JPWO2008044737A1 - Electrostatic spraying apparatus and electrostatic spraying method - Google Patents

Abstract

In a method using electrostatic spraying in which a biologically active substance is adhered to an insulating substrate 1 in a desired shape, a conductive layer 2 having a desired shape is formed on the substrate 1, and a predetermined distance from the substrate 1 is formed. A capillary 40 having an electrode wire disposed therein, a high-voltage power supply 15 for applying a predetermined high voltage to the electrode wire to electrostatically spray the bioactive substance, and a substrate between the substrate 1 and the capillary 40 1 and a cover 10 having a predetermined distance and a through hole corresponding to the shape of the conductive layer 2, and aligning the cover 10 corresponding to the portion on which the bioactive substance is to be deposited on the conductive layer 2. Then, a bioactive substance having a desired pattern shape is formed by electrostatic spraying.

Description

  The present invention relates to an electrostatic spraying apparatus and electrostatic spraying method for applying a solution (a solution containing a biologically active substance or the like) or the like on a substrate, a biochip manufacturing technique, and application of the solution or the like onto the substrate. Provide technology.

  As a method for forming a biologically active substance having a desired shape on a substrate while electrostatically spraying a solution containing a biologically active substance (substance having biological activity) and maintaining the biological activity, Japanese Patent Laid-Open Publication No. There is a method disclosed in Japanese Patent No. -281252. In the following description, Japanese Patent Laid-Open No. 2001-281252 is abbreviated as Patent Document 1. 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 having substantially the same shape as the shape of the through-hole formed in the mask is applied on the substrate. 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 and compared with methods other than electrostatic spraying. The coating uniformity is excellent.

  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 charged state of the mask can be controlled to some extent by controlling the humidity and removing electricity with an ionizer. However, it has been difficult to control the charged state to the extent that it does not affect the shape and uniformity of the bioactive substance. 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.

  The present invention solves the conventional problems, and aims to provide a substrate having a high uniformity and a bioactive substance applied in a predetermined shape, and a method for manufacturing such a substrate. The purpose is to provide.

  In order to solve the conventional problems, an electrostatic spraying apparatus according to the present invention is an electrostatic spraying apparatus that deposits a solution in a desired pattern shape on an insulating substrate, and a conductive layer having a desired pattern shape on the substrate. A capillary having an electrode wire disposed at a predetermined distance from the substrate, a high voltage power source for electrostatically spraying the solution by applying a predetermined high voltage to the electrode wire, A cover disposed between the substrate and the capillary in the vicinity of the substrate and having a through hole corresponding to the shape of the conductive layer, and corresponding to a portion on which the solution is to be deposited on the conductive layer. Then, the cover is aligned and the solution is electrostatically sprayed to form a substrate having a desired pattern shape.

  Further, the electrostatic spraying method of the present invention is the electrostatic spraying method in which a solution is deposited on an insulating substrate in a desired pattern shape, and a conductive layer having a desired pattern shape is formed on the substrate, and the conductive layer A cover having a through hole having a shape corresponding to the shape of the substrate is disposed at a predetermined distance from the substrate, and a reference mark serving as a reference position of a desired pattern shape to be formed on the substrate is formed on the reference mark. The position of the through hole of the cover is aligned based on this, and the solution is stacked by electrostatic spraying on the conductive layer on the substrate through the cover to form a substrate having a desired pattern shape It is.

  Further, the electrostatic spraying method of the present invention is an electrostatic spraying method in which a positive high voltage is applied to adhere a solution on an insulating substrate in a desired pattern shape. A plurality of conductive layers are formed, a cover having a through-hole having a shape corresponding to the shape of the conductive layer is arranged at a predetermined distance from the substrate, and the conductive to be electrostatically sprayed among the plurality of conductive layers A layer is set to a ground potential or a negative potential, and another conductive layer is set to be higher than the potential, and a conductive layer set to the ground potential or the negative potential is sequentially selected, and the solution is placed on the plurality of conductive layers. Electrospraying is performed to form a substrate having a desired pattern shape.

  According to the electrostatic spraying apparatus and the electrostatic spraying method of the present invention, it is possible to apply a solution containing a bioactive substance or the like on a substrate in a desired shape with high uniformity.

The figure which shows the structure of the board | substrate manufactured by the electrostatic spraying apparatus of Example 1 which concerns on this invention. The figure for demonstrating the process sequence of the electrostatic spraying method of Example 1 which concerns on this invention. The figure which shows the film-forming apparatus for forming the conductive layer in Example 1 which concerns on this invention The figure which shows the structure of the electrostatic spraying apparatus of Example 1 which concerns on this invention. The top view which shows the cover in the electrostatic spraying apparatus of Example 1 which concerns on this invention. The figure which shows the measurement result of the fluorescence intensity of the board | substrate manufactured by the electrostatic spraying apparatus of Example 1 which concerns on this invention. The figure for demonstrating the fluorescence measurement position in the board | substrate manufactured with the electrostatic spraying apparatus of Example 1 which concerns on this invention. The figure for demonstrating board | substrate manufacture performed by applying a bias voltage to a conductive layer in the electrostatic spraying apparatus of Example 1 which concerns on this invention. The top view which shows the board | substrate arrange | positioned in the electrostatic spraying apparatus of FIG. 8A. The figure which shows the structure of the board | substrate manufactured by the electrostatic spraying apparatus of Example 2 which concerns on this invention. The top view which shows the exposure mask of the board | substrate in the electrostatic spraying apparatus of Example 2 which concerns on this invention.

  Hereinafter, preferred embodiments of a method for manufacturing a substrate coated with a predetermined solution according to the present invention will be described in detail with reference to the accompanying drawings. Here, the solution is a solution having a biologically active substance. The biologically active substance itself contains a liquid, a solution mixed with a diluent, or a biologically active substance test reagent, and is simply referred to as a biologically active substance in the following description. 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 shows the structure of a substrate on which a bioactive substance is formed using the electrostatic spraying apparatus and electrostatic spraying method of Example 1 according to the present invention.

  In FIG. 1, a substrate 1 uses glass as a material, and an electrically conductive 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. In Example 1, a fluorescent dye-labeled antibody protein (Alexa Fluor 568 labeled Anti-goat IgG manufactured by Molecular Probes) was used as the biologically active substance 3. The biologically active substance 3 is formed on the conductive layer 2 in a shape that substantially matches the conductive layer 2. As shown in FIG. 1, all of the ten conductive layers 2 are connected so as to be electrically conductive by the connection lines 4, and the potentials of the connection lines 4 and the conductive layers 2 are applied to the connection portions 5. It is configured to be equal to the generated potential. In FIG. 1, the inside of a circular broken line is a cross-sectional view showing the conductive layer 2 and the bioactive substance 3 on the substrate.

  A method for processing the substrate 1 shown in FIG. 1 will be described in detail with reference to FIG.

(1) Film Forming Process First, in the film forming process, a copper thin film is formed on the substrate 1 in the film forming process as shown in FIG. In Example 1, although the case where copper is used for the conductive layer 2 is described, as the material of the conductive layer 2, for example, metals other than copper, such as gold, silver, platinum, and aluminum, indium tin oxide (ITO Even non-metals such as) can be used if they are conductive. In particular, in biochip applications, the bioactive substance 3 attached to the substrate 1 may be optically observed from both the front and back sides of the substrate 1. In such applications, transparent and conductive ITO is used as a thin film material. It is necessary to use a material such as

  The conductive layer 2 was formed using the film forming apparatus shown in FIG. In FIG. 3, the board | substrate 1 is fixed to the board | substrate holder 32 comprised with a conductor. The substrate holder 32 is connected to a high frequency power source for applying high frequency power to the substrate holder 32. The vacuum chamber 31 is connected to the exhaust device 34 via the main valve 35 in order to exhaust to a predetermined degree of vacuum. In the vacuum chamber 31, a vapor deposition port 36 for mounting a vapor deposition material 37 that is heated and vapor-deposited on the substrate 1 at a predetermined degree of vacuum is provided. The vacuum chamber 31 has a gas introduction pipe 38 for introducing a predetermined gas, and the housing of the vacuum chamber 31 is connected to the ground 39.

First, after the substrate 1 is placed on the substrate holder 32, the vacuum chamber 31 is evacuated. The pressure in the vacuum chamber 31 is set to 1.0 × 10 ⁻³ Pa or less.
Next, the glass substrate surface of the substrate 1 is subjected to plasma treatment. The plasma treatment is performed for the purpose of roughening the surface of the substrate 1. During plasma processing, nitrogen gas having a purity of 99.9% or more is introduced into the vacuum chamber 31, the pressure is set to 5.0 × 10 ⁻² Pa, and high frequency power is applied to the substrate holder 32 supporting the substrate 1 by the high frequency power source 33. To do. By applying the high frequency power, glow discharge is generated in the vacuum chamber 31 and nitrogen plasma is generated.

In FIG. 3, with the generation of nitrogen plasma, a first negative bias voltage is induced on the substrate 1, and the substrate 1 is subjected to nitrogen plasma treatment. In Example 1, the first negative bias voltage was set to 300V. After the nitrogen plasma treatment, the pressure in the vacuum chamber 31 is evacuated to 1.0 × 10 ⁻⁴ Pa or less. Argon gas is introduced after evacuation, the pressure in the vacuum chamber 31 is set to 1.0 × 10 ⁻² Pa, copper is evaporated from the vapor deposition boat 36, and a high frequency voltage is applied to the substrate holder 32 that supports the substrate 1. . Application of a high frequency voltage causes glow discharge in the vacuum chamber 31 to generate argon and copper plasma. Since the second negative bias voltage is induced in the substrate 1 with the generation of the argon and copper plasma, the argon ions and the copper ions in the plasma are accelerated by the second negative bias voltage to the surface of the substrate 1. It is accelerated toward.

  In Example 1, the second negative bias voltage was set to 400 V to form a copper film. A copper thin film is formed on the surface of the substrate 1 by the effect of copper ions colliding with the surface of the substrate 1 to form a copper thin film and the effect of vacuum deposition of non-ionized copper. 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 500 nm under the above conditions.

(2) From the resist coating process to the resist removal process Next, the resist coating process to the resist removal process will be described as shown in FIG. 2 (b) to FIG. 2 (f).
A resist 10 is applied by spin coating on the substrate 1 on which the copper thin film is formed. In Example 1, OFPR800 (viscosity 30 cp) was used as the resist 10. Next, in the exposure step, the resist 10 is irradiated with light 8 through the mask 7. Since the resist 10 used in Example 1 is a positive resist, the mask 7 has a shape that matches the shape of the resist to be formed on the conductive layer 2 and is configured to shield the light 8. Since the area | region which shields the light 8 of the mask 7 can be designed freely at the time of preparation of the mask 7, the resist 10 can be exposed to a desired shape.

  After the resist 10 was exposed with the light 8 as described above, it was immersed in the developer 11 and developed. NMD-3 was used as the developer. After the development, the resist 10 on the conductive layer 2 has a shape corresponding to the shape of the region where the light 8 is melted and the light 8 is shielded as shown in FIG. The resist 10 remains on the surface.

After development, the substrate 1 is immersed in the etching solution 12. As the etching solution 12, a ferric chloride solution was used. By etching, as shown in the etching step of FIG. 2E, the conductive layer 2 in the portion where the resist 10 is not formed is dissolved and removed from the substrate 1. That is, the conductive layer 2 is processed into a shape that matches the shape of the region where the light 8 is shielded in the mask 7.
After the etching, the resist 10 was dissolved and peeled by immersing the substrate 1 in the resist stripping solution 13 (see FIG. 2 (f)).

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 of the mask 7 where the light 8 is shielded. Since the shape of the area of the mask 7 where the light 8 is shielded can be freely produced when the mask 7 is designed and manufactured, the conductive layer 2 having any shape can be formed on the substrate 1 by the above procedure.
Unlike the first embodiment, when a negative resist is used for the resist 10, the mask 7 is manufactured so that the portion of the mask 7 that transmits light 8 matches the resist shape to be formed on the conductive layer 2. Thus, it is possible to produce the substrate 1 on which the conductive layer 2 having a desired shape is formed.

(3) Spraying Step 1 and Spraying Step 2 Next, as shown in FIG. 2 (g) to FIG. 2 (h), from the resist coating step of FIG. 2 (b) to FIG. 2 (f). The substrate 1 produced up to the resist removal step was set in the electrostatic spraying device shown in FIG. 4, and the bioactive substance 3 was applied on the conductive layer 2 of the substrate 1.

In FIG. 4, a high voltage line 28, which is an electrode line inserted into the capillary 40, is connected to the positive electrode side of the high voltage power supply 14, and the negative electrode side of the high voltage power supply 14 is connected to the substrate 1 after the resist removal process. Is done. Thus, the electrostatic spraying device is configured such that the high voltage power supply 14 generates a potential difference between the capillary 40 and the substrate 1 after the resist removal process. A controller 45 is connected to the high voltage power supply 14 and controls the voltage generated by the high voltage power supply 14. Further, the cover 9 is arranged at a predetermined distance X from the substrate 1. The cover 9 is formed with a substantially identical through-hole 48 that is slightly larger than the shape of the pattern on which the bioactive substance is applied.
FIG. 5 is a plan view of the cover 9 used in the first embodiment. In Example 1, as shown in FIG. 5, the width dimension Y of the through-hole 48 was 1.5 mm, and the length dimension Z of the through-hole 48 was 14 mm.

  The cover 9 is disposed between the capillary 40 and the substrate 1 after the resist removal step, and is aligned so that a portion to which the bioactive substance 3 on the surface of the substrate 1 after the resist removal step is to adhere is overlapped with the through hole 48. . An effective method for aligning the cover 9 and the substrate 1 will be described in Example 2 described later.

  In Example 1, a voltage of 3 kV was applied via the electrode line 27 and the high voltage line 28 between the capillary 40 and the substrate 1 after the resist removal process placed on the substrate holder 46 by the high voltage power supply 14. The distance from the tip of the capillary 40 to the surface of the substrate 1 after the resist removal process was 35 mm, and the tip of the capillary 40 had an outer shape of 50 μm. As a material for the cover 9, glass epoxy having a thickness of 0.1 mm was used. The amount of spray was 1 μlitre sprayed by measuring the liquid height of the solution in the capillary with a CCD camera. In Example 1, as shown in the spraying process 1 of FIG. 2G, the bioactive substance 3 was attached to only one portion of the conductive layer 2. Further, the distance X between the cover 9 and the surface of the conductive layer 2 was arranged to be 0.05 mm or less. According to the experiments by the inventors, it was found that when the distance X 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 considered the above 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 9 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 the first embodiment, the cover 9 is made of glass epoxy and is an insulating material. Therefore, it is considered that the cover 9 is positively charged during spraying. The cover 9 is positively charged, and the droplet passing through the through hole 48 of the cover 9 is also positively charged. Therefore, the droplet receives an electric repulsive force from the cover 9 and adheres onto the conductive layer 2 in a shape smaller than the through hole 48. When the distance from the cover 9 to the conductive layer 2 is long, the droplet receives a repulsive force due to the charging of the cover 9 at a position away from the conductive layer 2 and changes due to the electric repulsive force from the cover 9. In the orbit, the aircraft travels a long distance before reaching the conductive layer 2. 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 48 compared to the case where the distance from the cover 9 to the conductive layer 2 is short. 2 is considered to adhere to the surface.

  In addition, it is desirable that the width dimension Y and the length dimension Z in the through hole 48 of the cover 9 are larger than the dimension of the conductive layer 2. This is because if the conductive layer 2 and the width dimension Y and the length dimension Z are the same, the sprayed bioactive substance 3 adheres onto the conductive layer 2 with a smaller dimension than the conductive layer 2. This is probably because the cover 9 is charged. In order to cope with this, in Example 1, the width dimension Y formed on the cover 9 corresponding to the pattern of the conductive layer 2 is 1.5 mm and the length dimension Z is 14 mm, whereas the conductive layer 2 The dimensions are 0.8 mm and 11 mm, and the width dimension Y and the length dimension Z are made larger than the shape dimension of the conductive layer 2.

  The result of having observed the surface of the board | substrate 1 produced until the spraying process 1 shown to (g) of FIG. 2 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 results were designated as sample 1, sample 2, and sample 3, respectively. FIG. 9 shows the fluorescence intensities measured as Sample 1, Sample 2, and Sample 3.

  FIG. 7 shows the shape of the conductive layer 2 of the manufactured sample. The measurement points A to G are measured by dividing the conductive layer 2 on the side close to the connection line 4 as the measurement point A and the measurement point G on the side far from the connection line 4 in FIG. Points B to F were set. In FIG. 7, the position PE near the connection line 4 is the measurement point A, and the position DE far from the connection line 4 is the measurement point G. In three experiments, a CV value of 2.1% to 3.2% was obtained. To the best of the inventors' knowledge, no report has been received that a uniformity of about 3% CV value has been obtained by conventional 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 a bioactive substance is applied onto the substrate in a shape corresponding to the shape of the through-hole. Yes.

  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 has been found that the charged state of the mask affects the sprayed solution, and the larger the charge of the mask, the thinner the shape of the bioactive substance formed. For this reason, in order to form a highly-accurate shape and highly uniform bioactive substance on the substrate, it is necessary to manage the charged state of the mask to be always constant. The charged state of the mask can be controlled to some extent by controlling the humidity or removing the charge with an ionizer, but it is not possible to control the charged state so as not to affect the shape and uniformity of the bioactive substance. It was difficult.

  In the method of the present invention, the conductive layer 2 having a shape that matches the shape to which the biologically active substance 3 is to be applied is formed on the surface of the substrate 1, and the conductive layer 2 is positively charged by being grounded or a negative potential. The droplet containing the biologically active substance converges on the conductive layer 2, and a biologically active substance having a shape corresponding to the shape of the conductive layer 2 can be formed on the substrate. The cover 9 in the electrostatic spraying apparatus of the present invention is used to prevent a very small amount of adhesion to the entire surface of the substrate 1. Unlike the mask described in Patent Document 1, a cover 9 is formed on the substrate. Does not determine the shape of the active substance. 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 9 in the present invention has a through-hole in the cover 9 to the extent that the effect of charging is sufficiently small compared to the shape of the bioactive substance 3 to be deposited on the conductive layer 2. 48 is formed in a large shape, and its cover 9 is disposed on the substrate 1. Since the charging of the cover 9 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 of the present invention does not cause variation in coating uniformity due to variation in the charged state, and therefore, excellent coating uniformity can be obtained.

  In the present invention, since the width dimension Y and the length dimension Z of the through hole 48 of the cover 9 are larger than the dimension of the conductive layer 2, the bioactive substance 3 may be attached to a portion not covered with the cover 9. However, almost no adhesion was found in the results of experiments by the inventors. The effect of converging the charged particles in the conductive layer 2 works stronger as it is closer to the conductive layer 2, so even if the width dimension Y and the length dimension Z of the through hole 48 of the cover 9 are slightly larger than the dimension of the conductive layer 2. Most of the droplets that have passed through the through hole 48 of the cover 9 converge on the conductive layer 2. As a result, it is considered that the bioactive substance hardly adheres around the conductive layer 2. That is, the biologically active substance adheres on the conductive layer 2 and the biologically active substance hardly adheres to the portion 6 on the substrate without 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 determined by the conductive layer 2. This is 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 biologically active 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. These through holes are usually machined. For this reason, the method of Patent Document 1 causes a processing error of about several tens of μm. 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 the mask described in Patent Document 1 can also obtain high accuracy by forming a through hole by photolithography and etching, the mask needs to have a certain rigidity for the convenience of handling the mask. It is necessary to make the thickness thick. For this reason, it is necessary to remove a considerable thickness by etching in the process of forming the through hole, and the processing time becomes long, so 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 attached on the substrate 1 is as described above. So is very accurate. For this reason, in the electrostatic spraying device of the present invention, the spray amount controlled with high accuracy can be sprayed into a predetermined highly accurate shape, and the shape and application of the bioactive substance formed by one spray In addition to uniformity, it is possible to apply the coating while suppressing variation in shape and variation in application uniformity among a plurality of biologically active substances formed by a plurality of sprays.

  When producing a biochip in which a plurality of different bioactive substances are applied on a substrate using the method of the present invention, after the spraying step 1 of FIG. 2 (g), the spray of FIG. Repeat step 2 as many times as necessary. In the spraying step 2, spraying is performed after the substrate 1 is moved relative to the cover 9. 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 biochip in which a desired material is formed in a desired shape.

  In the method of the present invention, it is necessary to arrange the substrate 1 by aligning the through hole 48 of the cover 9 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 48 of the cover 9 approaches the conductive layer 2, and the influence of the charging of the cover 9 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 48 of the cover 9 covers the conductive layer 2, the bioactive substance 3 is not applied to the portion covered with the cover 9.

  In the present invention, alignment between the cover 9 and the substrate 1 is important, and if the required accuracy for alignment can be relaxed, the cost can be reduced by simplifying the alignment mechanism and the alignment time can be shortened. Is obtained. In order to ease the required accuracy for alignment, it is effective to make the through hole 48 of the cover 9 large in size. If the dimension of the through hole 48 is large, even if an alignment error occurs, a large gap can be provided between the edge of the cover 9 and the conductive layer 2 as compared with the case where the dimension of the through hole 48 is small. Therefore, the influence of the charging of the cover 9 on the application uniformity of the bioactive substance 3 can be suppressed to a small level. However, when the size of the through hole 48 of the cover 9 is increased, it is 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 size of the through hole 48, it is desirable to reduce the through hole 48 in order to reduce the adhesion amount.

  That is, in order to increase the size of the through hole 48 and ease the requirement for the alignment accuracy of the cover 9 and the substrate 1, even if the through hole 48 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 have invented the bioactive substance adhesion suppression 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 48 is increased. did.

  Hereinafter, the details of the method for suppressing the adhesion of the bioactive substance to the substrate around the conductive layer will be described with reference to FIGS. 2, 4 and 5, and FIGS. 8A and 8B. FIG. 8A is a diagram for explaining substrate fabrication by applying a bias voltage to the conductive layer in the electrostatic spraying apparatus of Example 1, and FIG. 8B is a substrate disposed in the electrostatic spraying apparatus of FIG. 8A. FIG.

  A conductive layer 2 is formed on the substrate 1 by the same procedure as the film forming step shown in FIG. Next, the conductive layer 2 having the shape shown in FIG. 8B is processed by the same procedure from the resist coating process shown in FIG. 2B to the resist removal process shown in FIG. In FIG. 8B, five conductive layers 17 to 21 are formed on the substrate 1. As the resist 10, a positive resist OFPR800 (viscosity 30 cp) is used. As shown in the conductive layers 17 to 21 in FIG. 8B, the regions of the mask 7 that shield the light 8 are the same.

  Through the above procedure, the first conductive layer 17 to the fifth conductive layer 21 were formed on the substrate 1.

Next, a procedure for attaching the bioactive substance 3 from the first conductive layer 17 to the fifth conductive layer 21 will be described.
The substrate 1 on which the first conductive layer 17 to the fifth conductive layer 21 after the resist removing step are formed is placed below the capillary 40 of the electrostatic spraying device shown in FIG. 8A, when the target conductive layer is, for example, the third conductive layer 19, the third conductive layer 19 is disposed facing the inside of the through hole 48, and the negative voltage of the high voltage power source 14 is set. Connect to the pole. Since the negative pole of the high voltage power supply 14 is connected to the ground 16, the third conductive layer 19 becomes the ground potential. The remaining first, second, fourth, and fifth conductive layers 17, 18, 20, and 21 were connected to the positive pole of the DC power supply 15, and the negative pole of the DC power supply 15 was connected to the ground 16. The first, second, fourth, and fifth conductive layers 17, 18, 20, and 21 are applied with a positive potential that is higher than the ground potential by the voltage of the DC power source 15, that is, a positive bias voltage. By configuring in this way, the biologically active substance 3 becomes difficult to adhere to the third conductive layer 19. Further, as shown in FIG. 5, the cover 9 in FIG. 4 has a through hole 48, and the cover 9 has the third conductive layer 19 disposed at a position facing the inside of the through hole 48. So that they are aligned. The width dimension Y of the through hole 48 was 2.0 mm, the length dimension Z was 14 mm, and the center of the through hole 48 was aligned with the center of the third conductive layer 19. By aligning in this way, the first, second, fourth, and fifth conductive layers 17, 18, 20, and 21 are covered to some extent by the cover 9, and the bioactive substance 3 sprayed from the capillary 40 is attached. Is prevented. The capillary 40 shown in FIG. 4 is held using a capillary holder 41 that adjusts the position in the X-axis direction, a capillary holder 42 that adjusts the position in the Y-axis direction, and a pressing member 43 that fixes the capillary holders 41 and 42. Has been. Thus, the capillary 40 is securely fixed and held at predetermined positions in the X-axis direction and the Y-axis direction with respect to the chamber outer wall 47. Here, the X-axis direction is a direction orthogonal to the paper surface of FIG. 4, and the Y-axis direction is the left-right direction in FIG. 4 orthogonal to the X-axis direction.

  With the configuration as described above, a voltage of 3 kV is applied to the capillary 40 whose position has been adjusted, and a voltage of +140 V is applied to the first, second, fourth, and fifth conductive layers 17, 18, 20, and 21, and electrostatic spraying is performed. went. The distance from the tip of the capillary 40 to the third conductive layer 19 as the target conductive layer was 35 mm. After the electrostatic spraying performed in this way, the fluorescence intensity on the substrate 1 around the conductive layer 2 was measured. As in the case where the dimension of the through hole 48 was 1.5 mm, the bioactive substance 3 was hardly adhered. Not observed. For this reason, even if the size of the through hole 48 of the cover 9 is increased, a positive bias voltage is applied to the first, second, fourth, and fifth conductive layers 17, 18, 20, and 21, so that It was found that a predetermined amount of the biologically active material 3 can be reliably applied only on the third conductive layer 19 under the condition that the adhesion of the biologically active material 3 is suppressed. As described above, in the present invention, since the size of the through hole 48 can be increased, the requirement for the alignment accuracy between the substrate 1 and the cover 9 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 40, and only the conductive layer to be sprayed is set to the ground potential. The spraying process is performed by setting the layer to a predetermined positive bias potential, and a desired bioactive substance can be sequentially formed on each of the plurality of conductive layers by the above method.

  In the above embodiment, the third conductive layer 19 as the target conductive layer is set to the ground potential. However, by setting the target conductive layer to a negative potential, the sprayed bioactive substance is positively applied to the conductive layer. Therefore, the effect of further improving the utilization efficiency of the bioactive substance can be expected.

  When mass-producing the substrate 1 manufactured using the cover 9 described in the first embodiment, there is an efficient method for accurately adjusting and fixing the position of the substrate 1 before performing the electrostatic spraying process. I need it. The reason is that when the cover 9 is used, the position adjustment error of the cover 9 is an error between the through hole 48 and the position where the bioactive substance 3 is to be attached. If the error is large, the adjacent conductive layer 2 is not necessary. This causes 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 the second embodiment, in order to shorten the position adjustment time and improve the adjustment accuracy, a position adjustment mark for changing the electrical or optical property of the substrate surface is formed. A method of forming will be described below.

  FIG. 9 is a diagram illustrating the structure of the substrate 100 on which the position adjustment mark 52 according to the second embodiment is formed. The substrate 100 of the second embodiment has a structure in which a position adjustment mark 52 is formed on the surface of the substrate 1 of FIG. 1 described in the first embodiment. The position adjustment mark 52 is a copper thin film and has a thin linear shape. The position adjustment mark 52 was formed simultaneously with the processing of the conductive layer 2 in the steps from the exposure step to the resist removal step of the processing method shown in FIGS. Specifically, using the mask 53 having the shape shown in FIG. 10 as an exposure mask, the resist 10 is electrically conductive not only in the portion where the bioactive substance 3 is attached but also in the portion where the position adjustment mark 52 is formed. It was formed on layer 2. Etching is then performed to dissolve and remove the copper thin film other than the portion where the resist 10 is formed. Finally, the resist 10 is removed, and in addition to the portion to which the bioactive substance 3 is attached, the position adjustment mark 52 was formed on the substrate. In FIG. 10, the hatched portion of the mask 53 is fabricated so as to block light.

  Since the position adjustment mark 52 and the conductive layer 2 of the manufactured substrate 100 are processed by photolithography as described above, the position error is suppressed to several μm or less. In the second embodiment, since the position adjustment mark 52 is formed of a copper thin film, the position adjustment mark 52 reflects light. Since the periphery of the position adjustment mark 52 is a glass surface, it transmits light. By recognizing the position adjustment mark 52 using this property, it is possible to automatically perform position adjustment with high accuracy. As a specific method of aligning with the cover 9, image recognition is performed on a part of the edge of the through hole 48 of the cover 9, and the position adjustment mark 52 is adjusted to a predetermined position from the edge of the through hole 48. Thus, the position error can be reduced to several μm or less. In the second embodiment, the optical position adjustment method has been described. For example, since the position adjustment mark 52 is conductive, the substrate is scanned with a measuring needle for measuring a change in electric resistance value. Even when the position adjustment mark 52 is recognized and the position is adjusted with respect to the image recognition result of the edge of the cover 9, high position adjustment accuracy can be obtained.

  The electrostatic spraying device and electrostatic spraying method according to the present invention are capable of uniformly applying a material in a fine shape, and need to apply a bioactive substance to a predetermined shape on a substrate. Useful as.

Claims (7)

In an electrostatic spraying apparatus for depositing a solution in a desired pattern shape on an insulating substrate,
A capillary having a predetermined distance from the substrate on which a conductive layer having a desired pattern shape is formed and having an electrode wire inside;
A high-voltage power supply for electrostatically spraying the solution by applying a predetermined high voltage to the electrode wire;
A cover disposed between the substrate and the capillary close to the substrate and having a through hole corresponding to the shape of the conductive layer;
With
The electrostatic spraying device is configured such that the cover is disposed corresponding to a portion on the conductive layer to which the solution is to be attached, and the solution is electrostatically sprayed to the substrate through the cover.   The electrostatic spraying device according to claim 1, wherein a distance between the substrate and the cover is 0.05 mm or less.   The electrostatic spraying device according to claim 2, wherein the through hole formed in the cover is formed to be larger than a corresponding shape of the conductive layer. In an electrostatic spraying method in which a solution is deposited in a desired pattern shape on an insulating substrate,
Forming a conductive layer having a desired pattern shape on the substrate;
Disposing a cover having a through hole having a shape corresponding to the shape of the conductive layer at a predetermined distance from the substrate;
Forming a reference mark serving as a reference position of a desired pattern shape to be formed on the substrate, aligning the position of the through hole of the cover based on the reference mark, and on the conductive layer on the substrate A step of laminating the solution by electrostatic spraying through the cover. In an electrostatic spraying method in which a high voltage of positive polarity is applied to adhere a solution on an insulating substrate in a desired pattern shape,
Forming a plurality of conductive layers having a desired pattern shape on the substrate;
Disposing a cover having a through hole having a shape corresponding to the shape of the conductive layer at a predetermined distance from the substrate;
Of the plurality of conductive layers, a step of setting a conductive layer to be electrostatically sprayed to a ground potential or a negative potential and setting another conductive layer higher than the potential, and a conductivity set to the ground potential or the negative potential. And electrostatic spraying the solution onto the plurality of conductive layers by sequentially selecting layers.   The electrostatic spraying method according to claim 4 or 5, wherein the predetermined distance is 0.05 mm or less.   The electrostatic spraying method according to claim 6, wherein the through hole formed in the cover is formed larger than the shape of the conductive layer.

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