JP5751187B2 - Injection molding micro chemical device - Google Patents

Description

The present invention relates to an injection-molded microchemical device , and more particularly to an injection-molded microchemical device in which a large number of microwells holding cells are integrated.

  In the case of observing cells using a microchemical device, the conventional technology can provide a device in which hundreds of thousands of microwells having a minimum diameter of 10 μm are integrated as well shapes suitable for the cells to be observed. In order to produce a microchemical device by molding, at least the distance between the wells is at least twice the diameter of the well (2 wells). In such a situation, although the number of wells integrated in one device is several hundred thousand, the number of wells in one field of view is limited in observations such as fluorescence microscope observation and laser microscope observation, and the observation sample is manufactured. Depending on the conditions, precious cells may not enter the well and may exist between the wells.

  In order to manufacture a microchemical device by injection molding with a synthetic resin, it is necessary to manufacture a mold insert in which the pattern shape of the device (well shape in the case of a well device) is reversed. In the current mold manufacturing technology, due to limitations coming from the processing tool, limitations coming from the processing machine, limitations coming from the device shape, etc., the radius that occurs at the corner that is not desired in the well shape manufacturing is zero, and the well diameter is from 10 μm to several Wells could not be arranged within a distance of within 1 well of a device having a well shape of 100 μm.

  In the optical field such as imprints, especially microlenses and diffusion sheets, there are actual examples in which the concave shape of the nano pattern is arranged at a distance of one pattern or less. However, since the concave shape is at the nano level, nickel-based steel is used. In some cases, it is possible to manufacture a mold insert. However, in the bio-related field that handles cells and proteins as in the present invention, the pattern size is 10 μm or more and several hundreds μm or less. It is difficult to manufacture a similar mold insert. Furthermore, pattern arrangement is also important. Even if the well shape and the distance between wells are the same, if the pattern arrangement is staggered, it cannot be processed by the current mold processing technology.

  Patent Document 1 describes that screening from a large amount of library is recently required for screening microorganisms, cells, proteins, etc., but the well size is 50 μm in diameter and the distance between adjacent wells is 80 μm. Furthermore, the cross-sectional shape of the well is a hemispherical shape, and the distance between wells cannot be reduced within one well at the conventional technology level.

  In Patent Document 2, the concave shape, the convex shape, and the hole shape with a size of 0.1 μm to 200 μm are described. Considered more than a minute. Furthermore, the side walls of the well are almost vertical, and no device is fabricated by injection molding.

  In addition, as a technique for giving a specific inclination angle to the well sidewall, a single crystal silicon wet etching method is well known. In this technique, an alkaline solution of (100) plane and (111) plane of the silicon crystal structure is used. By utilizing the difference in the wet etching rate due to (anisotropic etching) and wet etching the (100) surface of silicon, the (111) surface appears with an inclined surface of 54.74 degrees, but 54.74 degrees. There arises a restriction that only the inclined surface can be provided.

JP 2006-061023 A JP 2006-192535 A

Therefore, in view of the above-described situation, the present invention intends to solve the problem by reducing the distance between adjacent wells to 1 well or less by using an X-ray lithography method and a nickel electroforming method, so that the well side wall has 5 to 25 degrees. The object of the present invention is to provide a highly integrated injection-molded microchemical device having an arbitrary tilt angle suitable for the purpose of using wells.

In order to solve the above-mentioned problems, the present invention is a microchemical system in which hundreds of thousands of microwells for holding microorganisms, cells or proteins to be observed are integrated and used for observation with an optical microscope, fluorescence microscope or laser microscope. A device,
When irradiating the X-ray from the X-ray source, the X-ray mask and the resist substrate are relatively translated by a calculated arbitrary distance, whereby the X-ray dose irradiated to the resist substrate through the pattern shape on the X-ray mask. And a tilt angle of 5 degrees to 25 degrees corresponding to the relative translation between the X-ray mask and the resist substrate is provided on the side wall of the well formed on the resist substrate. A mold insert is manufactured by nickel electroforming after conducting a conductive treatment, and by injection molding of synthetic resin using the mold insert, the shape of one microwell is 10 μm to 300 μm, and the well sidewall is inclined. The angle is in the range of 5 degrees to 25 degrees, the well aspect ratio is 0.7 to 1, and the distance between adjacent wells is accumulated at a distance equal to or smaller than the well dimension of one well. An injection molded microchemical device was constructed.

According to the injection-molded microchemical device of the present invention as described above, hundreds of thousands of microwells for holding microorganisms, cells, or proteins to be observed are integrated and observed with an optical microscope, a fluorescence microscope, or a laser microscope. When the X-ray source irradiates the X-ray from the X-ray source, the X-ray mask and the resist substrate are moved relative to each other by the calculated distance, thereby allowing the pattern shape on the X-ray mask to pass through. Then, using a processing method for controlling the X-ray dose irradiated to the resist substrate, an inclination angle of 5 to 25 degrees corresponding to the relative translation of the X-ray mask and the resist substrate is provided on the side wall of the well formed on the resist substrate. Produced a mold insert by nickel electroforming after conducting a conductive treatment based on the resist substrate, and injection of synthetic resin using the mold insert Depending on the shape, the shape of one microwell is 10 to 300 μm, the well sidewall inclination angle is in the range of 5 to 25 degrees, the well aspect ratio is 0.7 to 1, and the distance between adjacent wells Can be provided at a distance less than the size of the frontage of one well, so that the degree of integration of wells per unit area can be maximized, and thereby a microorganism that can be screened with a single microchemical device, The number of cells, proteins, etc. can be increased, and when observing cells in a well with an observation device such as an optical microscope, laser microscope, or fluorescence microscope, the number of wells existing in the field of view increases even at the same magnification. Efficiency is greatly improved. In addition, since the distance between adjacent wells is small, cells or the like do not adhere between adjacent wells, and valuable samples can be used effectively.

  By making the well side wall tilt angle of the microwell in the range of 5 degrees to 25 degrees, the melt can completely fill the well pattern when the device is manufactured by injection molding, and the cells are smoothly in the well. Can be introduced.

It is explanatory drawing which shows the basic principle which forms a well shape in a positive resist by X-ray lithography. It is explanatory drawing which shows the example which manufactures the well which has a side wall inclination angle by reciprocatingly moving an X-ray mask. It is explanatory drawing which shows the example which manufactures the well which has a side wall inclination angle by reciprocatingly translating the X-ray mask different from FIG. It is explanatory drawing for demonstrating the preferable range of a well side wall inclination angle. The preferred basic shape of the well is shown, (a) is a square square frustum-shaped well, (b) is a rectangular quadrangular frustum-shaped well, (c) is a central sectional view of (a) and (b), (D) is a hexagonal frustum-shaped well having a regular hexagonal shape, (e) is a hexagonal frustum-shaped well having a horizontally long hexagonal shape, (f) is a central sectional view of (d) and (e), and (g) is a cone. A trapezoidal well, (h) shows a central sectional view of (g). FIG. 5 is a partial plan view showing a lattice-like close-packed layout in which square wells each having a side of 100 μm are used, the distance between adjacent wells is zero, and the wells are arranged vertically and horizontally. FIG. 10 is a partial plan view showing a staggered close-packed layout in which square wells having a side of 100 μm are used, the distance between adjacent wells is zero, and the wells are arranged in a staggered manner. FIG. 5 is a partial plan view showing a staggered close-packed layout in which square wells with a side of 100 μm are used, the distance between adjacent wells is 20 μm, and the wells are arranged in a staggered manner. FIG. 5 is a partial plan view showing a close-packed layout using a regular hexagonal well having a side of a well opening of 100 μm and a distance between adjacent wells of zero. FIG. 5 is a partial plan view showing a close-packed layout using a regular hexagonal well having a side of a well opening of 100 μm and a distance between adjacent wells of 20 μm. FIG. 6 is a partial plan view showing a close-packed layout using a regular hexagonal well having a well-to-well dimension (distance between two parallel sides facing each other) of 100 μm and a distance between adjacent wells of zero. FIG. 6 is a partial plan view showing a close-packed layout in which a regular hexagonal well having a well-to-well dimension (distance between two parallel sides facing each other) of 100 μm is used and a distance between adjacent wells is 20 μm. FIG. 6 is a partial plan view showing a close-packed filling layout using a regular octagonal well having a well opening dimension (distance between diagonal vertices) of 140 μm and a distance between adjacent wells of zero. FIG. 6 is a partial plan view showing a close-packed layout in which a regular octagonal well having a well-to-well dimension (distance between diagonal vertices) of 140 μm is used and a distance between adjacent wells is 20 μm. FIG. 6 is a partial plan view showing a close-packed layout using a circular well having a well opening dimension (diameter) of 100 μm and a distance between adjacent wells of zero.

  Next, the present invention will be described in more detail based on the embodiments shown in the accompanying drawings. FIG. 1 shows a part of the manufacturing method of the present invention and is a principle diagram of X-ray lithography using synchrotron radiation. By this X-ray lithography, a concave shape that is the same as the shape of the well is formed on the substrate, a mold insert (convex shape) is created by nickel electroforming using the substrate, and it is set in an injection mold. Then, a microchemical device is manufactured by injection molding with a synthetic resin.

  As shown in FIG. 1, X-ray lithography using synchrotron radiation means that an arbitrary amount of X-ray mask 2 is translated relative to a PMMA (polymethyl methacrylate resin) substrate 3 when irradiated with synchrotron radiation 1. Thus, the X-ray dose irradiated to the PMMA substrate 3 which is a positive resist is controlled. By controlling the X-ray dose, it is possible to form the well 4 having an arbitrary inclination on the PMMA substrate 3. The X-ray mask 2 includes an X-ray transmission part 2A and an X-ray non-transmission part 2B. The shape of the well 4 formed on the PMMA substrate 3 is determined by the shape of the X-ray transmission part 2A.

  That is, after determining the shape of one well including the side wall inclination angle, an X-ray mask is manufactured. By controlling the pattern shape of the X-ray mask and the X-ray mask movement amount, The distance can be in the range from zero to one well or less. Therefore, the well layout may be a lattice shape or a staggered shape.

  Furthermore, it becomes possible to manufacture a large number of microwells at a distance between adjacent portions of 1 well or less with a nano-level shape accuracy over the entire X-ray irradiation amount. Furthermore, when a convex shape having a shape of 10 μm to several hundred μm is processed directly from the mold steel material, a processing radius is attached to the side wall corner portion, but in this processing process, there is a feature that the processing radius is not attached. However, since the X-ray mask 2 and the PMMA substrate 3 are relatively moved, it is necessary to provide a movement clearance between them. Even when X-rays having a sub-nanometer wavelength are used, depending on the pattern size formed on the PMMA substrate 3, this clearance causes X-ray diffraction, and the formed pattern portion is rounded. Therefore, when a pattern having a pattern size of about several tens of μm, which is the pattern size of the present application, is formed, the moving clearance needs to be about 500 μm at maximum.

  2 and 3, when the X-ray transmission part 2A has the same square shape and the same pitch, but the X-ray mask movement amount is changed using the X-ray mask 2 having a different size, the PMMA is changed. The shape and size of the well 4 formed on the substrate 3 are shown. The X-ray mask 2 in FIG. 2 has a small X-ray transmission part 2A, and the X-ray mask 2 in FIG. 3 has a large X-ray transmission part 2A.

First, in FIG. 2, the initial position is set to L ₀ with reference to the edge position of the pattern of the X-ray mask, and it is set between L ₁ and L _{2 in} the left-right direction (this direction is the X-axis direction) (L ₁ − L ₂ corresponds to the movement distance of the X-ray mask), and the X-ray mask 2 is moved in the X-axis direction and the Y-axis direction respectively by a certain constant-speed circular motion, and is indicated by a solid line on the PMMA substrate 3 Such a well 4 is formed. By controlling the amount of movement of the X-ray mask 2 at a constant speed with high accuracy, the inclined surface of the well side wall becomes linear. In this case, the well 4 is formed with a flat bottom and has a quadrangular pyramid shape as a whole. The depth of the well 4 can be controlled by the X-ray dose (Dose). Then, when the X-ray mask 2 is moved in the X-axis direction and the Y-axis direction by constant-velocity circular motion between L ₃ and L ₄ , a well 4 as shown by a dotted line is formed. L ₄ represents the adjacent X-ray transmitting portions 2A, corresponds to an intermediate position 2A, in this case, the spacing between adjacent wells 4,4 formed becomes zero. Further, L ₃ is as corresponding to the center line of the X-ray transmitting portion 2A, the bottom of the well 4 becomes one point, well 4 becomes quadrangular pyramid. The X-ray mask 2 is formed of an X-ray absorbing material (for example, gold, copper, tantalum, tungsten) on a substrate made of an X-ray transmitting material (for example, silicon carbide, silicon nitride, carbon, silicon, polymer film). Any shape may be used as long as the shape 2 is patterned.

Next, in FIG. 3, similarly, the X-ray mask 2 is moved in the X-axis direction and the Y-axis direction by arbitrary constant-speed circular movements between L ₁ and L ₂ in the left-right direction around the initial position L _0. By moving, a square pyramid shaped well 4 is formed on the PMMA substrate 3 as indicated by a solid line. Further, when the amount of movement of the X-ray mask 2 is increased and moved between L ₃ and L ₄ in the X-axis direction and the Y-axis direction by constant-velocity circular motion, the well 4 having a quadrangular pyramid shape as shown by the dotted line Is formed. L ₄ represents the adjacent X-ray transmitting portions 2A, corresponds to an intermediate position 2A, in this case, the spacing between adjacent wells 4,4 formed becomes zero. In this case, since L ₃ does not reach the center line of the X-ray transmission part 2 A, a flat bottom is always formed in the well 4.

  In general, if the dose of X-rays (Dose) is the same, the greater the amount of movement of the X-ray mask 2, the greater the well sidewall inclination angle (angle relative to the plane perpendicular to the surface of the substrate 3). As the X-ray irradiation amount (Dose) increases, the well sidewall inclination angle decreases and the depth increases. Accordingly, when the distance between adjacent wells, the well sidewall inclination angle, and the well depth are determined, the shape and movement amount of the X-ray transmission part 2A of the X-ray mask 2 and the amount of X-ray irradiation are controlled accordingly. Get shape. Further, the well well pattern can be formed on the PMMA substrate by moving the X-ray mask with high accuracy by the same method when the planar shape of the well is circular or polygonal. However, since PMMA belongs to the category of positive resist, in order to form a well on a PMMA substrate, a predetermined amount of X-rays is irradiated onto the PMMA substrate, and then this PMMA substrate is separately mixed with a morpholine-alcohol mixed solvent. It is necessary to soak for a predetermined time.

  On the other hand, the nickel electroforming method using the PMMA substrate obtained by the X-ray lithography method as a master can be dealt with by using a nickel electroforming process using a general nickel sulfamate bath. In this process, the electroconductive thin film is formed on the fine pattern surface of the PMMA substrate by electroless plating, vapor deposition, or sputtering while paying attention to the film thickness particularly when the pattern size is several tens of μm. Do.

  In the case of pure nickel, the surface hardness of the finished nickel electroforming mold insert is about 300 Hv (Vickers hardness), but the surface hardness can be obtained by alloying with Ni such as Ni-Fe, Ni-P, Ni-Co, etc. The hardness can be increased from 700 Hv to 1000 Hv, and the durability of the mold insert is improved.

  The size of the well depends on the size of the microorganism, cell, or protein to be observed. For example, the size of various cells is 10 μm or less for yeast cells, 20 μm or less for CHO cells, several μm for E. coli, 7-8 μm for red blood cells and 20-30 μm for white blood cells, and mammalian mature egg cells are Approximately 150 μm, ES cells are several hundred μm, and Hela cells are 20 to 40 μm. Therefore, in the present invention, the size of the microwell is 10 to 500 μm, preferably 10 to 300 μm.

  Regarding the well pattern, in order to develop the same shape on the two-dimensional plane on the entire two-dimensional surface without a gap, there are a triangle, a square, and a hexagon. Accordingly, the same number of triangles, squares, and hexagons can be formed within the unit area if they have the same area geometrically. When the distance between wells (distance between adjacent wells) becomes long, the number of wells existing in the field of view even at the same magnification when observing cells in the well with an observation device such as an optical microscope, laser microscope, or fluorescence microscope And more cells that are not in the wells (between the wells). If the number of cells increases between the wells, there is an increased risk that the cells will be washed away by various solution treatments when observing the cells, and there is a problem that cells that are not in a predetermined position cannot be analyzed. For this reason, there is an increasing demand for devices having zero distance between adjacent wells. When manufacturing a microchemical device in which the distance between adjacent wells is reduced to one well or less by injection molding of synthetic resin, the setting of the sidewall inclination angle of the well becomes more important as the distance between adjacent wells becomes shorter. . This sidewall inclination angle is the optimum inclination angle for the melt to completely fill the well pattern when the device is produced by injection molding, and the optimum inclination angle for smoothly introducing the cells into the well. .

  In particular, in order to smoothly introduce cells into the well, the hydrophilic treatment of the device is required. By setting the side wall inclination angle of the well in the range of 5 degrees to 25 degrees, the hydrophilicity level on the device May be controlled in the range of 60 degrees to 40 degrees in the evaluation of the water droplet contact angle using water.

  As described above, when forming a square well over the entire plane, the bottom of the well depends on the size of the square formed on the X-ray mask, the arrangement of the squares (distance between pitches), and the amount of movement of the X-ray mask. Can be changed from a flat shape to a vertex shape of a quadrangular pyramid. Of course, by controlling the amount of movement of the X-ray mask, the distance between adjacent wells can be changed from zero (adhesion) to one well.

  Synthetic resins that can be used in the present invention are materials such as polystyrene, acrylic resin, polycarbonate, and cyclic olefin polymers, which are typically cured in the mold by melting the polymer (pellets) in the cylinder of an injection molding machine. As long as there is no restriction on the use, it is possible to use the polymer alone, and even a composite material to which graphite (graphite) is added can be used as long as the fluidity of the base polymer is not significantly reduced.

  ΦX and φY in Table 1 are the frontage dimensions of the truncated conical well 4 shown in FIG. That is, φX is a frontage dimension when the well side wall inclination angle is zero, and φY is a frontage dimension when the well side wall inclination angle is not zero. FIG. 4 shows a state in which spherical cells C are fitted in the well 4 without a gap. When the well depth is the same as the frontage dimension (well aspect ratio: 1), the well depth is 70 which is the frontage dimension. % (Well aspect ratio: 0.7) is also shown.

  When the well aspect ratio is 0.7, when the well side wall inclination angle is larger than 25 degrees, the frontage dimension is increased by 30% compared to the zero inclination angle. Furthermore, when the well aspect ratio is 1, when the well inclination angle is 30 degrees, it is about 1.7 times that when the well inclination angle is zero, and when the well aspect ratio is 1.2, it is about twice. It will spread. In addition, widening the well opening has the advantage of facilitating the entry of cells into the well, but there is also a risk that the cells will exit the well during cell washing. Therefore, the upper limit of the well sidewall inclination angle is set to 25 degrees. On the other hand, when the tilt angle is less than 5 degrees, the device cannot be smoothly released from the mold due to the effect of cooling and shrinkage of the melt during injection molding. Therefore, the lower limit of the well sidewall inclination angle is set to 5 degrees. That is, the well sidewall inclination angle is optimally in the range of 5 to 25 degrees.

  In addition, when the depth of the well becomes shallower than the diameter of the cell, the upper part of the cell is exposed from the well, which is preferable because solution processing and pick-up of cells and the like are easy to perform, but if the depth of the well becomes too shallow The cells cannot be stably held in the well.

  FIG. 5 shows a preferred basic shape of the well. (A) is a square square frustum-shaped well, (b) is a rectangular quadrangular frustum-shaped well, and (c) is a central sectional view of both. Further, (d) is a hexagonal frustum-shaped well having a regular hexagonal shape, (e) is a hexagonal frustum-shaped well having a horizontally long hexagonal shape, and (f) is a central sectional view of both. (G) shows a truncated cone-shaped well, and (h) shows a central sectional view thereof. If the dimensions in the figure are used, a range of A × 0.2 <d <A × 1.2 is preferable, and B is arbitrary (preferably within 300 μm). Here, d is the depth of the well. Further, the range of C × 0.2 <d <C × 1.2 is preferable, and E is arbitrary (preferably within 300 μm). And the range of Fx0.2 <d <Fx1.2 is preferable.

  Next, examples of layouts using basic wells are shown in FIGS. Each well has a depth of 100 μm and a well sidewall inclination angle of 20 degrees. 6 to 8 are layouts using square wells having a well opening dimension of 100 μm and a bottom dimension of 28 μm per side. 6 is a grid-like close-packed layout in which the distance between adjacent wells is zero and the wells are arranged vertically and horizontally, FIG. 7 is a staggered close-packed packing layout in which the distance between adjacent wells is zero and the wells are staggered, and FIG. A staggered close-packed layout in which the distance between adjacent wells is 20 μm and the wells are arranged in a staggered manner is shown.

  9 and 10 are layouts using regular hexagonal wells with a side of the well opening of 100 μm, FIG. 9 shows a close-packed layout with zero distance between adjacent wells, and FIG. 10 shows the distance between adjacent wells. A 20 μm close-packed layout is shown.

  11 and FIG. 12 are layouts using regular hexagonal wells having well well dimensions (distance between two parallel sides facing each other) of 100 μm, and FIG. 11 shows a close-packed layout with zero distance between adjacent wells. FIG. 12 shows a close-packed layout with a distance between adjacent wells of 20 μm.

  13 and 14 are layouts using regular octagonal wells having a well opening dimension (distance between diagonal vertices) of 140 μm, and FIG. 13 shows a close-packed layout with zero distance between adjacent wells. Indicates a close-packed layout with a distance between adjacent wells of 20 μm. However, in the case of this layout, a rectangular flat portion is formed at a position surrounded by four wells, but the intervals are smaller than the well opening dimensions.

  Finally, FIG. 15 shows a close-packed layout in which the distance between adjacent wells is zero with a layout using circular wells having a well opening dimension (diameter) of 100 μm.

DESCRIPTION OF SYMBOLS 1 Synchrotron radiation 2 X-ray mask 2A X-ray transmissive part 2B X-ray opaque part 3 Substrate 4 Well C cell

Claims (1)

Hundreds of thousands of microwells for holding microorganisms, cells or proteins to be observed are integrated, and are microchemical devices for observation with an optical microscope, a fluorescence microscope or a laser microscope,
When irradiating the X-ray from the X-ray source, the X-ray mask and the resist substrate are relatively translated by a calculated arbitrary distance, whereby the X-ray dose irradiated to the resist substrate through the pattern shape on the X-ray mask. And a tilt angle of 5 degrees to 25 degrees corresponding to the relative translation between the X-ray mask and the resist substrate is provided on the side wall of the well formed on the resist substrate. A mold insert is manufactured by nickel electroforming after conducting a conductive treatment, and by injection molding of synthetic resin using the mold insert, the shape of one microwell is 10 μm to 300 μm, and the well sidewall is inclined. The angle is in the range of 5 degrees to 25 degrees, the well aspect ratio is 0.7 to 1, and the distance between adjacent wells is accumulated at a distance equal to or smaller than the well dimension of one well. Injection molded micro chemical device.

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