JP6390707B2 - Biological material capturing filter and biological material capturing system - Google Patents

Description

  The present invention relates to a biological material capturing filter and a biological material capturing system that can efficiently capture biological materials such as circulating tumor cells (hereinafter referred to as CTC).

  Research and clinical significance of cancer cell enrichment is extremely great, and if cancer cells in blood can be concentrated, it can be applied to cancer diagnosis. For example, the most important factor in cancer prognosis and treatment is the presence or absence of cancer cell metastasis at the first visit and treatment. Detecting CTC is a useful means of determining the progression of cancer pathology when the initial spread of cancer cells extends into peripheral blood. However, since blood components such as red blood cells and white blood cells are overwhelmingly present in blood, it is difficult to detect a very small amount of CTC.

  In recent years, a method for efficiently detecting a small amount of CTC by using a resin filter using parylene has been proposed (Patent Document 1).

  Alternatively, a method of improving the strength of the filter by using a filter using a metal instead of a resin, and separating the leukocytes from the difference in leukocyte and cancer cell deformability has been proposed (Patent Document 2).

International Publication No. 2010/135603 JP 2013/42689 A

  The inventors found that the following problems existed while studying a filter using a metal.

  Since metal has low biocompatibility, blood components such as white blood cells and red blood cells are easily adsorbed, and clogging easily occurs. Furthermore, since the metal surface has high water repellency and easily repels water, bubbles are likely to be generated on the filter.

  At this time, it is conceivable that a polymer having high biocompatibility is treated in advance. However, it is difficult to treat the surface of the polymer other than the noble metal due to the influence of the oxide film on the surface. In addition, the metal oxide film varies depending on the sample, and it is difficult to chemically bond the metal oxide film and the polymer firmly. Furthermore, even if the polymer is treated, a sufficient effect cannot be obtained if the bonding property is insufficient.

  The present invention improves a conventional biological material capturing filter, and maintains a pressure resistance while preventing cell adsorption to the filter and efficiently capturing the biological material and biological body A material capture system is provided.

  In order to achieve the above object, a biological material capturing filter according to an embodiment of the present invention includes a metal filter made of gold, platinum, palladium, or an alloy thereof, and biocompatibility chemically bonded to the surface of the metal filter. And a polymer.

  Here, the contact angle of pure water on the filter surface may be 60 degrees or less.

  Moreover, it can be set as the aspect whose ratio of the metal in the said metal filter surface calculated | required by the measurement by a X-ray photoelectron spectrometer is 10 at% or less.

  The biocompatible polymer may be a 2-methacryloyloxyethyl phosphorylcholine homopolymer (MPC) or a copolymer containing the MPC.

  The biocompatible polymer and the metal filter surface may be bonded by an amide bond.

  The copolymer containing the MPC has a carboxyl group and can be combined with the metal filter having an amino group on the surface.

  The metal filter having an amino group on the surface may be an embodiment of the metal filter in which a polymer having an amino group is adsorbed on the surface.

  The polymer having an amino group may be an polyethyleneimine.

  The polymer having an amino group may be bonded to the metal filter having a carboxyl group on the surface.

  The said metal filter which has the said carboxyl group can be made into the aspect which is the said metal filter by which the metal surface is processed by the thiol which has a carboxyl group.

  Moreover, it can be set as the aspect couple | bonded with the said metal filter which the copolymer containing the said MPC has an amino group and has a carboxyl group on the surface.

  The metal filter having a carboxyl group on the surface may be an embodiment of the metal filter in which a polymer having a carboxyl group adsorbed on the surface is adsorbed.

  The polymer having a carboxyl group may be polyacrylic acid.

  The polymer having a carboxyl group may be bonded to the metal filter having an amino group on the surface.

  The metal filter having an amino group on the surface can be an embodiment in which the metal surface is treated with a thiol having an amino group.

  In addition, a biological material capturing system according to one embodiment of the present invention is characterized by using the biological material capturing filter described above.

  ADVANTAGE OF THE INVENTION According to this invention, the biological material capture filter and biological material capture system which can prevent a cell adsorption | suction to a filter and can capture a biological material efficiently are provided, maintaining pressure resistance.

It is a figure explaining schematic structure of a cell capture cartridge. It is a figure explaining the manufacturing method of a filter. It is a schematic sectional drawing which shows the method of manufacturing a filter using a copper plate (when performing copper plating, Ni plate). It is a figure explaining the definition of the long hole diameter in a filter, a short hole diameter, and an aperture ratio.

  DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

  In the biological material capturing system according to this embodiment, filtration by a filter is performed. The configuration of the apparatus for performing the filtration is not particularly limited, but the case where a biological material capturing cartridge including a filter is used as an example will be described. As shown in FIG. 1, the biological material capturing cartridge 100 is connected to an inlet 130 to which an inflow pipe 125 into which a sample liquid containing biological material flows is connected, and an outflow pipe 135 from which the sample liquid containing biological material flows out. A housing 120 having an outflow port 140 and a filter 105 (a biological material capturing filter) disposed in the housing 120 and having a capturing region for capturing the biological material contained in the sample liquid.

  A “biological material” that is a capture target in the biological material capture system refers to a specific type of cell contained in a biological sample. Examples of such a biological substance include cells in a living body. Examples of cells in the living body include cells in blood. Among cells in blood, examples of cells that can be captured include blood circulating cancer cells (CTC: Circulating Tumor Cell) and circulating endothelial cells (CEC: Circulating Endothelial Cells). CTCs are cancer (cancer) cells in the blood. When cells in blood are capture targets, they can be captured even if the cells are alive. The biological material capturing system is suitably used for capturing cancer cells in blood including CTC. CEC is an endothelial cell of a blood vessel and becomes a mature cell that has been detached from the blood vessel wall by metabolism. CTC is said to increase in pathological conditions such as cardiovascular diseases (such as myocardial infarction). The biological material capture system has a function of selectively capturing specific types of cells contained in a biological sample such as CTC and CEC. When CTC or CEC is a capture target, blood or a liquid obtained by adding an additive such as a buffer to blood can be used as a sample liquid containing a biological substance.

  The housing 120 is a member for holding the filter 105 and includes an upper member 110 and a lower member 115. The shape of the housing 120 may be a rectangular parallelepiped or a cylinder, and is not particularly limited.

  For example, a processing solution such as a sample solution or a cleaning solution is connected upstream of the inflow pipe 125. Further, by connecting the pump P downstream of the outflow pipe 135, the sample liquid and the like are supplied from the inflow pipe 125 to the inside of the housing 120 by driving the pump P. Then, it is discharged from the outflow pipe 135 to the outside.

  A through hole 106 is formed in the filter 105. When blood is introduced into the biological material capture cartridge 100 as a sample solution, blood red blood cells and the like pass through the through-hole 106, but blood rare cells that are biological materials to be captured cannot pass through the through-hole 106, and the filter 105 It stays on the surface. Thereby, blood rare cells can be collected. The filter 105 includes a metal filter 105A made of gold, platinum, palladium, or an alloy thereof, and a biocompatible polymer 105B that is chemically bonded to the surface of the metal filter. This point will be described later.

  The shape of the biological material capturing cartridge 100 is not limited to the above. The configuration of the apparatus is not particularly limited as long as the biological material can be captured by filtration using a filter.

  Next, with reference to FIGS. 2 and 3, a method for producing the metal filter 105 </ b> A (see FIG. 1) serving as the base material of the biological material capturing filter according to the present embodiment will be exemplified, and the metal filter will be described. Do.

  FIG. 2 is a schematic cross-sectional view showing a method of manufacturing a metal thin film filter using a substrate in which peelable copper foil (Ni foil when copper plating is performed) is bonded to MCL.

  FIG. 2A shows a substrate in which peelable copper foil 2 (Ni foil when copper plating is performed) is laminated on MCL1. First, this substrate is prepared.

  The material used as the substrate for producing the filter is preferably copper (nickel when the plating is copper). Copper can be easily removed by chemical dissolution with a chemical solution, and is superior to other materials in adhesion to the photoresist.

  Next, as shown in FIG. 2B, a photoresist 3 is formed on the peelable copper foil 2 of the substrate. The thickness of this photoresist is preferably 1.0 to 2.0 times the thickness of the subsequent conductor. If this thickness is thin, resist peeling will be difficult later, and if it is thick, circuit formability will be difficult. Specifically, a thickness of 15 μm to 50 μm is preferable. Next, a photomask is overlapped to perform photoresist exposure. Next, the photoresist development and removal of the unexposed portion is performed with an alkaline solution or the like. Next, plating is performed on a portion not covered with the photoresist by pattern electroplating. This plated portion becomes the material of the filter. The photosensitive resin composition that is a photoresist is preferably a negative photosensitive resin composition. The negative photosensitive resin composition preferably contains at least a binder resin, a photopolymerizable compound having an unsaturated bond, and a photopolymerization initiator.

  Next, as shown in FIG. 2C, after exposing the photomask 4 on the photoresist 3, photoresist exposure is performed. Thereby, the exposure part 3a is formed in the area | region where the photomask 4 is not piled up.

  Next, as shown in FIG. 2D, the photoresist is removed from the unexposed portion 3b with an alkaline solution or the like. Thereby, the exposure part 3a remains in the position corresponding to the through-hole of the filter.

  Next, as shown in FIG. 2E, the plating layer 5 is formed by performing electroplating on the portion not covered with the photoresist. This plated portion becomes the material of the filter.

  Next, as shown in FIG. 2 (F), peelable copper foil 2 on which plating layer 5 is formed by electrolytic plating is peeled from MCL1.

  Next, as shown in FIG. 2G, the peelable copper foil 2 is removed by chemical dissolution with a chemical solution, and the free-standing film formed by the exposed portion 3a and the plating layer 5 is taken out.

  Next, as shown in FIG. 2H, the exposed portion 3 a made of the photoresist remaining in the free-standing film is removed, and a through hole 6 is formed in the plating layer 5.

  Next, as shown in FIG. 2 (I), electroless gold plating is performed to form a gold plating layer 7 on the filter surface. As described above, the filter 105 used in the biological material capturing cartridge 100 can be manufactured.

  FIG. 3 is a schematic cross-sectional view showing a method of manufacturing a metal thin film filter using a copper plate (Ni plate when copper plating is performed).

  The method shown in FIG. 3 is the same as the manufacturing method shown in FIG. 2 except that a copper plate (or Ni plate) 2 'is used instead of the MCL 1 and the peelable copper foil 2 in the method shown in FIG.

  That is, FIG. 3A shows a process of preparing a copper plate 2 'used as a substrate.

  FIG. 3B shows a process of laminating the photoresist 3 on the copper plate 2 '. FIG. 3C shows the photoresist exposure with the photomask 4 overlaid. FIG. 3D shows development removal of the photoresist in the unexposed portion 3b. FIG. 3E shows a step of forming a plating layer by electrolytic plating on a portion of the photoresist not covered with the exposed portion 3a. FIG. 3F shows a process of removing the copper plate 2 ′ by chemical dissolution (chemical etching) with a chemical solution and taking out a free-standing film formed by the exposed portion 3 a and the plating layer 5. FIG. 3G shows a step of removing the exposed portion 3 a of the photoresist remaining in the free-standing film and forming a through hole 6 in the plating layer 5. FIG. 3H shows a process of performing electroless gold plating to form a gold plating layer 7 on the filter surface.

  As described above, the metal filter 105A in the filter 105 used in the biological material capturing cartridge 100 can be manufactured by using either the method shown in FIG. 2 or the method shown in FIG.

  The material of the metal filter 105A in the filter 105 is metal. As the main component of the metal, any of nickel, silver, palladium, copper, and iridium is preferable. These metals can be electroplated.

  The main component of the metal is preferably nickel, silver, palladium, copper, iridium, or an alloy thereof. These metals can be electroplated. The “main component” indicates that the weight ratio of the component to the total weight of the metal filter 105A is 50% or more.

  Palladium and iridium have high redox potential, are hardly soluble and have good characteristics, but have the disadvantage of being expensive. Nickel has a lower oxidation-reduction potential than hydrogen, so it is easily dissolved but is inexpensive. Silver and palladium are noble metals and are relatively inexpensive compared to iridium.

  The material used as the substrate (copper plate 2 ': see Fig. 3) for producing the filter is preferably copper (nickel when the filter material is copper). Copper can be easily removed by chemical dissolution with a chemical solution, and is superior to other materials in adhesion to the photoresist. In addition, also about the metal foil 2 (refer FIG. 2) laminated | stacked with respect to MCL1, copper (when the filter material is copper, nickel) is preferable.

  Moreover, the formation of the plating layer 5 shown in FIGS. 2E and 3E is performed by electrolytic plating. For example, as electrolytic nickel plating, Watts bath (mainly nickel sulfate, nickel chloride, boric acid), sulfamic acid bath (mainly nickel sulfamate, boric acid), strike bath (mainly nickel chloride, hydrogen chloride) Etc.

  Examples of the electrolytic silver plating include a bath mainly composed of potassium silver cyanide or potassium tartrate.

  Examples of the electrolytic palladium plating include a bath made of a water-soluble palladium salt and a naphthalene sulfonic acid compound.

  Examples of the electrolytic iridium plating include a bath containing a soluble iridium salt containing a halogen and alcohols.

  Examples of the electrolytic copper plating include a bath mainly composed of copper sulfate, sulfuric acid, and chloride ions.

Electrolytic plating is performed using these plating baths. The current density during electroplating is in the range of 0.3~4A / dm ² is well, and more preferably in the range of 0.5~3A / dm ^2. By setting the current density to 4 A / dm ² or less, it is possible to suppress the occurrence of roughness, and by setting the current density to 0.3 A / dm ² or more, the metal crystal grains are sufficiently grown and the effect as a barrier layer is achieved. Therefore, the effect of this embodiment can be obtained satisfactorily.

  The location of the resist when plating is the location of the through hole. Examples of the opening shape of the through hole include a circle, an ellipse, a square, a rectangle, a rounded rectangle, and a polygon. From the viewpoint of efficiently capturing the target component, a circle, rectangle, or rounded rectangle is preferable. In addition, a rounded rectangle is particularly preferable from the viewpoint of preventing clogging of the filter.

  The hole diameter of the through hole 6 is set according to the size of the component to be captured. In the present specification, the hole diameter in a shape other than a circle such as an ellipse, rectangle, or polygon is the maximum value of the diameter of a sphere that can pass through each through hole. For example, when the opening shape is a rectangle or a rounded rectangle, as shown in FIG. 4, the short hole diameter on the short side and the long hole diameter on the long side can be defined, but the hole diameter of the through hole 6 is the short hole diameter. It becomes. Further, when the opening shape is a polygon, the diameter is the diameter of the inscribed circle of the polygon. When the opening shape is a rectangle or a rounded rectangle, a gap is formed in the opening in the long side direction of the opening shape even when the component to be captured is captured in the through hole 6. Since liquid can pass through the gap, clogging of the filter can be prevented. The length of the short pore diameter of the filter is preferably 5 μm to 15 μm, more preferably 7 μm to 9 μm.

  The average aperture ratio of the through holes of the metal filter 105A in the filter 105 is preferably 3% to 50%, more preferably 3% to 20%, and particularly preferably 3% to 10%. Here, the aperture ratio refers to the ratio of the area occupied by the through hole to the area of the region functioning as a filter (see FIG. 4). In other words, the area of the region functioning as a filter is a region (region A1 surrounded by a broken line in FIG. 4) obtained by connecting the outermost portions of the through holes among the plurality of through holes included in the filter. If the aperture ratio is too high, the pressure applied to the white blood cells decreases, and the white blood cell residue increases. If the aperture ratio is low, the amount of blood that can flow is reduced. Note that the aperture ratio of the filter 105 is smaller than that of the metal filter 105A due to adhesion of a biocompatible polymer described later, but it can be considered as an error level.

  The thickness of the metal filter 105A in the filter 105 is preferably 3 μm to 50 μm, more preferably 5 μm to 30 μm, and particularly preferably 8 μm to 20 μm. When the film thickness of the metal filter 105A is less than 3 μm, the strength of the filter may be lowered, and handling may be difficult. On the other hand, when the thickness exceeds 50 μm, there is a concern that productivity decreases due to a longer processing time, cost disadvantage due to unnecessarily consuming materials, and difficulty in fine processing itself, and leukocytes are difficult to escape. Note that the thickness of the filter 105 is larger than that of the metal filter 105A due to adhesion of a biocompatible polymer described later, but it can be considered as an error level.

  After forming the circuit, the resin layer is peeled off and the copper foil is etched (FIG. 2), or the copper plate is removed (FIG. 3), so that plating is performed as shown in FIG. 2 (H) or FIG. 3 (G). The filter with layer 5 is completed.

  Next, the resist (exposed portion 3a) remaining on the filter is removed with strong alkali. As the strong alkali, 0.1 wt% to 10 wt% NaOH or KOH aqueous solution is preferable. Monoethanolamine (1 vol% to 20 vol%) or the like may be added to promote peeling. When peeling is difficult, the resist can be removed with a solution obtained by adding alkali (0.1 wt% to 10 wt% NaOH or KOH) to sodium permanganate or potassium permanganate.

  Precious metal plating may be performed on the filter from which the resist has been removed.

  As the noble metal plating, gold, palladium, platinum, ruthenium, indium and the like are desirable.

  Among precious metal plating, gold is said to have the highest oxidation-reduction potential among all metals as described above and is not cytotoxic. There is almost no discoloration during long-term storage. In the following embodiments, a plating method in the case of performing gold plating will be described.

  Gold plating can be performed electrolessly or electrolytically. In the case of performing electrolysis, it is desirable to perform the electroless process because the thickness variation increases and the accuracy of the hole diameter of the filter 105 is easily affected. However, electrolytic gold plating can improve the coverage.

  Gold plating is effective only by displacement plating, but a combination of displacement plating and reduction plating is more effective.

  The surface of the filter before gold plating may be oxidized. Therefore, the oxide film is removed, but here it is preferable to wash with an aqueous solution containing a compound that forms a complex with a metal ion.

  Specifically, an aqueous solution containing cyanides, EDTAs or citric acids is preferable.

  Of these, citric acids are most suitable as a pretreatment for gold plating. Specifically, citric acid anhydride, citric acid hydrate, citrate or citrate hydrate may be used. Specifically, citric acid anhydride, citric acid monohydrate, Sodium citrate, potassium citrate and the like can be used. The concentration is preferably 0.01 mol / L to 3 mol / L, more preferably 0.03 mol / L to 2 mol / L, and particularly preferably in the range of 0.05 mol / L to 1 mol / L. preferable. By setting it as 0.01 mol / L or more, the adhesiveness of an electroless gold plating layer and a filter improves. Moreover, when exceeding 3 mol / L, an effect is not improved and it is economically unpreferable.

  Immersion in a solution containing citric acid is preferably performed at 70 to 95 ° C. for 1 to 20 minutes.

  In the solution containing citric acid, it is possible to add a buffer such as a reducing agent and a pH adjusting agent contained in the plating solution as long as the effects of the invention can be obtained. Desirably, an aqueous solution of citric acid alone is most preferred. The pH of the solution containing citric acid is preferably 5 to 10, more preferably 6 to 9.

  The pH adjuster is not particularly limited as long as it is an acid or an alkali. As the acid, hydrochloric acid, sulfuric acid, nitric acid and the like can be used. As the alkali, alkali metals such as sodium hydroxide, potassium hydroxide and sodium carbonate can be used. Or the alkaline-earth metal hydroxide solution is mentioned. As described above, it can be used as long as the effect of citric acid is not inhibited. Moreover, when the nitric acid is contained at a high concentration of 100 ml / L in the solution containing citric acid, the effect of improving the adhesiveness is reduced as compared with the case where the solution is treated with the solution containing only citric acid.

  The reducing agent is not particularly limited as long as it is reducible, and examples thereof include hypophosphorous acid, formaldehyde, dimethylamine borane, and sodium borohydride.

  Next, substitution gold plating is performed. The displacement gold plating includes a cyan bath and a non-cyan bath, but the non-cyan bath is desirable in view of environmental load or remaining cytotoxicity. Examples of the gold salt contained in the non-cyan bath include chloroaurate, gold sulfite, gold thiosulfate, and gold thiomalate. Only one type of gold salt may be used, or two or more types may be used in combination.

  Furthermore, since the cyan bath has an effect of dissolving the metal too much, depending on the metal, it tends to dissolve and easily generate pinholes. When the pretreatment is sufficiently performed as described above, a non-cyan plating bath is preferable.

  Gold sulfite is particularly preferred as the gold source. As the gold sulfite, sodium gold sulfite, potassium gold sulfite, gold ammonium sulfite and the like are preferable.

  The gold concentration is preferably in the range of 0.1 g / L to 5 g / L. If it is less than 0.1 g / L, gold is difficult to precipitate, and if it exceeds 5 g / L, the liquid tends to decompose.

The displacement gold plating bath may contain an ammonium salt or ethylenediaminetetraacetate as a gold complexing agent. Examples of the ammonium salt include ammonium chloride and ammonium sulfate. Examples of the ethylenediaminetetraacetate include ethylenediaminetetraacetic acid, sodium ethylenediaminetetraacetate, potassium ethylenediaminetetraacetate, and ammonium ethylenediaminetetraacetate. The concentration of the ammonium salt is preferably used in the range of 7 × 10 ⁻³ mol / L to 0.4 mol / L. If the concentration of the ammonium salt is outside this range, the liquid tends to become unstable. The concentration of ethylenediaminetetraacetate is preferably 2 × 10 ⁻³ mol / L to 0.2 mol / L. If the ammonium salt concentration is outside this range, the liquid tends to become unstable. .

  In order to keep the liquid stable, 0.1 g / L to 50 g / L of sulfite is preferably contained. Examples of the sulfite include sodium sulfite, potassium sulfite, and ammonium sulfite.

  When lowering the pH as a pH adjuster, it is preferable to use hydrochloric acid or sulfuric acid. Moreover, when raising pH, it is preferable to use sodium hydroxide, potassium hydroxide, or aqueous ammonia. The pH may be adjusted to 6-7. Outside this range, the stability of the solution or the appearance of the plating is adversely affected.

  Displacement plating is preferably used at a liquid temperature of 30 to 80 degrees, and outside this range adversely affects the stability of the liquid or the appearance of the plating.

  Although displacement plating is performed as described above, it is difficult to completely cover the metal by displacement plating. Therefore, next, reduction-type gold plating containing a reducing agent is performed. The thickness of the displacement plating is preferably in the range of 0.02 μm to 0.1 μm.

  As the gold salt of the reduction type gold plating, gold sulfite and thiosulfate are preferable, and the content thereof is preferably in the range of 1 g / L to 10 g / L as gold. If the gold content is less than 1 g / L, the gold precipitation reaction decreases, and if it exceeds 10 g / L, the stability of the plating solution decreases and the amount of gold consumed increases due to the removal of the plating solution. It is not preferable. The content is more preferably 2 g / L to 5 g / L.

  Examples of the reducing agent include hypophosphorous acid, formaldehyde, dimethylamine borane, sodium borohydride, and the like, and a phenyl compound-based reducing agent is more preferable. For example, phenol, o-cresol, p-cresol, o-ethylphenol, p-ethylphenol, t-butylphenol, o-aminophenol, p-aminophenol, hydroquinone, catechol, pyrogallol, methylhydroquinone, aniline, o-phenylene Examples thereof include diamine, p-phenylenediamine, o-toluidine, o-ethylaniline, p-ethylaniline, and one or more of these can be used.

  The content of the reducing agent is preferably 0.5 to 50 g / L. If the content of the reducing agent is less than 0.5 g / L, it tends to be difficult to obtain a practical deposition rate, and if it exceeds 50 g / L, the stability of the plating solution tends to decrease. . The content of the reducing agent is more preferably 2 to 10 g / L, and particularly preferably 2 to 5 g / L.

  The electroless gold plating solution may contain a heavy metal salt. From the viewpoint of promoting the deposition rate, the heavy metal salt is preferably at least one selected from the group consisting of thallium salt, lead salt, arsenic salt, antimony salt, tellurium salt and bismuth salt.

  Examples of the thallium salt include inorganic compound salts such as thallium sulfate salt, thallium chloride salt, thallium oxide salt, and thallium nitrate salt, and organic complex salts such as dithallium malonate salt. Lead salts include lead sulfate salt, nitric acid salt, and the like. Examples thereof include inorganic compound salts such as lead salts and organic acetate salts such as acetates.

  Examples of arsenic salts include inorganic compound salts such as arsenic salts and arsenic arsenate trioxide, and organic complex salts. Examples of antimony salts include organic complex salts such as antimony tartrate, antimony chloride salts, and antimony oxysulfate. And inorganic compound salts such as salts and antimony trioxide.

  Examples of the tellurium salt include inorganic compound salts such as tellurite and tellurate, and organic complex salts. Examples of the bismuth salt include bismuth sulfate (III), bismuth chloride (III), and bismuth nitrate (III). And organic complex salts such as bismuth (III) oxalate.

  One or more of the above heavy metal salts can be used, but the total amount of addition is preferably 1 ppm to 100 ppm, more preferably 1 ppm to 10 ppm, based on the total capacity of the plating solution. If it is less than 1 ppm, the effect of improving the deposition rate may not be sufficient, and if it exceeds 100 ppm, the stability of the plating solution tends to deteriorate.

  The electroless gold plating solution may contain a sulfur compound. By further including a sulfur compound in the electroless gold plating solution containing a phenyl compound-based reducing agent and a heavy metal salt, a sufficient deposition rate can be obtained even at a liquid temperature of about 60 to 80 ° C. In addition, the stability of the plating solution is particularly excellent.

  Examples of the sulfur compounds include sulfide salts, thiocyanates, thiourea compounds, mercaptan compounds, sulfide compounds, disulfide compounds, thioketone compounds, thiazole compounds, thiophene compounds, and the like.

  Examples of the sulfide salt include potassium sulfide, sodium sulfide, sodium polysulfide, potassium polysulfide, and the like. Examples of the thiocyanate include sodium thiocyanate, potassium thiocyanate, potassium dithiocyanate, and the like. Examples of the thiourea compound include thiourea, methylthiourea, dimethylthiourea and the like.

  Mercaptan compounds include 1,1-dimethylethanethiol, 1-methyl-octanethiol, dodecanethiol, 1,2-ethanedithiol, thiophenol, o-thiocresol, p-thiocresol, o-dimercaptobenzene, m -Dimercaptobenzene, p-dimercaptobenzene, thioglycol, thiodiglycol, thioglycolic acid, dithioglycolic acid, thiomalic acid, mercaptopropionic acid, 2-mercaptobenzozoimidazole, 2-mercapto1-methylimidazole, 2- Examples include mercapto-5-methylbenzimidazole.

  Examples of the sulfide compound include diethyl sulfide, diisopropyl sulfide, ethyl isopropyl sulfide, diphenyl sulfide, methyl phenyl sulfide, rhodanine, thiodiglycolic acid, thiodipropionic acid, and the like. Examples of the disulfide compound include dimethyl disulfide, diethyl disulfide, disulfide. A propyl disulfide etc. can be illustrated.

  Furthermore, examples of the thioketone compound include thiosemicarbazide, and examples of the thiazole compound include thiazole, benzothiazole, 2-mercaptobenzothiazole, 6-ethoxy-2-mercaptobenzothiazole, 2-aminothiazole, 2,1,3- Examples include benzothiadiazole, 1,2,3-benzothiadiazole, (2-benzothiazolylthio) acetic acid, 3- (2-benzothiazolylthio) propionic acid and the like, and examples of thiophene compounds include thiophene and benzothiophene. It can be illustrated.

  A sulfur type compound may be used independently and may use two or more types. The content of the sulfur compound is preferably 1 ppm to 500 ppm, more preferably 1 ppm to 30 ppm, and particularly preferably 1 ppm to 10 ppm. If the content of the sulfur-based compound is less than 1 ppm, the deposition rate is reduced, poor plating adhesion occurs, and the coating appearance deteriorates. If it exceeds 500 ppm, the concentration management becomes difficult and the plating solution becomes unstable.

  The electroless gold plating solution preferably contains at least one of a complexing agent, a pH buffering agent and a metal ion concealing agent in addition to the above-described gold salt, reducing agent, heavy metal salt and sulfur compound. Etc. are more preferable.

  The electroless gold plating solution of the present invention preferably contains a complexing agent. Specific examples include non-cyanide complexing agents such as sulfites, thiosulfates, and thiomalates. The content of the complexing agent is preferably 1 g / L to 200 g / L based on the total capacity of the plating solution. When the content of the complexing agent is less than 1 g / L, the gold complexing power decreases and the stability decreases. If it exceeds 200 g / L, the plating stability is improved, but recrystallization occurs in the solution, which is not economically good. The content of the complexing agent is more preferably 20 g / L to 50 g / L.

  The electroless gold plating solution preferably contains a pH buffer. The pH buffering agent has an effect of keeping the deposition rate constant and stabilizing the plating solution. A plurality of buffering agents may be mixed. Examples of pH buffering agents include phosphates, acetates, carbonates, borates, citrates, sulfates, etc. Among these, boric acid and sulfates are particularly preferable.

  The content of the pH buffering agent is preferably 1 g / L to 100 g / L based on the total capacity of the plating solution. When the content of the pH buffering agent is less than 1 g / L, there is no pH buffering effect, and when it exceeds 100 g / L, recrystallization may occur. A more preferable content is 20 g / L to 50 g / L.

  The gold plating solution preferably contains a masking agent. As the masking agent, a benzotriazole-based compound can be used, and examples of the benzotriazole-based compound include benzotriazole sodium, benzotriazole potassium, tetrahydrobenzotriazole, methylbenzotriazole, and nitrobenzotriazole.

  The content of the metal ion concealing agent is preferably 0.5 g / L to 100 g / L based on the total capacity of the plating solution. When the content of the metal ion concealing agent is less than 0.5 g / L, there is a tendency that the effect of concealing impurities is small and sufficient liquid stability cannot be ensured. On the other hand, if it exceeds 100 g / L, recrystallization may occur in the plating solution. In view of cost and effect, a range of 2 g / L to 10 g / L is most preferable.

  The pH of the gold plating solution is preferably in the range of 5-10. When the pH of the plating solution is less than 5, sulfite or thiosulfate, which is a complexing agent of the plating solution, may be decomposed to generate toxic sulfite gas. When the pH exceeds 10, the stability of the plating solution tends to decrease. In order to improve the deposition efficiency of the reducing agent and obtain a fast deposition rate, the pH of the electroless gold plating solution is preferably in the range of 8-10.

  As a method of electroless plating, gold plating is performed by immersing the filter after the replacement gold plating.

  The temperature of the plating solution is preferably 50 ° C to 95 ° C. If it is less than 50 ° C., the deposition efficiency is poor, and if it is 95 ° C. or more, the liquid tends to become unstable.

  The outermost gold plating layer 7 thus formed is preferably made of gold having a purity of 99% by weight or more. When the gold purity of the gold plating layer 7 is less than 99% by weight, the cytotoxicity of the contact portion is increased. From the viewpoint of improving reliability, the purity of the gold layer is more preferably 99.5% by weight or more.

  The thickness of the gold plating layer 7 is preferably 0.005 μm to 3 μm, more preferably 0.05 μm to 1 μm, and still more preferably 0.1 μm to 0.5 μm. By making the thickness of the gold layer 0.005 μm or more, metal elution can be suppressed to some extent. On the other hand, even if the thickness exceeds 3 μm, the effect is not greatly improved. Therefore, it is preferably 3 μm or less from the economical viewpoint.

  The gold surface (metal surface) of the metal filter 105A formed as described above has no cytotoxicity and is stable in the atmosphere or most aqueous solutions containing blood. However, since the gold surface is relatively hydrophobic and has low biocompatibility, a treatment for improving biocompatibility is performed. Hereinafter, in order to improve biocompatibility, an example of the process for chemically bonding biocompatible polymer 105B to the surface of metal filter 105A is shown.

  White blood cells, red blood cells, and platelets, which are components in blood, show rejection against foreign substances. Therefore, it is preferable to pre-treat the metal surface. In this case, it is preferable that the biocompatible polymer is chemically and strongly adsorbed to the surface of the metal filter 105A (the surface of the gold plating layer 7).

  Examples of biocompatible polymers include vertebrate albumin or artificial synthetic polymers, and artificial synthetic polymers are preferred in view of filter storage stability and lot differences in polymer properties. When vertebrate albumin is used, it is necessary to perform a filter treatment immediately before blood treatment, which complicates the work. In particular, when blood cells are immobilized (when biologically dead), the artificial synthetic polymer has better characteristics.

Examples of the artificial synthetic polymer include silicone, various polyurethanes, polyphosphazene, and the like. Among them, 2-methacryloyloxyethyl phosphorylcholine (abbreviation: MPC) homopolymer or a copolymer containing MPC is particularly excellent. The structural formula is shown below.

  In addition, although the said Chemical formula shows MPC polymer, an alkyl group, hydrogen, an amino group, a hydroxyalkyl group, etc. are applicable to R.

  Commercially available MPC polymers can also be used. Examples of commercially available MPC polymers include Biolipideure 103, Biolipidure 203, Biolipidure 206, Biolipidure 405, Biolipidure 502, Biolipidure 702, Biolipidure 802, Biolipidure 1002, Biolipidure 1201, Biolipidure 1301, and the like.

  Among them, among MPC polymers, those in which R is hydrogen in the chemical formula or those containing an amino group are preferable because the binding property to the filter is improved. That is, when the biocompatible polymer contains an amino group or a carboxyl group, an alternating lamination method using electrostatic adsorption can be used and the cytotoxicity is low.

  The surface of the gold plating layer 7 of the filter (the surface of the metal filter 105A) and the biocompatible polymer are preferably bonded by an amide bond. In order to form an amide bond between the gold plating layer 7 and the biocompatible polymer, first, the gold plating layer 7 is treated with a compound having a mercapto group (thiol group). Thereby, a mercapto group is formed on the surface of the gold plating layer 7. The mercapto group on the surface of the gold plating layer 7 is reacted with a biocompatible polymer containing a structural unit having an amide group at its terminal. Thereby, the surface of the gold plating layer 7 and the biocompatible polymer are bonded by an amide bond. In this case, the connectivity between the two is improved.

  Here, for example, a method of forming a polymer having a carboxyl group or an amino group on the surface of the gold plating layer 7 of the filter 105 (the surface of the metal filter 105A: the same applies to other materials such as palladium or platinum) will be described.

  The gold surface can be modified with a compound having any of a mercapto group, a sulfide group, and a disulfide group that forms a coordination bond with gold.

  Specifically, the compounds used for modifying the gold surface include 2-aminoethanethiol, o-fluorobenzenethiol, m-hydroxybenzenethiol, 2-methoxybenzenethiol, 4-aminobenzenethiol, cysteamine, cysteine , Dimethoxythiophenol, furfuryl mercaptan, thioacetic acid, thiobenzoic acid, thiosalicylic acid, or dithiodipropionic acid.

  A method for modifying the gold surface using the above compound is not particularly limited, but a compound such as mercaptoacetic acid is dispersed in an organic solvent such as methanol or ethanol in an amount of about 10 mmol / L to 100 mmol / L, and gold is contained therein. Conductive particles having a surface are dispersed.

  Next, in order to increase the coverage, it is desirable to coat with a polymer or the like. The polymer may be coated using electrostatic interaction.

  Such a method is called an alternating lamination method (Layer-by-Layer assembly). The alternate lamination method is described in G.H. This is a method for forming an organic thin film published in 1992 by Decher et al. (Thin Solid Films, 210/211, p831 (1992)). In this method, a polycation adsorbed on a substrate by electrostatic attraction by alternately immersing the base material in an aqueous solution of a polymer electrolyte having a positive charge (polycation) and a polymer electrolyte having a negative charge (polyanion). A combination of polyanions is laminated to obtain a composite film (alternate laminated film).

  In the alternating layering method, the film is grown by attracting the charge of the material formed on the substrate and the material having the opposite charge in the solution by electrostatic attraction, so that the adsorption proceeds and the charge is neutralized. When this occurs, no further adsorption occurs. Therefore, when reaching a certain saturation point, the film thickness does not increase any more.

  Examples of such a polymer include polyethylene glycol, polyhydroxylmethacrylate 2-hydroxyethyl, polyacrylic acid, polyethyleneimine, and polyallylamine, and are not particularly limited. The polymer may be copolymerized with acrylic acid or methacrylic acid. From the viewpoint of charge density and cost, polyethyleneimine is preferable for a cation, and polyacrylic acid is preferable for an anion.

  These polymers cannot be generally determined depending on the type, but generally those of about 500 to 1,000,000 are preferable, and the range of 5,000 to 200,000 is more preferable. In general, the concentration of the polymer electrolyte in the aqueous solution of the polymer electrolyte is preferably about 0.01% to 10% (weight). Further, the pH of the polymer electrolyte solution is not particularly limited.

  The coverage can be controlled by adjusting the type, molecular weight, and concentration of the polymer electrolyte thin film. The concentration of the polymer in the aqueous polymer electrolyte solution is preferably in the range of 0.1% to 5.0%.

  Thus, after coating a cationic or anionic polymer, it is good to coat | cover with the biocompatible polymer which has a carboxyl group or an amino group at the end.

  When the filter surface is coated with a polymer having an amino group, it is preferable to coat a biocompatible polymer having a carboxyl group. Conversely, when the filter surface is coated with a polymer having a carboxyl group, a biocompatible polymer having an amino group may be coated.

  When increasing the adsorption thickness of the biocompatible polymer, a biocompatible polymer having a carboxyl group may be coated on the biocompatible polymer having an amino group.

  The thickness of the biocompatible polymer thus formed is desirably 20 mm (angstrom) or more. The thickness of the biocompatible polymer can be controlled by the treatment concentration and the number of treatments.

  When the thickness of the biocompatible polymer is 20 mm or less, the effect tends to be insufficient.

  There is no particular upper limit on the thickness of the biocompatible polymer. However, if the thickness exceeds 0.1 μm, the pore size of the filter is affected, and leukocytes are difficult to escape.

  By treating the biocompatible polymer, the water contact angle on the metal surface is reduced. The contact angle may be measured with an apparatus conforming to JIS R3265 “Testing method for wettability of substrate glass surface”. The contact angle on the filter surface after treatment with the biocompatible polymer is preferably 90 degrees or less, and more preferably 60 degrees or less.

  Generally, the contact angle of pure water is said to be wet when it is less than 90 degrees, but it is not perfect. When filtration is performed with poor wettability, bubbles are generated and a part of the filter tends to become unusable.

  The biocompatible polymer should completely cover the metal surface of the filter. Specifically, when analysis is performed using XPS (X-ray Photoelectron Spectroscopy), the lower the surface metal (Au, Pd, Pt) ratio, the better. Desirably, it is more desirable that it is 5 at% or less. When the proportion of surface metal is high, the coverage of the biocompatible polymer is low and the effect is reduced.

  The measurement result by XPS is also strictly different depending on the measuring device or the like. Therefore, in this embodiment, it shall measure on the conditions shown in Table 1.

  In this way, blood is passed through the filter treated with the biocompatible polymer (filtration). Examples of the blood collection tube include an EDTA blood collection tube that preserves cells alive and a fixed blood collection tube, but a fixed blood collection tube is preferable. Examples of such a blood collection tube include Cyto-Cex, Cell-Free-DNA, (trade name, manufactured by Streck). In the present invention, it is preferable to use a fixed blood collection tube because a fixed blood collection tube can be used for a long time after blood collection compared to an EDTA blood collection tube.

  As filtration, the filter 105 is attached to a device such as the biological material capturing cartridge 100 to allow blood to pass therethrough. The blood may be treated with a negative pressure from below the filter, may be treated with pressure from above the filter, or may be treated with a centrifugal force like centrifugation. In either method, it is important to control the linear velocity at which blood passes through the pores of the filter.

  The linear velocity (blood volume / total pore area) through which the blood passes through the pores of the filter is preferably in the range of 0.5 cm / min to 100 cm / min, and in the range of cm / min to 5 cm / min. Is more desirable.

  Rare cells such as CTC concentrated as described above can be concentrated. By applying a biocompatible polymer to the filter chemically and firmly, it is possible to exclude components such as red blood cells, white blood cells, and plasma plates.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and a comparative example, this invention is not limited to a following example at all.

(Filter 1)
Photosensitive resin composition (PHOTEC RD-1225: thickness 25 μm, manufactured by Hitachi Chemical Co., Ltd.) 250 mm square substrate (MCL-E679F: substrate in which peelable copper foil is bonded to the surface of MCL, manufactured by Hitachi Chemical Co., Ltd.) Laminated on one side. Lamination conditions were performed at a roll temperature of 90 ° C., a pressure of 0.3 MPa, and a conveyor speed of 2.0 m / min.

Next, a glass mask having a light-transmitting portion with a rounded rectangular shape, a size of 8.8 × 30 μm, and a pitch of 60 μm in both the minor axis and major axis directions was allowed to stand on the photoresist laminate surface of the substrate. In this embodiment, a glass mask in which rounded rectangles facing in the same direction are arranged at a constant pitch in the major axis and minor axis directions was used. Subsequently, under a vacuum of 600 mmHg or less, ultraviolet rays having an exposure amount of 30 mJ / cm ² were irradiated from above the substrate on which the glass mask was placed by an ultraviolet irradiation device.

  Next, development was performed with a 1.0% aqueous sodium carbonate solution to form a resist layer with a rectangular photoresist standing vertically on the substrate. Plating was performed so that the copper exposed portion of the resist-coated substrate had a temperature of 55 ° C. in a nickel plating solution adjusted to a pH of 4.5 and a thickness of about 20 μm for about 20 minutes. Table 2 shows the composition of the nickel plating solution.

  Next, the obtained nickel plating layer is peeled off together with the peelable copper foil of the substrate, and this peelable copper foil is chemically dissolved by a chemical solution in a stirring process at a temperature of 40 ° C. for about 120 minutes (MEC BRIGHT SF-5420B, MEC stock). The self-supporting membrane (20 mm × 20 mm) to be a metal filter was removed by removing it by the company.

  Finally, the photoresist remaining in the self-supporting film was removed by resist stripping (P3 Poleve, Henkel) by ultrasonic treatment at a temperature of 60 ° C. for about 40 minutes to produce a metal filter having fine through holes.

  As a result, there was no damage such as wrinkles, creases, scratches, curls, etc., and a metal filter having through holes with sufficient accuracy was produced.

  Next, the metal filter was immersed in acidic degreasing solution Z-200 (manufactured by World Metal: trade name), and organic substances on the metal filter were removed (40 ° C. for 3 minutes).

  After washing with water, pretreatment for substitution gold plating was performed at 80 ° C for 10 minutes with a solution obtained by removing gold sulfite as a gold supply source from HGS-100 (trade name, manufactured by Hitachi Chemical Co., Ltd.), a non-cyan electroless Au plating. It was.

  Next, it was immersed in HGS-100 (trade name, manufactured by Hitachi Chemical Co., Ltd.), which is a non-cyan substitution type electroless Au plating, for 20 minutes at 80 ° C. to perform substitution gold plating. The thickness of the displacement gold plating was 0.05 μm.

  After washing with water, it was immersed in HGS-5400 (trade name, manufactured by Hitachi Chemical Co., Ltd.), which is non-cyan reduced electroless Au plating, at 65 ° C. for 10 minutes, gold-plated, washed with water and dried. The total thickness of the gold plating was 0.2 μm. Thereby, a metal filter is created.

(surface treatment)
A reaction solution was prepared by dissolving 8 mmol of dithiodipropionic acid having a carboxyl group in the molecule in 200 mL of methanol. Next, a metal filter after gold plating was added to the reaction solution, reacted at room temperature for 2 hours, and then washed with methanol to prepare a filter having a carboxyl group on the surface.

  A filter having an amino group on the surface was prepared by immersing in a metal filter having a carboxyl group in a 0.3 wt% polyethyleneimine 0.3 wt% aqueous solution having a large number of amino groups in the molecule for 15 minutes and washing.

  The filter having an amino group on the surface is immersed for 15 minutes in a methanol solution containing 0.3 wt% of a copolymer polymer BL405 (manufactured by NOF CORPORATION: trade name) of an MPC monomer and a carboxyl group-containing monomer. Finally, a 30 minute treatment at 80 ° C in a vacuum dryer facilitates dehydration condensation of carboxyl groups and amino groups, and captures biological materials in which biocompatible polymers are firmly chemically bonded to the surface of the metal filter. A filter 1 was prepared.

(Filter 2)
A biological material capturing filter 2 was produced under the same conditions as the filter 1 except that the treatment concentration of BL405 was 0.1 wt%.

(Filter 3)
A biological material capturing filter 3 was produced under the same conditions as the filter 1 except that the treatment concentration of BL405 was changed to 0.03 wt%.

(Filter 4)
A filter 4 for capturing a biological material was produced under the same conditions as the filter 1 except that the surface treatment step was changed to the method shown below.

(Surface treatment of filter 4)
A reaction solution was prepared by dissolving 8 mmol of 2-aminoethanethiol having an amino group in the molecule in 200 mL of methanol. Next, a metal filter after gold plating was added to the reaction solution, reacted at room temperature for 2 hours, and then washed with methanol to prepare a filter having an amino group on the surface.

  A filter having a carboxyl group on the surface was prepared by immersing in a metal filter having a carboxyl group in a 0.3 wt% polyacrylic acid 0.3 wt% aqueous solution having a number of carboxyl groups in the molecule for 15 minutes and washing. .

  The filter having a carboxyl group on the surface is immersed for 15 minutes in a methanol solution containing 0.3 wt% of a copolymer polymer BL502 (manufactured by NOF Corporation: trade name) of a monomer having an MPC monomer and an amino group for washing. Finally, by performing a treatment at 80 ° C. for 30 minutes in a vacuum dryer, it promotes dehydration condensation of carboxyl groups and amino groups, and for capturing biological substances in which the biocompatible polymer is firmly chemically bonded to the filter surface. Filter 4 was produced.

(Filter 5)
A filter was produced in the same manner as Filter 1 except that one-stage electroless platinum plating was performed instead of the two-stage gold plating. Rectres Pt100 (Nippon Electroplating Engineers Co., Ltd. product name) was used as the electroless platinum plating solution. The thickness of platinum was 0.1 μm.

(Filter 6)
A filter 6 was prepared in the same manner as the filter 1 except that one-stage electroless palladium plating was performed instead of the two-stage gold plating. Paradex Strike 3 (manufactured by Nippon Electroplating Engineering Co., Ltd .: trade name) was used as the electroless palladium plating solution. The thickness of palladium was 0.1 μm.

(Filter 7)
A filter 7 was produced in the same manner as the filter 1 except that electrolytic palladium plating was used instead of electrical Ni plating. Paradix LF-5 (Nippon Electroplating Engineers Co., Ltd .: trade name) was used as the electroplating plating solution. Plating is performed under the same conditions as filter 1 except that the plating temperature is 50 ° C., the current density is 1 A / dm 2, and the electroplating is performed to be about 20 μm under the condition of about 4.2 minutes / μm. went.

(Filter 8)
A peelable nickel foil was used in place of the MCL peelable copper foil (the nickel foil was removed after electroplating). Further, a surface-treated filter 8 was produced in the same manner as the filter 1 except that electrolytic copper plating was used instead of electrical Ni plating. As the electrolytic copper plating solution, Microfab Cu200 (Nippon Electroplating Engineers Co., Ltd .: trade name) was used. Plating is performed under the same conditions as filter 1 except that the plating temperature is 25 ° C. and the current density is 3 A / dm 2, and the electroplating is performed so as to be about 20 μm at about 1.5 minutes / μm. went.

(Filter 9)
A filter 9 was produced in the same manner as the filter 1 except that the surface treatment was not performed.

(Filter 10)
A filter 10 was produced in the same manner as the filter 1 except that gold plating was not performed.

(Filter 11)
A filter 11 was prepared in the same manner as the filter 1 except that CM5206 (manufactured by NOF Corporation: trade name), which is an MPC-containing polymer having no hydrophobic and reactive functional group, was used instead of BL405.

(Filter 12)
Filter 12 was prepared in the same manner as filter 1 except that the dithiodipropionic acid treatment was omitted.

(Filter 13)
A filter 13 was prepared in the same manner as the filter 1 except that the dithiodipropionic acid treatment and the polyethyleneimine treatment were omitted.

(Filter 14)
A filter 14 was prepared in the same manner as the filter 1 except that BL203 (manufactured by NOF Corporation: trade name), which is a pure MPC polymer having no hydrophobic and reactive functional groups, was used instead of BL405.

(Experiment)
(Adjustment of non-small cell carcinoma cell lines)
NCI-H358 cells, a non-small cell carcinoma cell line, were statically cultured in an RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37 ° C. and 5% CO 2. Cells were detached from the culture dish by trypsin treatment and recovered, washed with phosphate buffered saline (PBS), and then washed with 10 μM CellTracker Red CMTPX (Life Technologies Japan, Inc.) at 37 ° C. for 30 minutes. NCI-H358 cells were stained by allowing to stand. Thereafter, the cells were washed with PBS and allowed to stand at 37 ° C. for 3 minutes by trypsin treatment to dissociate the cell mass. Thereafter, the trypsin treatment was stopped in the medium, washed with PBS, and then suspended in PBS containing 2 mM EDTA and 0.5% bovine serum albumin (BSA) (hereinafter referred to as 2 mM EDTA-0.5% BSA-PBS). did. PBS is phosphate buffered saline, and product code 166-23555 manufactured by Wako Pure Chemical Industries, Ltd. was used. BSA used was manufactured by SIGMA-ALDRICH (Product Name: Albumin from bovine serum-Lyophilized powder, Bio Reagent for cell culture). As EDTA, 2Na (ethylenediamine-N, N, N ′, N′-4 acetic acid disodium salt dihydrate) (product code 345-01865 manufactured by Wako Pure Chemical Industries, Ltd.) was used.

(Concentration of CTC in blood sample)
(Evaluation example 1)
An experiment was conducted using a CTC recovery device: CT6000 (trade name, manufactured by Hitachi Chemical Co., Ltd.) in which the filter 1 is set in a cartridge. The CTC recovery device has a flow channel for introducing a blood sample and a reagent, and the inlet of the flow channel is connected to a reservoir produced by processing a syringe. The cartridge in which the filter is set corresponds to the biological material capturing system in the above embodiment. By sequentially putting blood samples and reagents into this reservoir, operations such as CTC capture, staining, and washing can be performed easily and continuously.

  A blood sample was introduced into the CTC recovery device to concentrate the cancer cells. A sample containing 1000 cancer cells per mL of blood was used in healthy human blood collected in Cell Free DNA (manufactured by Streck: trade name), which is a vacuum blood collection tube containing a cell stabilizer as a blood sample. As a cancer cell, the above human non-small cell lung cancer cell line NCI-H358 was used. The blood used was 6 hours after blood collection.

  To the reservoir, 1 mL of 2 mM EDTA-0.5% BSA-PBS (hereinafter referred to as a washing solution) was introduced to fill the filter. Continuously, liquid feeding was started using a peristaltic pump at a flow rate of 200 μL / min. Thereafter, 1 mL of blood was added. After about 5 minutes, 2 mL of 2 mM EDTA-0.5% BSA-PBS was introduced into the reservoir to wash the cells.

  Next, a solution obtained by dissolving 4% PFA in the washing solution was placed in the cartridge, and the cells were immersed for 15 minutes.

  After washing, a solution obtained by dissolving 0.2% Triton X in the washing solution was put in the cartridge, and the cells were immersed for 15 minutes.

  After another 10 minutes, the pump flow rate was changed to 20 μL / min, 600 μL of cell staining solution (Hoechst 33342: 30 μl, Wash buffer: 300 mL) was introduced into the reservoir, and the cancer cells or leukocytes on the filter were fluorescently stained. The cells captured on the filter were stained for 30 minutes, and then 1 mL of 2 mM EDTA-0.5% BSA-PBS was introduced into the reservoir to wash the cells.

  Subsequently, the filter was observed using a fluorescence microscope (BX61, Olympus Corporation) equipped with a computer-controlled electric stage and a cooled digital camera (DP70, Olympus Corporation), and the number of cancer cells and leukocytes on the filter was determined. I counted.

  In order to observe fluorescence derived from Hoechst 33342 and CellTracker Red CMTPX, images were acquired using WU and WIG filters (Olympus Corporation), respectively. Lumina Vision (Mitani Corporation) was used as image acquisition and analysis software. The results are shown in Table 1. Cell recovery rate (%) = number of cancer cells collected in filter / number of cancer cells mixed in blood sample × 100%. The number of leukocytes was calculated by subtracting the number of cancer cells from the number of cells stained with Hoechst 33342. The experiment according to Evaluation Example 1 was performed by the above method.

(Evaluation Examples 2 to 14)
Experiments related to Evaluation Examples 2 to 14 were performed under the same conditions as in Evaluation Example 1 except that filters 2 to 14 were used instead of filter 1 used in Evaluation Example 1.

(Measurement of XPS)
Elemental analysis of the surface of the filter 1 was performed under the conditions shown in Table 3. An example of the measurement results is shown in Table 4. As shown in Table 4, it was confirmed that Au was almost covered. Hereinafter, the measurement of the filters 2 to 14 was performed while paying attention to Au.

(Contact angle)
For each of Evaluation Examples 1 to 14, the contact angle of the filter was measured. The measurement was performed with an apparatus (DM701, manufactured by Kyowa Interface Science Co., Ltd., trade name) based on JIS R3265 “Testing method for wettability of substrate glass surface”. The droplet size was 2 μL, and the time from dropping to measurement was 30 seconds.

(result)
The results are shown in Tables 5 and 6. Table 5 shows information related to the types, materials, and surface treatments 1 to 3 according to each evaluation example. Table 6 shows the results of elemental analysis on contact angle, Au, white blood cell count, and NCI-H358 recovery rate. And the result of the evaluation which concerns on a bubble is shown. Note that “A” related to bubbles indicates that almost no bubbles were generated in the cartridge. “B” indicates that bubbles are generated in a part (less than 5% by volume) of the cartridge. “C” indicates that more bubbles than the result B (5% by volume or more in the cartridge) were generated.

  As shown in Evaluation Example 1, when a biocompatible polymer is chemically bonded onto a gold-plated filter (metal filter), the number of white blood cells is relatively small and the recovery rate of cancer cells is high. In this case, the coverage of the polymer on the metal filter is also high (the gold element ratio is low). Since the contact angle is also low, the generation of bubbles can be suppressed.

  In Evaluation Examples 2-3, the polymer concentration during BL405 treatment is reduced, and the adsorption thickness by the biocompatible polymer is reduced. In this case, the polymer coverage is low (the gold element ratio is high). If the gold element ratio exceeds 5 at%, the white blood cell count increases and the recovery rate decreases (Evaluation Example 3).

  In Evaluation Example 4, the treatment order of cations and anions is changed, and a biocompatible polymer is chemically bonded onto the gold plating filter. In this case as well, as in Evaluation Example 1, the number of white blood cells is relatively small, and the recovery rate of cancer cells is high.

  In Evaluation Examples 5 and 6, Pt and Pd plating are performed instead of gold plating. In the case of Pt and Pd, an effect close to that of Au plating can be obtained.

  In Evaluation Examples 7 and 8, the bulk metal is changed from Ni to Pd and Cu. When the bulk metal is Pd or Cu, the characteristics are slightly better than Ni.

  Evaluation Example 9 is a case where the surface treatment of the metal filter was not performed. In this case, the white blood cell count increases and the recovery rate decreases. The hydrophobicity was high and bubbles were generated.

  Evaluation Example 10 is a case where Au plating was not performed. In this case, the polymer coverage is low (the gold element ratio is high) due to the influence of the Ni oxide film, and the white blood cell count increases and the recovery rate decreases. Evaluation Example 11 is a case where MPC polymer (CM5206) having no reactive functional group is coated. In this case, the contact angle becomes extremely high and bubbles are likely to be generated. The white blood cell count increases and the recovery rate decreases.

  Evaluation examples 12 and 13 are cases where some or more of the surface treatments are omitted. In particular, the evaluation example 13 has low chemical adsorption, and the polymer is easily peeled off. Evaluation Example 14 is a case where a homopolymer of MPC was used. The chemical reactivity with the filter was slightly reduced, and the polymer coverage was low (the gold element ratio was high).

  As described above, it was confirmed that the characteristics were improved as compared with the conventional filter by chemically reacting the biocompatible polymer with the surface of the noble metal.

DESCRIPTION OF SYMBOLS 1 ... MCL, 2 ... Peelable copper foil, 2 '... Copper plate, 3 ... Photoresist, 3a ... Photoresist exposure part (exposure part), 3b ... Photoresist image development part, 4 ... Photomask, 5 ... Plating layer, 6 ... Through hole, 7 ... gold plating layer.

Claims (7)

A metal filter made of gold, platinum, palladium, or an alloy thereof;
A biocompatible polymer chemically bonded to the surface of the metal filter;
A biological material capturing filter comprising :
The metal filter has an amino group on its surface by adsorbing polyethyleneimine to the carboxyl group on the metal surface,
The biocompatible polymer is a copolymer of 2-methacryloyloxyethyl phosphorylcholine homopolymer (MPC) and a monomer having a carboxyl group,
The biological material capturing filter, wherein the metal filter and the biocompatible polymer are bonded by an amide bond . The biological material capturing filter according to claim 1, wherein the metal filter having a carboxyl group is the metal filter in which a metal surface is treated with a thiol having a carboxyl group. A metal filter made of gold, platinum, palladium, or an alloy thereof;
A biocompatible polymer chemically bonded to the surface of the metal filter;
A biological material capturing filter comprising :
The metal filter has a carboxyl group on its surface by adsorbing polyacrylic acid to the amino group on the metal surface,
The biocompatible polymer is a copolymer of 2-methacryloyloxyethyl phosphorylcholine homopolymer (MPC) and a monomer having an amino group,
The biological material capturing filter, wherein the metal filter and the biocompatible polymer are bonded by an amide bond . The said metal filter which has an amino group on the surface is the said metal filter by which the metal surface is processed with the thiol which has an amino group, The biological material capture filter of Claim 3 . The biological material capture filter according to any one of claims 1 to 4, wherein a contact angle of pure water on the surface of the biological material capture filter is 60 degrees or less. The biological material capture filter according to any one of claims 1 to 5, wherein a ratio of metal on the surface of the biological material capture filter obtained by measurement with an X-ray photoelectron spectrometer is 10 at% or less. The biological material capture system using the filter for biological material capture as described in any one of Claims 1-6 .

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