and the possibility of tailor-made medicine that provides medications and treatments tailored to individual genetic characteristics.

  Methods for injecting genes into cells include electrical methods using high-pressure pulses (electroporation method), chemical methods using endocytosis and membrane fusion (lipofection method), and organisms using viruses. General methods (virus vector method), mechanical methods using microneedles (hereinafter referred to as microinjection method), optical methods using laser light (laser injection method), etc. are generally well known. .

However, the electrical method has a large damage to cells, the chemical method has poor gene injection efficiency, and the biological method cannot introduce all injection materials (restriction of viruses). .
On the other hand, at the present time, a mechanical method, that is, a microinjection method has attracted attention as the safest and high injection efficiency method.

  In order to use this microinjection method, it is necessary to capture and fix cells. For this reason, a cell fixing plate having a concave structure with a diameter equal to or smaller than the cell diameter for capturing cells (generally, A silicon chip), and by sucking a culture solution (liquid medium) containing cells from a capture hole provided at the bottom of the recess structure, the cell can be adsorbed and fixed to the surface portion of the hole on the cell fixing plate. It has been proposed (see, for example, Patent Documents 1 to 5).

  Alternatively, a concave plate along the shape of a fertilized egg is provided in a porous plate such as microporous glass, and a fertilized egg having a diameter of several hundreds of μm is captured in the concave portion by suction through a micropore included in the porous plate. It has also been proposed (see, for example, Patent Document 6).

  In addition, as other means for immobilizing cells, a high frequency is applied between the electrodes, and the force of the electric field acting on the cells is used to immobilize the cells on the microelectrode on the cell immobilization plate, or the cells have affinity for cells. For example, a cell-affinity substance is placed on a cell-fixing plate, and cells are fixed on the cell-affinity substance.

Capturing and fixing cells by any of these means, inserting a microinjection needle (capillary) into the fixed cells, and sequentially injecting (injection processing), enabling injection into a large number of cells (For example, see Patent Document 3).
JP-A 63-129980 Japanese Patent Laid-Open No. 01-112976 JP 2002-027969 A JP 2000-023657 A Japanese Patent Laid-Open No. 01-144970 JP-A-10-165166

  However, the cells targeted by the above-described conventional microinjection method were relatively large cells such as egg cells having a diameter of several hundred μm, but recently, regenerative medicine / regeneration using stem cells having a diameter of 5 to 10 μm. Medical research and treatment are becoming more and more demanding for microinjection of stem cells.

  These stem cells are very small compared to egg cells, etc., and in order to reliably align and fix these small-diameter cells on a fixed substrate, the cells in the above-mentioned structure using the conventional process technology There is a problem in that microinjection is difficult because of a problem of passing through.

  Moreover, in the case of the above-mentioned Patent Document 3, since the cells are partially drawn into the suction holes and sucked and trapped, damage to the cells becomes a problem.

  Furthermore, the method of forming fine suction holes by etching in a substrate with a silicon substrate cannot practically produce fine suction holes for sucking and fixing small diameter cells such as stem cells having a diameter of 5 to 10 μm. There is a problem.

  If a fine suction hole shape suitable for small-diameter cells such as stem cells is to be realized by using the current process, an expensive process device is required, and the suction hole and the concave portion for holding the cells are used. Since the alignment with the structure is difficult and the yield decreases, there is a problem that the manufacturing cost increases as a result.

Accordingly, an object of the present invention is to form a fine suction hole without using lithography, and to reliably capture and immobilize a fine small-diameter cell body without damaging it.

FIG. 1 is a diagram illustrating the basic configuration of the present invention. Means for solving the problems in the present invention will be described with reference to FIG.
See FIG. 1 In order to solve the above-mentioned problem, the present invention includes a concave structure layer 6 for capturing and fixing a small-sized cell body 8 made of either a cell or a microcapsule, and a suction hole 5 penetrating in one direction. a suction hole layer 4 having the with a support substrate 1 for supporting the concave structure layer 6 and the suction holes layer 4, the suction holes 5 in the recess structure layer 6 is arranged at least two or more, and, the suction holes 5 is a small-diameter cell body capturing chip whose diameter is 1/100 to 1/10 of the average diameter of the small-diameter cell body 8 , wherein the suction hole layer 4 is made of an alumite pore film, and the suction hole 5 is anodized It is characterized by comprising pores that are self-organized .

Thus, by forming the small-diameter cell body capturing structure separately into the concave structure layer 6 and the suction hole layer 4 having the suction hole 5 penetrating in one direction, the diameter of the suction hole 5 is reduced to the small-diameter cell body 8. It becomes easy to form the suction hole layer 4 having an average diameter of 1/100 to 1/10, and it becomes easy to dispose at least two suction holes 5 in the concave structure layer 7, Since the diameter of the suction hole 5 is 1/100 to 1/10 of the average diameter of the small-diameter cell body 8, the small-diameter cell body 8 made of either cells such as stem cells or microcapsules is not damaged, and Thus, it is possible to reliably capture without being drawn into the suction hole 5.

In this case, the suction hole layer 4 is an anodized pore film, and the suction hole 5 is a pore self-organized by anodic oxidation, so that an average diameter of the small-diameter cell body 8 is reduced to 1/100 or more without using an etching process. It becomes possible to reliably form the suction hole layer 4 of 1/10 by a simple process.

Further, the concave structure layer 6 and the suction hole layer 4 are desirably optically transparent to the extent that microscopic observation of the small-diameter cell body 8 is possible, thereby allowing injection of a drug solution or virus into the small-diameter cell body 8. This can be performed while observing under a microscope.

In the case of producing a small-diameter cell body capturing chip, the step of forming the anodized pore film by anodizing the aluminum plate, the support substrate having the opening 2 on the surface of the aluminum plate on which the anodized pore film is formed 1, a step of forming a suction hole layer 4 made of an alumite pore film by thinning an aluminum plate, and a plurality of pores provided in the suction hole layer 4 on the suction hole layer 4. And the step of providing the recess structure layer 6 with the recesses 7 formed thereon. Since the suction holes 5 are arranged on one surface of the suction hole layer 4, alignment accuracy with the recess structure layer 6 is not necessary. Therefore, it is possible to prevent a decrease in yield.

  In this case, before the step of providing the recess structure layer 6, a step of removing the exposed portion of the adhesive layer used in the step of bonding the surface of the aluminum plate on which the anodized pore film is formed to the support substrate 1 having the opening 2 is performed. It is necessary to have.

Alternatively, a step of forming an aluminum layer on the support substrate 1, a step of anodizing the aluminum layer to form an alumite pore film to form the suction hole layer 4, a step of forming the opening 2 in the support substrate 1, and And a step of providing a recess structure layer 6 in which a recess 7 is formed so as to include a plurality of pores provided in the suction hole layer 4 on the suction hole layer 4. It becomes possible to manufacture a small-diameter cell body capturing chip by using it, and it is possible to reduce the cost of the small-diameter cell body capturing chip.

  In this case, in the step of forming the aluminum layer, it is desirable to form the aluminum layer on the support substrate 1 via the intervening layer 3 in which the aluminum layer is formed on the support substrate 1, so that when the pore is penetrated The support substrate 1 is not damaged, and the anodized pore film is not damaged when the opening 2 is formed in the support substrate 1.

  In this case, the intervening layer 3 is desirably formed by thermally oxidizing the support substrate 1, and the process of forming the intervening layer 3 is simplified.

  In addition, in the step of forming the alumite pore film, a nanoimprint method established in the field of patterned media production may be used, whereby the pores can be formed at an arbitrary pitch and an arbitrary diameter in the range of 50 nm to 1000 nm. Can be formed.

According to the present invention, large-scale and high-speed injection into small-diameter cell bodies consisting of either cells such as stem cells or microcapsules, which has been impossible with conventional structures, is possible, and damage stress due to cell deformation By reducing, accurate evaluation and observation of cellular responses becomes possible.

In addition, since the anodized pore film formed by anodization is used as a suction hole layer in the manufacturing process, not only an expensive process device is used, but also an improvement in chip yield is expected, and a small diameter suitable for small diameter cells such as stem cells. It becomes possible to easily provide a cell body capturing chip.

The present invention relates to a suction hole layer having a suction hole penetrating in one direction in order to capture and fix a small diameter cell body having a diameter of 10 μm or less such as a stem cell or a microcapsule, in particular, the average diameter of the small diameter cell body by anodic oxidation. An alumite pore film having suction holes with a size of 1/100 to 1/10 and a recess structure layer formed so that at least two suction holes are arranged in the recess structure are laminated on the support substrate. It is what I supported.

When manufacturing such a small-diameter cell body capture chip, an anodized aluminum film is formed by anodizing the aluminum plate, and the surface of the aluminum plate on which the anodized pore film is formed serves as a negative pressure suction port. After a substrate is bonded to a support substrate such as a silicon substrate having a portion by a substrate bonding technique using an adhesive or SiO ₂ film, an aluminum plate is thinned to form a suction hole layer made of an alumite pore film, and then a suction hole layer What is necessary is just to provide the recessed part structure layer which formed the recessed part so that the several pore provided in the suction hole layer may be included on it.

Alternatively, an aluminum layer is formed on a support substrate such as a silicon substrate through a barrier film such as a SiO ₂ film formed by thermal oxidation, and then the aluminum layer is anodized to form a suction hole layer made of an alumite pore film. Then, after forming an opening in the support substrate, the exposed portion of the barrier layer is removed, and then a recess structure layer in which a recess is formed on the suction hole layer so as to include a plurality of pores provided in the suction hole layer. Should be provided.

  Further, in the step of forming the alumite pore film, the pores may be regularly formed by normal self-organization accompanying anodic oxidation, or the pores may be formed at an arbitrary pitch and 50 nm to 1000 nm using a nanoimprint method. You may make it form in arbitrary diameter in the range.

Here, with reference to FIG. 2 thru | or FIG. 4, the manufacturing process of the cell capture chip | tip of Example 1 of this invention is demonstrated.
See Figure 2
First, an Al plate 11 having a purity of 99.99% or more and a mirror-finished thickness of 100 μm, for example, is prepared, and an anodizing solution made of, for example, a 0.3 M / liter dilute sulfuric acid bath is used. A porous alumina film, that is, an alumite pore film 12 is formed by anodizing.

Although not shown here, since the protective film is provided on one surface of the Al plate 11, the anodic oxidation proceeds from the other surface, and the unoxidized Al layer 13 is formed on the protective film side. Remains.
At this time, the pores 14 are formed in the alumite pore film 12 by regular arrangement in a hexagonal close-packed structure by self-organization, and the pitch is
Pore pitch [nm] = DC voltage during anodization [V] x 2.5
Therefore, the pore pitch and therefore the pore density can be controlled by the DC voltage.

  In this case, when the cell diameter is large, the desired diameter cannot be achieved only by anodic oxidation. Therefore, it is possible to enlarge the diameter by performing pore widening using 4% phosphoric acid as necessary. .

On the other hand, after the SiO ₂ films 22 and 23 are formed by thermal oxidation on the surface of the silicon substrate 21 serving as the support substrate, an opening 24 having a side of, for example, 1.7 mm is formed in one SiO ₂ film 23 using hydrofluoric acid. Form.

Next, for example, an exposed portion 25 of the negative pressure suction is formed by etching the exposed portion of the silicon substrate 21 using an etching solution such as KOH, and then the SiO ₂ films 22 and 23 are etched using hydrofluoric acid. Remove.

See Figure 3
Next, the Al plate 11 on which the alumite pore film 12 is formed is bonded to the silicon substrate 21 using an adhesive (not shown) so that the alumite pore film 12 and the silicon substrate 21 face each other.

  Next, the Al layer 13 is completely removed by polishing from the Al layer 13 side, and the suction hole 16 is passed through the pore 14 by polishing until the thickness of the alumite pore film 12 becomes 5 μm (or 10 μm), for example. Then, the adhesive (not shown) exposed in the opening 25 is removed.

  Next, after applying a resist to be the recess layer 17, exposure and development are performed to form a recess to be a cell trapping portion 18 having a diameter of 5 μm to 20 μm, and by leaving the resist as it is, the thickness becomes, for example, By setting the concave layer 17 to 5 μm, the basic structure of the cell trapping chip of Example 1 of the present invention is completed.

See Figure 4
FIG. 4 is a diagram illustrating the configuration of the cell trapping chip according to the first embodiment of the present invention. In the figure, the number of cell trapping portions 18 for trapping the cells 20 is 16 (4 × 4). When the chip size is 3.5 mm □, for example, about 1000 are formed.

  Further, as shown in the lower diagram of FIG. 4, a plurality of suction holes 16 are exposed in the cell trapping portion 18, and depending on the pore pitch defined by the DC voltage in anodization, for example, about 80 are exposed. Thus, by sucking negative pressure from the suction hole 16 through the opening 25, the flowing cell 20 is sucked and captured by the cell trapping part 18.

  Thus, in the present invention, since the cell trapping structure is divided into the concave layer 17 provided with the cell trapping part 18 and the suction hole layer 15 provided with the suction hole 16, the size corresponding to each characteristic is obtained. Can be formed.

  Further, the suction hole layer 15 having the suction holes 16 is formed by utilizing self-organization by anodization established in the patterned media field, so that fine suction can be performed without requiring an advanced etching technique. The holes 16 can be formed at an arbitrary pitch, and the suction holes 16 are uniformly exposed on the surface of the suction hole layer 15, thereby forming the concave layer 17 provided with the cell trapping portion 18. Alignment is not necessary.

  Further, the size of the suction hole 16 in this case is appropriately determined according to the size of the cell 20 to be captured, and can be 1/100 to 1/10 of the average diameter of the cell 20. When sucking and capturing the cells 20, the cells 20 are not drawn into the suction holes 17, and stable capturing is possible without damaging the cells 20.

  In addition, since alumina is transparent to visible light, even if irregular reflection occurs due to minute suction holes, when injecting a drug solution into captured cells, the drug solution can be injected while observing with an optical microscope. It is transparent.

Next, with reference to FIG.5 and FIG.6, the manufacturing process of the cell trapping chip of Example 2 of this invention is demonstrated.
See Figure 5
First, after forming SiO ₂ films 32 and 33 by thermal oxidation on the surface of a silicon substrate 31 having a thickness of, for example, 525 μm, for example, an Al film having a thickness of, for example, 5 μm using a sputtering method. 34 is formed.

Next, for example, the Al layer 34 is converted to the alumite pore film 35 by performing anodization using an anodizing solution composed of a 0.3 M / liter dilute sulfuric acid bath.
After the anodic oxidation, an alumina barrier film 37 is formed on the SiO ₂ film 32 side.

  In this case, when the cell diameter is large, the desired diameter cannot be achieved only by anodic oxidation. Therefore, it is possible to enlarge the diameter by performing pore widening using 4% phosphoric acid as necessary. .

Next, an opening 38 having a side of, for example, 1.7 mm is formed on the SiO ₂ film 33 formed on the back surface of the silicon substrate 31 using hydrofluoric acid, and then the silicon substrate 31 is etched using an etching solution such as KOH. The exposed portion is etched to form a negative pressure suction opening 39.

See FIG.
Next, the SiO ₂ film 33 and the SiO ₂ film 32 exposed to the opening 39 are removed by etching with a BHF solution, and then the alumina barrier film 37 is removed using an ion milling method or the like to penetrate the pores 36, thereby sucking holes. The suction hole layer 40 is provided with 41.

  Next, after applying a resist to be the recess layer 42, exposure and development are performed to form a recess to be a cell trapping portion 43 having a diameter of 5 μm to 20 μm, and the resist is left as it is. By forming the concave layer 42 of 5 μm, the basic structure of the cell trapping chip of Example 2 of the present invention is completed.

  In Example 2 as well, when the chip size is 3.5 mm □, for example, about 1000 cell trapping portions 43 are formed, and the cell trapping portion 43 has about 80 pieces. A suction hole 41 is included.

  As described above, in the present invention, since the cell trapping structure is formed by combining semiconductor technology and anodization, it is possible to form the fine suction holes 41 at an arbitrary pitch without requiring an advanced etching technique. In addition, since the suction holes 41 are uniformly exposed on the surface of the suction hole layer 40, alignment is not necessary when the concave layer 42 provided with the cell trapping portion 43 is formed.

Next, the manufacturing process of the cell trapping chip according to the third embodiment of the present invention will be described with reference to FIG. 7. Since the basic process is the same as that of the second embodiment, only the anodic oxidation process is illustrated. To do.
See FIG.
First, as in the second embodiment, after the SiO ₂ films 32 and 33 are formed on the surface of a silicon substrate 31 having a thickness of, for example, 525 μm by thermal oxidation, the thickness becomes a thickness using, for example, a sputtering method. For example, an Al film 34 of 5 μm is formed.

Next, the SiC stamper 50 having the regularly arranged protrusions 51 is pressed against the Al film 34 to form a recess 44, and then the Al layer 34 is formed by anodizing using, for example, an anodizing solution made of an oxalic acid bath. The alumite pore film 35 is converted.
At this time, the recess 44 is deeply etched to form the pore 36.

  Also in this case, if the cell diameter is large, the desired diameter cannot be achieved only by anodic oxidation. Therefore, if necessary, the diameter can be increased by performing pore widening using 4% phosphoric acid. is there.

Next, after forming an opening 39 for negative pressure suction in the silicon substrate 31, the SiO ₂ film 33 and the SiO ₂ film 32 exposed to the opening 39 are removed by etching, and then an alumina barrier is used using an ion milling method or the like. By removing the film 37 and penetrating the pores 36, the suction hole layer 40 having the suction holes 41 is obtained.

  Next, after applying a resist to be the recess layer 42, exposure and development are performed to form a recess to be a cell trapping portion 43 having a diameter of 5 μm to 20 μm, and the resist is left as it is. By setting the concave layer 42 to 5 μm, the basic structure of the cell trapping chip of Example 3 of the present invention is completed.

  In the third embodiment, since the nano-imprint method is used to form the pores 36, the pitch of the pores can be arbitrarily and more regularly arranged regardless of the applied voltage.

Next, the manufacturing process of the cell trapping chip according to the fourth embodiment of the present invention will be described with reference to FIG. 8. Since the basic process is the same as that of the first embodiment, only the bonding process is illustrated.
See FIG.
As in Example 1 above, first, an Al plate 11 having a purity of 99.99% or more and a mirror-finished thickness of 100 μm, for example, is prepared, for example, 0.3 M / liter dilute sulfuric acid A porous alumina film, that is, an alumite pore film 12 is formed by anodizing using an anodizing solution comprising a bath.

  Also in this case, if the cell diameter is large, the desired diameter cannot be achieved only by anodic oxidation. Therefore, if necessary, the diameter can be increased by performing pore widening using 4% phosphoric acid. is there.

On the other hand, after the SiO ₂ films 22 and 23 are formed by thermal oxidation on the surface of the silicon substrate 21 serving as the support substrate, an opening 24 having a side of, for example, 1.7 mm is formed in one SiO ₂ film 23 using hydrofluoric acid. Then, the exposed portion of the silicon substrate 21 is etched using, for example, an etching solution such as KOH, thereby forming the negative pressure suction opening 25.

Next, after the SiO ₂ film 19 is formed on the surface of the alumite pore film 12 using the CVD method, the SiO ₂ film 22 and the SiO ₂ film 19 are opposed to each other in the same manner as in the substrate bonding technique of the silicon substrate. By heating in this state, the Al plate 11 and the silicon substrate 21 are bonded together.

  Next, the Al layer 13 is polished to remove the Al layer 13 completely, and the alumite pore film 12 is polished until the thickness of the anodized pore film 12 becomes 5 μm, for example, and penetrated through the pores 14 so as to have the suction holes 16 The pore layer 15 is used.

Then, by dry etching, after removing the SiO ₂ film 22 and the SiO ₂ film 19 is exposed to the SiO ₂ film 23 and the opening 24, the diameter by a resist comprising a recess layer 17 is applied, exposed and developed By forming a recess to be a cell capture portion 18 of 5 μm to 20 μm and leaving the resist as it is, the thickness of the recess layer 17 is, for example, 5 μm, so that the cell capture chip of Example 4 of the present invention is used. The basic structure is completed.

As described above, in Example 4 of the present invention, since the cell trapping structure is bonded to the support substrate with SiO _{2 having} low chemical reactivity by using the substrate bonding technique, compared to the case where an adhesive is used. Thus, there is no need to consider the influence on the adhesive such as the chemical solution or the culture solution used in the injection process, and conversely the influence of the adhesive component on the cells or the chemical solution.

  The embodiments of the present invention have been described above. However, the present invention is not limited to the configurations, conditions, and numerical values shown in the embodiments, and various modifications are possible. For example, in the above embodiments, The chip size, the number of cell traps, etc. shown are completely arbitrary, and can be changed as needed.

In each of the above embodiments, the recess layer is formed of a resist. However, when there is a risk that the resist components may give cells, chemicals, or the like, or when the hydrophilicity or hydrophobicity of the surface becomes a problem. May be replaced with other inorganic substances having low chemical reactivity such as SiO ₂ and SiN.

  In each of the above-described embodiments, the support substrate is formed of a silicon substrate with a fine processing technology, but is not limited to a silicon substrate, and other substrates such as a glass substrate and a sapphire substrate are used. It is good.

  In Example 3 described above, the position of the pore is controlled by nanoimprint technology using a SiC master substrate. However, the pores are recessed on the aluminum surface by the etching technique of the opening resist portion by electron beam lithography or X-ray lithography. Then, an anodized pore film having pores corresponding to the depressions may be formed by anodic oxidation in which a voltage is applied in an acidic electrolyte solution such as oxalic acid.

Here, referring to FIG. 1 again, the detailed features of the present invention will be described again.
Again see Figure 1
(Supplementary note 1) Recess structure layer 6 for capturing and fixing small cell bodies 8 made of either cells or microcapsules, suction hole layer 4 having suction holes 5 penetrating in one direction, and recess structure layer 6 And the support substrate 1 for supporting the suction hole layer 4, and at least two suction holes 5 are disposed in the concave structure layer 6, and the diameter of the suction hole 5 is an average diameter of the small-diameter cell body 8. 1/100 to 1/10 of the small-sized cell body capturing chip, wherein the suction hole layer 4 is made of an alumite pore film, and the suction hole 5 is made of a pore that is self-organized by anodization. A small-diameter cell body capture chip.
(Appendix 2) The endoplasmic reticulum trapping chip according to appendix 1, wherein the concave structure layer 6 and the suction hole layer 4 are optically transparent to such an extent that the small-diameter cell body 8 can be observed with a microscope.
(Additional remark 3) The process of forming an alumite pore film | membrane by anodizing an aluminum plate, the process of bonding the surface in which the alumite pore film | membrane of the said aluminum plate was formed to the support substrate 1 which has the opening part 2, the said aluminum plate And forming a suction hole layer 4 made of an alumite pore film, and a cell in which a recess 7 is formed on the suction hole layer 4 so as to include a plurality of pores provided in the suction hole layer 4 Or a step of providing a concave structure layer 6 for capturing and fixing a small-diameter cell body made of either of microcapsules .
(Additional remark 4) Before the process of providing the said recessed part structure layer 6, the exposed part of the contact bonding layer used for the process of bonding the surface which formed the alumite pore film | membrane of the said aluminum plate to the support substrate 1 which has the opening part 2 is removed. The manufacturing method of the small diameter cell body capture | acquisition chip | tip of additional statement 3 characterized by having the process to do.
(Additional remark 5) The process of forming the aluminum layer on the support substrate 1, The process of forming the suction hole layer 4 which anodizes the said aluminum layer, and consists of an alumite pore film | membrane, The process of forming the opening part 2 in the said support substrate 1 And a recess for capturing and fixing a small-diameter cell body made of either a cell or a microcapsule having a recess 7 formed so as to include a plurality of pores provided in the suction hole layer 4 on the suction hole layer 4 And a step of providing a structural layer 6.
(Appendix 6) In the step of forming the aluminum layer on the support substrate 1, the aluminum layer is formed on the support substrate 1 through the intervening layer 3 formed on the support substrate 1, and the recess structure is formed. 6. The method for producing a small-diameter cell body capturing chip according to appendix 5, wherein the exposed portion of the intervening layer 3 is removed before the step of providing the layer 6.
(Additional remark 7) The said intervening layer 3 is formed by thermally oxidizing the said support substrate 1, The manufacturing method of the small diameter cell body trapping chip of Additional remark 6 characterized by the above-mentioned.
(Appendix 8) In the step of forming the alumite pore film, a resist film is formed on the surface of the aluminum layer, and the openings 2 having a diameter of 50 nm to 1000 nm regularly arranged in the resist film by a nanoimprint method are formed. 8. The method for producing a small-diameter cell body-capturing chip according to any one of appendices 3 to 7, comprising a step of anodizing the aluminum layer exposed to the opening 2.

  As an application example of the present invention, a cell capture chip in the case of injecting into stem cells is typical, but the capture target is not limited to stem cells, and other cells larger than stem cells may be used, Furthermore, the capture target is not limited to living cells, but can also be applied to capture microcapsules for experimenting with various pharmaceutical reactions and chemical reactions.

It is explanatory drawing of the fundamental structure of this invention. It is explanatory drawing of the manufacturing process to the middle of the cell capture chip | tip of Example 1 of this invention. It is explanatory drawing of the manufacturing process after FIG. 2 of the cell trapping chip of Example 1 of this invention. It is a structure explanatory drawing of the cell capture chip | tip of Example 1 of this invention. It is explanatory drawing of the manufacturing process to the middle of the cell capture chip | tip of Example 2 of this invention. It is explanatory drawing of the manufacturing process after FIG. 5 of the cell trapping chip of Example 2 of this invention. It is explanatory drawing of the manufacturing process of the cell capture chip | tip of Example 3 of this invention. It is explanatory drawing of the manufacturing process of the cell capture chip | tip of Example 4 of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Support substrate 2 Opening part 3 Intervening layer 4 Suction hole layer 5 Suction hole 6 Recessed structure layer 7 Recessed part 8 Small-diameter cell body 11 Al plate 12 Anodized pore film 13 Al layer 14 Pore 15 Suction hole layer 16 Suction hole 17 Recessed layer 18 Cell Trapping portion 19 SiO ₂ film 20 Cell 21 Silicon substrate 22, 23 SiO ₂ film 24 Opening portion 25 Opening portion 31 Silicon substrate 32, 33 SiO ₂ film 34 Al film 35 Anodized pore film 36 Pore 37 Alumina barrier film 38 Opening portion 39 Opening Part 40 Suction hole layer 41 Suction hole 42 Recessed layer 43 Cell trapping part 44 Recess 50 SiC stamper 51 Projection part

Claims (4)

  A concave structure layer for capturing and fixing small cell bodies composed of either cells or microcapsules, a suction hole layer having a suction hole penetrating in one direction, and a support substrate for supporting the concave structure layer and the suction hole layer A small-diameter cell body trapping chip in which at least two suction holes are disposed in the recess structure layer, and the diameter of the suction holes is 1/100 to 1/10 of the average diameter of the small-diameter cell bodies The small-diameter cell body capturing chip, wherein the suction hole layer is made of an alumite pore film, and the suction hole is made of a pore that is self-organized by anodization.   The small-diameter cell body capturing chip according to claim 1, wherein the concave structure layer and the suction hole layer are optically transparent to such an extent that the microscopic observation of the small-diameter cell body is possible. A step of forming an alumite pore film by anodizing the aluminum plate; a step of bonding the surface of the aluminum plate on which the anodized pore film is formed to a support substrate having an opening; and thinning the aluminum plate to anodize the aluminum plate A step of forming a suction hole layer made of a pore film, and a step of providing a concave structure layer for capturing and fixing a small-sized cell body made of either a cell or a microcapsule on the suction hole layer. A method for producing a small-diameter cell body capturing chip. Forming an aluminum layer on a support substrate, forming an aspiration hole layer made of an alumite pore film by anodizing the aluminum layer, forming an opening in the support substrate, and on the suction hole layer And a step of providing a concave structure layer for capturing and fixing small cell bodies composed of either cells or microcapsules .

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