JP4656038B2 - Electrostatic spraying method and microfluidic chip - Google Patents

Description

  The present invention relates to an electrostatic spraying method for forming a bioactive substance in a desired pattern on a substrate and a microfluidic chip formed by bonding a flow path member to the substrate.

  As a method for forming a biologically active substance having a desired shape on a substrate in a state where the biological activity is maintained by electrostatically spraying a solution containing the biologically active substance, a method described in Patent Document 1 is disclosed. In Patent Document 1, in order to form a bioactive substance in a desired shape on a substrate, a member in which a through hole called a mask is formed is used. A bioactive substance is applied onto the substrate in substantially the same shape as the shape of the through hole formed in the mask. There are several techniques for applying a biologically active substance on a substrate other than the technique using electrostatic spray described in Patent Document 1, but in the method described in Patent Document 1, the biologically active substance is applied in a dry state. It has the feature that it can be applied to the substrate surface.

In methods other than electrostatic spraying, it has been found that since the bioactive substance is applied onto the substrate in an incompletely dried state or in a solution state, uneven coating due to uneven drying occurs after coating. In the method by electrostatic spraying disclosed in Patent Document 1, since the biologically active substance in a dry state can be coated on the substrate, drying unevenness after coating does not occur, compared with methods other than electrostatic spraying. , It is characterized by excellent coating uniformity.
JP 2001-281252 A

  However, in the method of Patent Document 1, it is known that the charged state of a mask made of an insulating material affects the sprayed solution, and the biological activity formed on the substrate is increased as the mask is charged more. The shape of the material becomes thin, and the coating uniformity also varies. In order to obtain a highly accurate shape and high coating uniformity, it was necessary to control the charged state of the mask to be always constant. The charge state of the mask can be controlled to some extent by controlling the humidity and removing electricity with an ionizer, but it was difficult to control the charge state to the extent that the shape and uniformity of the bioactive substance were not affected. . As a general method for measuring coating uniformity, there is a method in which coating uniformity is evaluated by coating a solution containing a fluorescent substance on a substrate and measuring the fluorescence value after coating. The coating uniformity is generally represented by a numerical value called a coefficient of variation (CV) value. In this specification, the CV value is used as a value representing the coating uniformity. In this specification, the CV value is a value obtained by dividing the standard deviation of the fluorescence measurement value by the average of the fluorescence measurement value and multiplying by 100. That is. It can be said that the smaller the CV value, the better the coating method with less coating unevenness and shape defects.

  The biochip produced using the method of Patent Document 1 has insufficient coating uniformity, and improvement has been desired.

  As a means for improving the coating uniformity, there is a method using a substrate in which a conductive layer 2 is formed on the surface of the substrate 1 as shown in FIGS. 13A and 13B, and the conductive layer 2 is processed by photolithography and etching. By doing so, highly accurate patterning is possible, and since the bioactive substance can be applied in a shape that matches the conductive layer patterned with high accuracy, the application uniformity can be improved. However, in the substrates shown in FIGS. 13A and 13B, the conductive layer 2 has a convex shape by the level difference 62, and therefore, in a fluid chip application that needs to be bonded to the flow path member 6, the conductive layer There was a possibility that a gap 61 was generated around 2 and the liquid flowing into the flow path from the gap 61 leaked out. In actual use, the conventional substrate having the convex conductive layer shown in FIGS. 13 (a) and 13 (b) also uses polydimethylsiloxane as the flow path member. Since it is easily deformed like rubber, it is possible to prevent leakage of the liquid flowing in the flow path because the flow path member itself is deformed and is in close contact with the substrate if there are some steps. . However, when the interval 10 between the channels formed in the channel member 6 is narrowed, the thickness of the wall separating the adjacent channels is reduced, and the gap 61 between the substrate and the channel member 6 is partially adjacent. There was a problem that the flow paths connected to each other were connected. For this reason, when a liquid is caused to flow through the flow path, liquid leakage may occur in the adjacent flow path.

  The present invention solves the conventional problems, and provides a substrate that applies a biologically active substance in a predetermined shape with good uniformity and does not cause leakage of liquid flowing in the flow path even when combined with the flow path member. The purpose is to do. A method for manufacturing a substrate is also provided.

  In order to solve the conventional problems, the electrostatic spraying method of the present invention includes a step of applying a resist to form a predetermined pattern layer on a substrate and exposing and developing through a mask having the predetermined pattern. A step of blasting the developed substrate, and forming a conductive layer in a stepped groove pattern formed between the base material surface of the resist-coated surface and the base material surface of the non-resist-coated surface by the blast processing. And a step of removing the resist after film formation of the conductive layer and electrostatically spraying a biologically active substance through a cover having a through hole corresponding to the shape of the conductive layer. The bioactive substance can be formed in a desired pattern with good uniformity.

  Further, the microfluidic chip of the present invention is a microfluidic chip for inspecting a biologically active substance, and a flow path member comprising a groove portion that is arranged at both ends and is connected to the injection port to form a flow path, Using the electrostatic spraying method according to claim 1, the bioactive substance is bonded to a coated substrate.

  When the electrostatic spraying method described in the present invention is used, it is possible to form a biologically active substance on a substrate in a desired shape with good uniformity, and a liquid that has flowed through the flow path even when combined with the flow path member. Will not leak.

  Hereinafter, fabrication of a microfluidic chip using the electrostatic spraying method of the present invention will be described in detail with reference to the drawings. Here, the biologically active substance itself is a liquid, or contains a solution mixed with a diluent or a test reagent for the biologically active substance, and hereinafter simply referred to as a biologically active substance. The biologically active substance generally refers to DNA, protein, and the like, but of course, it is naturally applicable to a solution containing a substance other than the biologically active substance.

FIG. 1 is a diagram showing an example of a microfluidic chip created using the electrostatic spraying method of the present invention. The illustrated microfluidic chip has a structure in which a flow path member 6 and a glass substrate 1 are bonded together. In FIG. 1, a substrate 1 uses glass as a material, and an electrically conductive layer 2 is formed on the substrate 1. Then, the bioactive substance 3 is applied on the conductive layer 2. As the substrate material, plastics other than glass, insulating resin materials, and the like can be used. And, a flow path 9 is formed on the bonding surface 11 of the flow path member 6 and is connected to the injection port 7 and the injection port 8. It has become. Further, the flow path 9 is not closed on the bonding surface 11 side. That is, it has a structure in which a groove is formed on the bonding surface 11, and by bonding to the substrate 1, a closed flow path connecting between the inlet 8 and the outlet 9 is formed. FIG. 2 is a view for explaining the characteristics of the microfluidic chip of FIGS. 1 (a) and 1 (b). The conventional microfluidic chip has a structure in which the conductive layer 2 is convex on the surface of the substrate 1 by a step 62, as shown in FIGS. 13 (a), 13 (b) and FIG. The substrate of the present invention has a structure in which the surface of the substrate 1 and the surface of the conductive layer 2 coincide as shown in FIG. That is, the surface that contacts the bonding surface 11 of the flow path member 6 is flat. Therefore, the gap 61 generated due to the step 62 of the conductive layer 2 as shown in FIGS. 13 and 14 does not occur in the substrate of the present invention. Therefore, there is no leakage to the adjacent flow path.

  In the microfluidic chip thus prepared, a predetermined reagent solution is injected from the injection port 7 or the injection port 8 and reacted with the biologically active material formed on the conductive layer 2 to cause the biologically active material to react. Can be inspected.

  FIG. 3 is a diagram showing a detailed structure of the glass substrate 1. In FIG. 3, reference numeral 1 denotes a substrate, which is made of glass. Reference numeral 2 denotes a conductive layer having electrical conductivity. 3 is a biologically active substance. In this example, fluorescent dye-labeled antibody protein (Alexa Fluor 568-labeled Anti-goat IgG manufactured by Molecular Probes) was used. The biologically active substance 3 is formed on the conductive layer 2 in a shape that substantially matches the conductive layer 2. Reference numeral 4 denotes a connection line, which is connected to all of the ten conductive layers 2 and the connection portion 5 so as to be electrically conductive. The potentials of the connection line 4 and the conductive layer 2 are applied to the connection portion 5. It is configured to be equal to the generated potential.

The substrate processing method shown in FIG. 3 will be described in detail with reference to FIG.
(1) From the resist coating process to the film forming process First, as shown in FIG. 4, the resist 13 is uniformly coated on the substrate 1 in the resist coating process. The resist 13 is applied by spin coating. In this example, OFPR800 (viscosity 30 cp) was used as the resist 13. Next, in the exposure step, the light 13 is irradiated to the resist 13 through the mask 14. Since the resist 13 used in this embodiment is a positive resist, the mask 14 is designed so as to be able to expose the resist 13 in a shape corresponding to the region to be blasted on the surface of the substrate 1. Since the region of the mask 14 that shields the light 15 can be freely designed when the mask 14 is manufactured, the resist 13 can be exposed to a desired shape.

  After the resist 13 was exposed to light 15 as described above, it was immersed in the developer 16 and developed. NMD-3 was used as the developer 16. After the development, as shown in the drawing of the development process, the resist 13 is melted at the portion irradiated with the light 15, and the resist 13 remains in a shape that matches the shape of the region where the light 15 is shielded. The thickness of the resist 13 was 10 μm.

  Blasting is performed on the surface of the substrate 1 on which the resist 13 has been formed after the development process.

  The surface of the substrate 1 on which the resist 13 is formed is processed by blasting so that a step 17 is formed between a portion where the resist 13 is not formed and a portion where the resist 13 is formed. In this example, silica particles having an average particle diameter of 7 μm were used for blasting, and the air pressure for ejecting the particles was 0.5 MPa. The processing time was 12 seconds. If the particles to be used and the air pressure are the same, the processing depth is substantially proportional to the processing time, and therefore it is possible to control the depth by controlling the processing time. Under the conditions of this example, the step 17 was about 0.2 μm.

  Further, at the time of blast processing, the surface roughness of the portion where the resist is not formed is processed so that the arithmetic average roughness (hereinafter abbreviated as Ra) is in the range of 0.7 nm to 100 nm in the measurement range of 1 μm square. And the conductive layer 2 was formed in the film-forming process after this. The means for forming the conductive layer 2 will be described later. An atomic force microscope was used to measure the surface roughness. When the surface roughness is set to Ra 0.7 nm or more, even if a peeling test is performed by attaching a tape to the surface of the conductive layer 2 after the conductive layer 2 is formed, no peeling of the conductive layer 2 is observed. It was found that a peel strength without problems can be obtained. When the surface roughness was Ra 0.7 nm or less, the conductive layer 2 was partially peeled. Conventionally, when the conductive layer 2 is formed on the substrate 1, an intermediate layer that can ensure adhesion is formed by the material of the conductive layer 2 and the substrate 1. For example, in indium tin oxide, silicon oxide was used as the intermediate layer. On the other hand, if the method of the present invention is used, an intermediate layer is not required, so that the process can be simplified and productivity can be improved.

  Further, it was found that when the surface roughness of the portion where the resist is not formed is rougher than 100 nm, the coating uniformity of the bioactive substance 3 is deteriorated. Since the portion where the resist is not formed becomes an underlayer of the conductive layer 2, when the surface roughness is 100 nm or more, large irregularities are generated on the surface of the conductive layer 2, and the bioactive substance 3 on the surface of the conductive layer 2 is formed. It is considered that the adhesion state is affected by large unevenness and becomes non-uniform, which causes the deterioration of uniformity. Therefore, it is desirable that the portion where the resist after blasting is not formed, that is, the portion where the conductive layer 2 is formed, is in the range of 0.7 nm to 100 nm in Ra.

  After the blast processing, the conductive layer 2 was formed using the apparatus shown in FIG. In FIG. 6, 37 is a vacuum chamber, 38 is a substrate holder made of a conductor for fixing the substrate 1, and 39 is a high frequency power source for applying high frequency power to the substrate holder 38 made of a conductor. , 40 is an exhaust device that exhausts the vacuum chamber 37 to a vacuum, 41 is a main valve that connects the vacuum chamber 37 and the exhaust device 40, 42 is a vapor deposition boat that heats and evaporates metal, and 43 is a vapor deposition material 44 is a gas introduction pipe for introducing a predetermined gas into the vacuum chamber 37, and 45 is a ground.

  In this embodiment, the case where copper is used for the vapor deposition material 43 and the conductive layer 2 is formed of copper is described. However, as the material of the conductive layer 2, for example, metal other than copper, such as gold, silver, platinum, and aluminum Alternatively, non-metals such as indium tin oxide (ITO) can be used if they are conductive. In particular, the bioactive substance 3 attached to the substrate 1 may be optically observed from both the front and back of the substrate 1, and in such applications, a transparent and conductive material such as ITO is used as a thin film material. There is a need.

As a procedure for forming the conductive layer 2, first, the substrate 1 is placed on the substrate holder 38, and then the vacuum chamber 37 is evacuated. The pressure in the vacuum chamber 37 is below 1.0 × 10 ^-3 Pa.
Next, the surface of the glass substrate 1 is subjected to plasma treatment. The plasma treatment is performed for the purpose of roughening the surface of the glass substrate 1. During the plasma processing, nitrogen gas having a purity of 99.9% or more is introduced into the vacuum chamber 37, the pressure is set to 5.0 × 10 ⁻² Pa, and the high frequency power is supplied to the substrate holder 38 supporting the glass substrate 1 by the high frequency power supply 39. Apply. By applying the high frequency power, glow discharge is generated in the vacuum chamber 37 and nitrogen plasma is generated.

In the substrate 1 shown in FIG. 6, the first negative bias voltage is induced on the substrate 1 with the generation of nitrogen plasma, and the substrate 1 is subjected to the nitrogen plasma treatment. In this embodiment, the first negative bias voltage is 300V. After the nitrogen plasma treatment, the pressure in the vacuum chamber 37 is evacuated to 1.0 × 10 ⁻⁴ Pa or less. Argon gas is introduced after evacuation, the pressure in the vacuum chamber 37 is set to 1.0 × 10 ⁻² Pa, copper is evaporated from the vapor deposition boat 42, and a high frequency voltage is applied to the substrate holder 38 that supports the substrate 1. . Glow discharge is generated in the vacuum chamber 37 by the application of the high-frequency voltage, and argon and copper plasma is generated. Since the second negative bias voltage is induced in the glass substrate 1 with the generation of the argon and copper plasma, the argon ions and copper ions in the plasma are accelerated by the second negative bias voltage, and the surface of the substrate 1 is accelerated. It is accelerated toward.

In this embodiment, the second negative bias voltage is set to 400 V and the copper film is formed. A copper thin film is formed on the surface of the substrate 1 due to the effect of copper ions colliding with the surface of the glass substrate 1 to form a copper thin film and the effect of non-ionized copper being vacuum deposited. In addition, the film formation rate was controlled to gradually increase from the start of film formation to the end between 0.1 nm / sec and 10 nm / sec. Film formation was performed so that the film thickness was 0.2 μm under the above conditions.
(2) About Resist Removal Step Next, the resist removal step will be described as shown in FIG. The substrate 1 on which the copper thin film is formed is immersed in a resist stripping solution 20. Acetone was used as the resist stripping solution 20. Simultaneously with the peeling of the resist 13, the copper thin film formed on the resist is removed, and the surface of the substrate 1 becomes a substantially flat surface. In the case of producing a complete plane, it can be realized by grinding the surface of the substrate 1 after the resist removing step.

  By the above procedure, a substrate on which the patterned conductive layer 2 was formed on the substrate 1 was produced. The conductive layer 2 is processed into a shape that matches the shape of the region through which the light 15 is transmitted in the mask 14. Since the shape of the region through which the light 15 is transmitted can be freely produced when the mask 14 is designed and manufactured, the conductive layer 2 having an arbitrary shape can be formed on the substrate 1 by the above procedure.

Unlike the present embodiment, when a negative resist is used for the resist 13, the mask 14 is produced so that the portion of the mask 14 where the light 15 is shielded coincides with the region to be blasted on the surface of the substrate 1. Thus, the substrate 1 on which the conductive layer 2 having a desired shape is formed can be manufactured.
(3) About the spraying process 1 and the spraying process 2 Next, in the process of FIG.4 (g) to FIG.4 (h), the board | substrate produced at the process to FIG.4 (f) is used for the apparatus shown in FIG. The bioactive substance 3 was applied on the conductive layer 2 of the substrate 1.

  Reference numeral 46 shown in FIG. 7 denotes a capillary, and reference numeral 47 denotes a substrate after the resist removing process, which is arranged below the capillary 46. Reference numeral 22 denotes a high voltage power source, and the negative pole of the power source is connected to the substrate 47 after the resist is removed. The positive electrode side of the high voltage power supply 22 is connected to an electrode line 36 inserted into the capillary 46. The high voltage power supply 22 is configured to generate a potential difference between the capillary 46 and the substrate 47 after resist removal. A controller 48 is connected to the high voltage power supply 22 and controls the voltage generated by the high voltage power supply 22. Reference numeral 21 denotes a cover having a through hole 55 as shown in FIG. The dimension 56 of the through hole 55 was 1.5 mm. The dimension 57 of the through hole 55 was 14 mm.

  The cover 21 is disposed between the capillary 46 and the substrate 47 after the resist is removed, and is aligned so that the portion to which the bioactive substance 3 on the surface of the substrate 47 after the resist is removed overlaps the through hole 55. An effective method for aligning the cover 21 and the substrate will be described in Example 2 described later.

  In this embodiment, a voltage of 3 kV was applied between the capillary 46 and the substrate 47 after resist removal by the high voltage power supply 22. The distance from the tip of the capillary 46 to the surface of the substrate 47 after resist removal was 35 mm, and the tip of the capillary 46 had an outer shape of 50 μm. As a material for the cover 21, glass epoxy having a thickness of 0.1 mm was used. The amount of spraying was 1 μl sprayed by measuring the liquid height of the solution in the capillary with a CCD camera and measuring the image. A solution is supplied into the capillary from the side opposite to the capillary tip. When the solution supply is continuously connected to the capillary by connecting the solution supply pipe, it becomes impossible to observe the change in the height of the solution in the capillary with a CCD camera. The liquid was supplied from the side opposite to the capillary tip, and the liquid height after the supply of the solution was made observable with a CCD camera.

  In this example, as shown in the spraying process 1 in FIG. 4, the bioactive substance 3 was attached to only one portion of the conductive layer 2. Further, the gap 54 between the cover 21 and the surface of the conductive layer 2 was arranged to be 0.05 mm or less. It was found that when the distance 54 is 0.05 mm or more, the shape of the bioactive substance 3 formed on the conductive layer 2 is smaller than the shape of the conductive layer 2.

  The inventors consider the cause as follows. When spraying is started, droplets of the positively charged bioactive substance 3 are generated. The charged droplets diffuse and adhere to the cover 21 and the outer wall of the apparatus. When the attached portion is insulative, the portion to which the droplet is attached is positively charged due to the positive charge of the droplet. In this embodiment, the cover 21 is made of glass epoxy and is an insulating material. Therefore, it is considered that the cover 21 is positively charged during spraying. Since the cover 21 is positively charged and the droplet passing through the through hole 55 of the cover 21 is also positively charged, the droplet receives an electric repulsive force from the cover 21 and is smaller than the through hole 55. It adheres on the conductive layer 2 in shape. When the distance from the cover 21 to the conductive layer 2 is long, the droplet receives a repulsive force due to charging of the cover 21 at a position away from the conductive layer 2, and changes due to the electric repulsive force from the cover 21. A long distance is required to reach the conductive layer 2 in the orbit. Therefore, when the sprayed droplets of the bioactive substance 3 reach the conductive layer 2, the conductive layer has a shape smaller than the shape of the through hole 55 compared to the case where the distance from the cover 21 to the conductive layer 2 is short. 2 is considered to adhere to the surface. The dimensions 56 and 57 are preferably larger than the dimensions of the conductive layer 2. This is because when the dimensions 56 and 57 are the same as those of the conductive layer 2, the sprayed bioactive substance 3 is deposited on the conductive layer 2 with a smaller dimension than that of the conductive layer 2. I think it is caused by electrification. In this example, while the dimension 56 is 1.5 mm and the dimension 57 is 14 mm, the dimensions of the conductive layer 2 are 0.8 mm and 11 mm, and the dimensions 56 and 57 are made large.

  The result of having observed the surface of the board | substrate produced by the spraying process 1 shown in FIG. 4 with the fluorescence microscope is shown in FIG. For fluorescence measurement, a fluorescent microscope BX51W1 manufactured by OLYMPUS was used. The observation magnification was 1 ×, the sample was irradiated with excitation light with a wavelength of 540 nm to 590 nm for 1 second at an output of 20 mW, and the fluorescence intensity of the sample was measured through a filter that transmitted light between 615 nm and 695 nm. . The experiment was performed three times, and the fluorescence intensity measured for each result as Sample 1, Sample 2, and Sample 3 is described. FIG. 12 shows the shape of the conductive layer 2 of the manufactured sample. In FIG. 12, the measurement points A to G are divided into equal intervals with the measurement point A on the conductive layer 2 closer to the connection line 4 and the measurement point G on the side far from the connection line 4. Points B to F were set. In FIG. 12, 59 is the measurement point A and 60 is the measurement point G.

  In addition, the inventors have made similar studies using a substrate having a structure shown in FIGS. 13 (a) and 13 (b). In the substrates shown in FIGS. 13A and 13B, the conductive layer 2 formed on the substrate surface has a convex shape of dimension 62. Such a substrate can be formed by etching after forming the conductive layer 2 on the substrate 1, and there is no need to process the surface of the substrate 1 before forming the conductive layer 2 as in the method of the present invention. Therefore, there is an advantage that the conductive layer 2 can be formed by a relatively simple procedure. However, since the conductive layer 2 has a convex shape corresponding to the dimension 62, a gap 61 is generated between the channel member 6 and connected to the adjacent channel by the gap 61, so that liquid flows into the channel. In such a case, the liquid may leak into the adjacent flow path. In the substrate of the present invention, the surface of the conductive layer 2 coincides with the surface of the substrate 1 and is flat. Therefore, it is considered that liquid leakage to the adjacent flow path, which has been a problem in the past, is eliminated. For comparison of the performance when a microfluidic chip is manufactured using the substrate shown in FIGS. 13 (a) and 13 (b) and the substrate of the present invention, the substrate shown in FIGS. 13 (a) and 13 (b) is used. Were also evaluated in the same manner as the substrate of the present invention. The results are shown in FIG.

  In the substrate in which the conductive layer 2 is formed in a convex shape on the surface of the substrate 1 shown in FIGS. 13A and 13B, the CV value is 2.1% to 3.2% in three experiments. The value is obtained. On the other hand, with the substrate having a flat surface of the substrate 1 according to the present invention, a CV value of 1.9% to 3.5% was obtained in three experiments. It has been found that the coating uniformity equivalent to that of the substrate on which the conductive layer 2 is formed so as to have a convex shape on the surface of the substrate 1 has little influence on the coating uniformity.

  As far as the inventors know, there is no report that uniformity of about 3% CV value was obtained by electrostatic spraying.

  The inventors consider the reason why the coating uniformity can be improved as compared with the conventional electrostatic spraying technique disclosed in Patent Document 1.

  In the conventional electrostatic spray technique described in Patent Document 1, the substrate surface is covered with a member having a through hole called a mask, so that the bioactive substance is applied onto the substrate in a shape corresponding to the shape of the through hole.

  By the method of Patent Document 1, it is possible to apply the bioactive substance in a shape corresponding to the shape of the through hole of the mask. However, it is known that the charged state of the mask affects the sprayed solution, and the higher the charge of the mask, the thinner the shape of the bioactive substance that is formed. In order to form the biologically active substance on the substrate, it was necessary to manage the charged state of the mask so as to be always constant. The charged state of the mask can be controlled to some extent by controlling the humidity and removing the charge with an ionizer, but it is difficult to control the charged state to the extent that it does not affect the shape and uniformity of the bioactive substance. It was.

  In the method of the present invention, the conductive layer 2 is formed in a shape that matches the shape to which the bioactive substance 3 is to be applied to the surface of the substrate 1, and the conductive layer 2 is charged positively by being grounded or at a negative potential. The droplets containing the bioactive substance converge on the conductive layer 2, and the bioactive substance can be formed on the substrate in a shape that matches the shape of the conductive layer 2. The cover 21 is used to prevent a minute amount of adhesion to the entire surface of the substrate 1, and unlike the mask described in Patent Document 1, it does not determine the shape of the bioactive substance formed on the substrate. In the present invention, the shape of the bioactive substance 3 formed on the surface of the substrate 1 is determined by the shape of the conductive layer 2. Therefore, unlike the mask described in Patent Document 1, the cover 21 in the present invention is different from the shape of the bioactive substance 3 to be deposited on the conductive layer 2 so that the influence of charging is sufficiently small. The shape is formed large and placed on the substrate 1. Since the charging of the cover 21 hardly affects the bioactive substance 3 formed on the substrate 1, the influence on the shape and coating uniformity of the bioactive substance 3 attached on the substrate 1 due to the variation in the charged state is not affected. rare. Compared with the method described in Patent Document 1, the method according to the present invention does not cause variations in coating uniformity due to variations in the charged state, and is considered to provide excellent uniformity.

  In the present invention, since the dimensions 56 and 57 of the cover 21 are larger than the dimensions of the conductive layer 2, the bioactive substance 3 may be attached to a portion not covered with the cover 21. It wasn't. The effect of converging the charged particles in the conductive layer 2 is stronger as the conductive layer 2 is closer to the conductive layer 2, so that even if the dimensions 56 and 57 of the cover 21 are slightly larger than the dimensions of the conductive layer 2, Since most of the passed droplets converge on the conductive layer 2, it is considered that the bioactive substance hardly adheres around the conductive layer 2.

  The method of the present invention is superior to the conventional method described in Patent Document 1 in addition to the improvement of coating uniformity. In the method of the present invention, since the shape of the bioactive substance 3 formed on the surface of the substrate 1 is determined by the shape of the conductive layer 2, the accuracy of the shape of the bioactive substance 3 attached on the substrate 1 is It will be determined by the processing accuracy. Since the shape of the conductive layer 2 is formed by photolithography and etching and has a very high accuracy, the shape of the bioactive substance 3 can be formed with a high accuracy. On the other hand, the method of Patent Document 1 attaches a droplet containing a bioactive substance that has passed through a through hole of a mask to a conductive layer in a shape corresponding to the shape of the through hole of the mask. Since the through hole is usually formed by machining, a machining error of about several tens of μm occurs. Since the shape of the through-hole of the mask determines the shape of the bioactive substance attached on the conductive layer, the accuracy of the shape of the bioactive substance has an error of several tens of μm. Although it is possible to obtain high accuracy by forming a through hole by photolithography and etching processing, the mask described in Patent Document 1 also needs a certain rigidity for the convenience of handling the mask, and has a thickness. Need to be made thick. Therefore, it is necessary to remove a considerable thickness by etching in the process of forming the through hole, and the processing time becomes long, resulting in a problem that the processing cost increases.

  Furthermore, in the present invention, since the liquid height in the capillary is image-recognized, the spray amount of the bioactive substance can be controlled with high accuracy, and the shape of the bioactive substance 3 adhered on the substrate 1 is as described above. Because of its extremely high accuracy, it is possible to spray a spray amount controlled with high accuracy into a predetermined highly accurate shape, and only with the shape and application uniformity of the bioactive substance formed by one spray. In addition, it is possible to perform coating while suppressing variation in shape and variation in coating uniformity among a plurality of biologically active substances formed by spraying a plurality of times.

  When producing a microfluidic chip in which a plurality of different biologically active substances are applied on a substrate using the method of the present invention, the spraying process 2 is repeated as many times as necessary after the spraying process 1 of FIG. In the spraying step 2, spraying is performed after the substrate 1 is moved relative to the cover 21. As the bioactive substance 3 used for spraying, different materials can be used for each spray, and the shape of the conductive layer 2 can be arbitrarily formed as described in the above-described exposure, development, and etching steps. By repeating Step 2, it is possible to produce a microfluidic chip in which a desired material is formed in a desired shape.

  In the method of the present invention, it is necessary to align and arrange the through hole 55 of the cover 21 and the conductive layer 2 to which the bioactive substance 3 is applied before spraying. If the alignment accuracy is poor, the edge portion of the through hole 55 of the cover 21 approaches the conductive layer 2, and the influence of charging of the cover 21 works strongly, so that the bioactive substance 3 applied on the conductive layer 2 Application uniformity deteriorates. In addition, if there is an alignment error such that the edge portion of the through hole 55 of the cover 21 covers the conductive layer 2, the bioactive substance 3 is not applied to the portion covered with the cover 21. In the present invention, the alignment of the cover 21 and the substrate 1 is important, and if the required accuracy for the alignment can be relaxed, there is an advantage that the alignment mechanism can be simplified and the cost can be reduced and the alignment time can be shortened. can get. In order to ease the required accuracy for alignment, it is effective to make the through hole 55 of the cover 21 large. If the size of the through hole 55 is large, even if an alignment error occurs, a large gap can be provided between the edge of the cover 21 and the conductive layer 2 as compared with the case where the size of the through hole 55 is small. Therefore, the influence of the charging of the cover 21 on the application uniformity of the bioactive substance 3 can be suppressed to a small level. However, when the size of the through hole 55 of the cover 21 is increased, it has been confirmed that the sprayed bioactive substance 3 adheres to the substrate 1 around the conductive layer 2. Since the adhesion amount is proportional to the dimension of the through hole 55, it is desirable to make the through hole 55 smaller in order to reduce the adhesion amount. In other words, in order to increase the size of the through hole 55 and alleviate the requirement for the alignment accuracy of the cover 21 and the substrate 1, even if the through hole 55 is enlarged, the biological material on the substrate 1 around the conductive layer 2 can be reduced. A method that can suppress the adhesion of the active substance is required. The inventors devised the method described below as a method for suppressing the adhesion of the bioactive substance on the substrate 1 around the conductive layer 2 even if the size of the through hole 55 is increased.

  Details will be described with reference to FIGS. 4, 5, 7, and 8.

  A resist 13 is applied on the substrate 1 in the resist application step shown in FIG. Next, the conductive layer 2 is processed into the shape shown in FIG. Since the positive resist OFPR800 (viscosity 30 cp) is used as the resist 13, the shape of the region of the mask 14 where the light 15 is shielded is the same as that of the conductive layers 25 to 29 in FIG.

  Conductive layers 25, 26, 27, 28 and 29 were produced on the substrate 1 by the above procedure.

  Next, a procedure for attaching the bioactive substance 3 onto the conductive layers 25 to 29 will be described. The substrate on which the conductive layers 25 to 29 after the resist removing process are formed is disposed below the capillary 46 shown in FIG. 47 is the substrate after the resist removing step. Here, as shown in FIG. 5A, the conductive layer 27 is connected to the negative pole of the high voltage power supply 22. Wiring from the positive electrode or the negative electrode of the high-voltage power source 22 was arranged between the cover 21 and the substrate 1, and a predetermined voltage was applied to the conductive layers 25 to 29. Although not carried out in the present embodiment, a conductive layer is formed on the surface of the cover 21 on the substrate 1 side, and the cover 21 is placed on the substrate 1 so that the conductive layer formed on the cover 21 becomes conductive on the substrate 1. A structure in which a predetermined voltage is supplied to the conductive layers 25 to 29 in contact with the layers 25 to 29 may be employed.

  Since the negative pole of the high voltage power supply 22 is connected to the ground 24, the conductive layer 27 is at ground potential. The remaining conductive layers 25, 26, 28 and 29 were connected to the positive pole of the DC power supply 23, and the negative pole of the DC power supply 23 was connected to the ground 24. The conductive layers 25, 26, 28, and 29 have a positive potential that is higher than the ground potential by the voltage of the DC power supply 23. Further, a through hole 55 is formed in the cover 21 in FIG. 7 as shown in FIG. 8, and the cover 21 is aligned so that the conductive layer 27 enters the through hole 55. The dimension 56 of the through hole 55 was 2.0 mm, the dimension 57 was 14 mm, and the center of the through hole 55 was aligned with the center of the conductive layer 27. By aligning in this way, the conductive layers 25 and 29 are covered with the cover 21, and adhesion of the bioactive substance 3 sprayed from the capillary 46 can be prevented.

  With the configuration described above, electrostatic spraying was performed by applying a voltage of 3 kV to the capillary 46 and applying a voltage of +140 V to the conductive layers 25, 26, 28, and 29. The distance from the capillary 46 to the conductive layer 27 is 35 mm. When the fluorescence intensity on the substrate 1 around the conductive layer 2 was measured, almost no adhesion of the bioactive substance 3 was observed as in the case where the dimension of the through hole 55 was 1.5 mm. For this reason, even if the size of the through hole 55 of the cover 21 is increased, by applying a positive voltage to the conductive layers 25, 26, 28, and 29, the adhesion of the bioactive substance 3 on the substrate 1 is suppressed. It was found that the bioactive substance 3 can be applied on the conductive layer 27. As described above, since the size of the through hole 55 can be increased, the requirement for the alignment accuracy between the substrate 1 and the cover 21 can be relaxed.

  In the case of attaching a plurality of different types of bioactive substances 3, the conductive layer to be electrostatically sprayed is moved directly below the capillary 46, only the conductive layer to be sprayed is set to the ground potential, and the other conductive layers are set to the ground potential. A spraying process is performed by setting to a predetermined positive potential to sequentially form a desired bioactive substance on the conductive layers 25 to 29.

  Further, although the conductive layer 27 is set to the ground potential under the above conditions, the effect of positively collecting the sprayed bioactive substance on the conductive layer is brought about by setting the conductive layer 27 to a negative potential. The effect of improving the use efficiency of can be expected.

  When the substrate 1 on which the biologically active substance is applied on the conductive layer 2 as described above is used by being bonded to the flow path member 6 as shown in FIG. 1, the conventional structure has a problem of 0.2 μm. The gap 61 with the flow path member 6 caused by the step 62 of the conductive layer 2 is eliminated, and the liquid does not leak even when the liquid to be inspected flows through the flow path member 6.

  Even in the conventional substrate having a structure in which a convex conductive layer is formed on the substrate surface shown in FIGS. 13A and 13B, polydimethylsiloxane is used for the flow path member, and the polydimethylsiloxane is a rubber. As described above, since the flow path member itself is deformed so as to be in close contact with the substrate if there are some steps, it is possible to prevent leakage of the liquid flowing through the flow path. However, when the interval 10 between the channels formed in the channel member 6 is narrowed, the thickness of the wall separating the adjacent channels is reduced, and the gap 61 between the substrate and the channel member 6 is partially adjacent. There was a problem that the flow paths connected to each other were connected. For this reason, when a liquid is caused to flow through the flow path, liquid leakage may occur in the adjacent flow path. In the method of the present invention, since there is no step on the substrate surface, even if the interval between the channels formed in the channel member is narrowed, no gap is formed between the substrate surface and the channel member, It is possible to eliminate liquid leakage.

  In other words, by using the substrate of the present invention, a microfluidic chip that does not leak liquid between the channels while maintaining the same performance as the conventional substrate in the application uniformity of the bioactive substance is produced. Is possible.

  When mass-producing a substrate to be manufactured using the cover 21 described in Example 1, an efficient method for accurately adjusting and fixing the position of the substrate is necessary before performing the electrostatic spraying process. . As a reason, when the cover 21 is used, the position adjustment error of the cover 21 is an error between the through hole 55 and the position where the bioactive substance 3 is to be attached. If the error is large, the cover 21 is attached to the adjacent conductive layer. Therefore, it becomes a problem in product quality. The position adjustment error needs to be kept small, but if the operator observes and adjusts for each spray, the adjustment takes time and is not efficient.

  In this embodiment, a method for forming an adjustment mark in which the electrical or optical properties of the substrate surface are changed in order to shorten the position adjustment time and improve the adjustment accuracy will be described below.

  FIG. 9 is a diagram showing the structure of the substrate on which the position adjustment mark 63 of the present embodiment is formed. The position adjustment mark 63 is formed on the surface of the substrate of FIG. Yes. The position adjustment mark 63 is a copper thin film and has a thin linear shape. The position adjustment mark 63 was formed simultaneously with the processing of the conductive layer 2 in the steps from the resist coating step to the resist removal step of the processing method shown in FIG. Specifically, the mask 58 having the structure shown in FIG. 10 is used as an exposure mask, and exposure and development are performed so that the resist 13 is not formed on the portion where the conductive layer 2 is formed and the portion where the position adjustment mark 63 is formed. And then blasting. Furthermore, the conductive layer 2 is formed in a subsequent film formation step, and the resist 13 is removed, whereby the substrate shown in FIG. 9 is manufactured.

  Since the position adjustment mark 63 and the conductive layer 2 of the manufactured substrate are processed by photolithography as described above, the position error is suppressed to several μm or less. In this embodiment, since the position adjustment mark 63 is formed of a copper thin film, the position adjustment mark 63 reflects light. Since the periphery of the position adjustment mark 63 is a glass surface, it transmits light. By utilizing this property and recognizing the image of the position adjustment mark 63, it is possible to automatically perform position adjustment with high accuracy. As a specific method of aligning with the cover 21, image recognition is performed on a part of the edge of the through hole 55 of the cover 21, and the position adjustment mark 63 is adjusted to a predetermined position from the edge of the through hole 55. Thus, the position error can be reduced to several μm or less. In this embodiment, the optical position adjustment method has been described. For example, since the position adjustment mark 63 is conductive, the substrate is scanned with a measuring needle for measuring a change in electric resistance value. Also, a high position adjustment accuracy can be obtained by the method of recognizing the position adjustment mark 63 and adjusting the position of the image recognition result of the edge of the cover 21.

  The operation and action of the substrate having a fine shape formed by electrostatic spraying as described above and a method for manufacturing the substrate will be described below.

  The method and product for forming a fine shape by the electrostatic spraying method according to the present invention can apply a material to a fine shape with good uniformity, and it is necessary to apply a bioactive substance on a substrate in a predetermined shape. Is useful as a biochip.

Configuration diagram of a microfluidic chip created using the electrostatic spraying method in Example 1 of the present invention The figure for demonstrating the characteristic of the microfluidic chip created using the electrostatic spraying method in Example 1 of this invention. The figure which shows the detailed structure of the board | substrate of the microfluidic chip produced using the electrostatic spraying method in Example 1 of this invention. The figure for demonstrating the process sequence of the board | substrate of the microfluidic chip produced using the electrostatic spraying method in Example 1 of this invention. The block diagram of the board | substrate which changed the electroconductive layer pattern of the microfluidic chip created using the electrostatic spraying method in Example 1 of this invention The figure which shows the electrostatic spraying apparatus (film-forming apparatus of a conductive layer) which performs the electrostatic spraying method in the Example of this invention Configuration diagram of an electrostatic spraying apparatus for performing an electrostatic spraying method in an embodiment of the present invention The figure which shows the cover used for the electrostatic spraying apparatus which performs the electrostatic spraying method in the Example of this invention. The block diagram of the board | substrate produced using the electrostatic spraying method in Example 2 of this invention The figure which shows the exposure mask of the board | substrate used for the electrostatic spraying method in Example 2 of this invention. The figure which shows the measurement result of the fluorescence intensity of the board | substrate created using the electrostatic spraying method in Example 1 of this invention. The figure for demonstrating the fluorescence measurement position of the electrostatic spraying method in Example 1 of this invention Configuration diagram of a conventional microfluidic chip The figure for demonstrating the characteristic of the conventional microfluidic chip | tip.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Board | substrate 2 Conductive layer 3 Bioactive substance 4 Connection line 5 Connection part 6 Channel member 7 Inlet 8 Outlet 9 Channel 10 Channel interval 11 Bonding surface 12 Non-conductive part 13 Resist 14 Mask 15 Light 16 Development Liquid 17 Step 18 Blast nozzle 19 Blast particle 20 Resist stripping liquid 21 Cover 22 High voltage power supply 23 DC power supply 24 Ground 25, 26, 27, 28, 29 Conductive layer 30, 31, 32, 33, 34 Switching section 35 Electrode wire 36 High voltage line 37 Chamber 38 Substrate holder 39 High frequency power supply 40 Exhaust device 41 Main valve 42 Deposition boat 43 Deposition material 44 Gas introduction tube 45 Ground 46 Capillary 47 Substrate after resist removal 48 Control device 49 Substrate holder 50 Chamber outer wall 51 Capillary holder (X)
52 Capillary holder (Y)
53 Holding member 54 Spacing 55 Through hole 56 Size 57 Size 58 Exposure mask 59 Measurement point A
60 measuring points G
61 Gap 62 Step 63 Mark for position adjustment

Claims (9)

Applying a resist to form a predetermined pattern layer on the substrate and exposing and developing through a mask having the predetermined pattern;
Blasting the substrate after development,
Forming a conductive layer in a stepped groove pattern formed between the base surface of the resist-coated surface and the base surface of the non-resist-coated surface by the blasting;
Removing the resist after film formation of the conductive layer and electrostatically spraying a biologically active substance through a cover having a through hole corresponding to the shape of the conductive layer;
An electrostatic spraying method comprising: The electrostatic spraying method according to claim 1, wherein the step is approximately 0.2 μm. 2. The electrostatic spraying method according to claim 1, wherein the blasting is performed so that a surface roughness of a resist non-coated surface is 0.7 nm to 100 nm. The step of forming the conductive layer includes
A capillary having a biologically active substance solution and an electrode line is disposed inside the substrate at a predetermined distance, and a predetermined high voltage is applied to the electrode line to pass the biologically active substance solution through the cover. The electrostatic spraying method according to claim 1, wherein electrostatic spraying is performed. The electrostatic spraying method according to claim 4, wherein a distance between the substrate and the cover is 0.05 mm or less. The electrostatic spraying method according to claim 5, wherein the through hole formed in the cover is formed to be slightly larger than the shape of the conductive layer. The conductive layer is formed by forming a plurality of the groove patterns, setting a groove pattern to be electrostatically sprayed to a ground potential or a negative potential, and setting another groove pattern higher than the potential. 2. The electrostatic spraying method according to 1. The electrostatic spraying method according to claim 1, further comprising a step of forming a position adjustment mark that serves as a reference position of a predetermined pattern layer to be formed on the substrate. A microfluidic chip for testing bioactive substances,
A flow path member comprising an inlet at both ends and a groove part connected to the inlet to form a flow path,
A microfluidic chip produced by attaching the bioactive substance to a substrate coated with the electrostatic spraying method according to claim 1.

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