WO2012160857A1

WO2012160857A1 - Paper-based chip for reaction and method for producing same

Info

Publication number: WO2012160857A1
Application number: PCT/JP2012/056086
Authority: WO
Inventors: 健人前島; 智明伊井; 鈴木　孝治; ダニエルチッテリオ
Original assignee: 学校法人慶應義塾
Priority date: 2011-05-20
Filing date: 2012-03-09
Publication date: 2012-11-29
Also published as: JPWO2012160857A1; JP5935153B2

Abstract

Disclosed are a method for producing a chip for reaction, which is capable of not only producing even a chip for reaction having a very minute flow path by a simple method without using a large quantity of volatile organic solvent but also producing the chip for reaction at low cost, and a chip for reaction produced by the method. A method for producing a paper-based chip for reaction comprises: a step for printing the outer edge of a desired flow path and/or reaction spot on paper with ultraviolet curable ink by an inkjet printer; a step for irradiating the ultraviolet curable ink discharged onto the paper with ultraviolet rays to cure the ink; and a step for coating the reaction spot with a reagent necessary for the reaction.

Description

Paper-based reaction chip and manufacturing method thereof

The present invention relates to a paper-based reaction substrate in which flow paths and reaction spots are formed on paper and a method for manufacturing the same.

Conventionally, various inspection chips have been used for sanitary inspection of drinking water and food and beverages, diagnosis of various infectious diseases, and the like. Many of these inspection chips that have been used in the past are provided with channels and reaction spots by forming grooves and recesses on a glass substrate or plastic substrate, and the reaction spots are coated with reagents necessary for inspection. It is.

However, these inspection chips are expensive and expensive to produce. In developing countries, the demand for hygiene tests and infectious disease diagnosis is particularly large, but many developing countries are not very economically economical and there is a strong demand for cheaper test chips.

As one method for providing an inexpensive inspection chip, a method has been proposed in which paper is used as a substrate and a flow path and reaction spots are printed on the paper using a printer. For example, Non-Patent Document 1 discloses a method for producing a paper-based microchannel chip that prints microchannels and reaction spots using a wax printer. However, since the wax printer requires heating after printing, the process becomes complicated and the cost increases. Also, with a wax printer, the width of the line that can be drawn is at least about 0.85 mm, and it is difficult to draw a fine microchannel. In addition, the reaction spot cannot be coated with a reagent with a wax printer.

In Non-Patent Document 2, paper was immersed in a polystyrene toluene solution, dried and coated with polystyrene on the entire surface, and a flow path and reaction spots were printed on the paper with an inkjet printer using toluene as an ink. A method for forming flow paths and reaction spots on paper by selectively etching polystyrene is described. However, this method requires a large amount of a volatile organic solvent, causes environmental pollution problems, and uses a large amount of a volatile organic solvent or polystyrene.

An object of the present invention is to produce a reaction chip having a high-definition flow path by a simple method without using a large amount of a volatile organic solvent, and thus produce a reaction chip at low cost. It is possible to provide a reaction chip manufacturing method and a reaction chip manufactured by the method.

As a result of diligent research, the inventors of the present application have printed an outer edge of a flow path or a reaction spot with ultraviolet curable ink on a paper by using an inkjet printer, thereby easily and low-cost without using a volatile organic solvent. The present invention has been completed by conceiving that it is possible to manufacture a reaction chip precisely.

That is, the present invention includes a step of printing an outer edge of a desired flow path and / or reaction spot on paper with ultraviolet curable ink by an inkjet printer, and irradiating the ultraviolet curable ink ejected on paper with ultraviolet rays. There is provided a method for producing a paper-based reaction chip, comprising a step of curing ink and a step of coating a reagent necessary for the reaction on the reaction spot. The present invention also provides a paper-based reaction chip manufactured by the method of the present invention.

According to the method of the present invention, since the ultraviolet curable ink can be cured simply by irradiating with ultraviolet rays, a reaction chip can be easily produced at low cost. Moreover, the inkjet printer currently marketed has a higher resolution than a wax printer, and can be produced even with a reaction chip having a relatively high-definition microchannel. Furthermore, according to the method of the present invention for curing an ultraviolet curable ink, it is not necessary to use a volatile organic solvent, and a polymer solution is applied to the entire surface of the paper as in Non-Patent Document 2, and a volatile organic solvent is applied from an inkjet printer. This method is advantageous in terms of environmental pollution and cost as compared with a method of selectively removing the polymer by etching by discharging a solvent. Furthermore, in a preferred embodiment, since both the flow path and the reaction spot and the reagent can be printed using an ink jet printer, a reaction chip can be produced more simply and at low cost.

It is a figure which shows an example of the flow path etc. of the paper base reaction chip | tip which can be utilized for the water quality test manufactured by the method of this invention. It is a figure which shows the pattern of the flow path etc. which were produced in the following Example. It is a figure which shows the pH measurement result using the pH measurement chip | tip produced in the following Example. It is a figure which shows the measurement result of lead ion using the lead ion detection chip produced in the following example. It is a schematic diagram showing the cross section of the flow path etc. which were produced in the following Example. It is a figure which shows the pattern of the flow path etc. which have the cover coat | covered on the surface of the flow path produced in another Example. It is a schematic diagram showing the cross section of the flow path etc. which are shown in FIG.

In the present invention, the “reaction chip” is provided with a channel and / or a reaction spot (sometimes referred to as “channel or the like” in the present specification) on a base material, and a reagent necessary for the reaction is further reacted. It means what is coated on the spot. Conventionally, these are provided on a slide glass or a plastic substrate, and are called “chips” such as microchannel chips, DNA chips, peptide chips, etc. However, in the present invention, since the base material is paper, the “chip” has flexibility. Moreover, since it prints on paper with an inkjet printer, it can be set as arbitrary two-dimensional shapes, and is not limited to a rectangular shape like the conventional slide glass. It can also be much larger than a glass slide, and this is also called a “chip”. In addition, that the base material is paper is called “paper base”.

In the present invention, printing is performed on paper. Here, the paper is not particularly limited as long as it can be printed by an ink jet printer, and examples thereof include filter paper, plain paper (normal copy paper), paper for an ink jet printer, a postcard, an envelope, and the like.

As described above, in the method of the present invention, an outer edge of a flow path or the like is printed on paper with ultraviolet curable ink by an inkjet printer. The width of the outer edge is not particularly limited, but usually it may be about 0.1 to 1.2 mm as set on the computer.

As the inkjet printer, a general-purpose printer that is commercially available as a peripheral device for a personal computer can be used. Commercially available commercially available inkjet printers are mainly of the piezo type and the thermal type. The piezo method is a method in which ink is ejected using a piezo element that deforms when a voltage is applied. It is used in Seiko Epson (Machjet (trade name) system) and Brother's inkjet printers. On the other hand, the thermal method is a method in which a part of the nozzle is heated and ink is ejected by the generated bubbles. It is used in Canon printers (called the Bubble Jet (registered trademark) system) and Hewlett-Packard ink-jet printers. In the method of the present invention, either a piezo method or a thermal method can be used. However, since the thermal method heats the ink, it is preferable to use a piezo ink jet printer if the reagents described below may cause problems due to heating. Since many biological substances such as antibodies and antigens and various chemical substances may be affected by heat, it is often preferable to use a piezo ink jet printer. The inner diameter of the nozzle of the ink jet printer is usually about 10 μm to 90 μm.

UV curable ink is cured by irradiating with ultraviolet rays to give a resin film, and is widely used for UV coating. The ultraviolet curable ink used in the present invention may be a commercially available product, or may be prepared by appropriately blending known components used in the ultraviolet curable ink.

In the method of the present invention, the outer edge of the flow path or the like is printed with the ultraviolet curable ink, and since the aqueous liquid is usually put in the flow path or the like, the ultraviolet curable ink is hydrophobic after being cured. High is preferred. Further, it is desired that the photopolymerization rate is high, the ink can be ejected by a printer (melting point and viscosity are appropriate), low odor, and low toxicity. Furthermore, characteristics such as high stability against fluctuations in humidity and the fact that the polymerization reaction does not proceed at the stage where ultraviolet rays are not irradiated are also desired. Those satisfying these are preferably those containing monomers or oligomers that undergo photoradical polymerization. The monomer for photo radical polymerization is particularly preferably an acrylic monomer having a high hydrophobicity after curing, a high photo polymerization rate, and a viscosity capable of being printed by an ink jet printer, and especially acrylic acid and about 14 to 18 carbon atoms. Of these, esters with normal saturated aliphatic alcohols are preferred. Specifically, tetradecyl acrylate, pentadecyl acrylate, hexadecyl acrylate, heptadecyl acrylate, octadecyl acrylate, and the like are preferable. Among these, since octadecyl acrylate is solid at room temperature, other liquids that are liquid at room temperature such as 1,10-bis (acryloyloxy) decane 別名 (also known as 1,10-decandiol diacrylate) (DDA) It can be used by dissolving in a radically polymerizable oligomer or monomer. The viscosity of ink that can be printed with an ink jet printer is usually about 0.4-20 mPa · s, although it varies depending on the nozzle diameter used. Moreover, the above-mentioned monomers and oligomers can be used alone or in combination of two or more.

The ultraviolet curable ink further contains a photopolymerization initiator. Various photopolymerization initiators used for UV coating are well known and are soluble in the above-mentioned photoradical polymerizable monomers (or mixtures of photoradical polymerizable monomers and photopolymerizable oligomers) that are liquid at room temperature. Possible ones can be selected and used as appropriate. Examples of preferred photopolymerization initiators include azobisisobutyronitrile (AIBN), benzophenone (BP), and benzyldimethyl ketal (BDK). These are all well known. Commercially available products can be preferably used. Examples of commercially available products include 2-hydroxy-2-methyl-1-phenyl-propan-1-one (trade name DAROCUR 1173), Bis (2,4,6-trimethylbenzoyl) ) -Phenylphosphine oxide (trade name IRGACUR 819, 2- (dimethylamino) -1- (4-morpholin-4-ylphenyl) -2- (phenylmethyl) butan-1-one (trade name IRGACUR 369) , 2,2-dimethoxy-2-phenylacetophenone (trade name IRGACUR 651), ethyl-4-dimethylaminobenzoate (trade name DAROCUR EDB), isopropylthioxanthone (trade name CIBACURE 2-ITX), phenylglyoxylic acid methyl ester (product) Name DAROCUR MBF) (all from Ciba Specialty Chemicals Inc.) and 2-ethylanthraquinone These photopolymerization initiators can be used alone or in combination.

The concentration of the photopolymerization initiator is usually about 5 to 25% by weight, preferably about 10 to 20% by weight in the ultraviolet curable ink.

There is no limitation on the shape of the flow path or the like printed on the paper, and any desired shape can be obtained. For example, a flow path having the shape shown in FIG. The flow channel shown in FIG. 1 has a circular sample spotting portion 10a at the center, from which eight flow channels 10 extend radially, and a circular reaction spot 12 is located at the end of each flow channel. Shape. The sample spot application part 10a can be considered as a part of the flow path. In the example shown in FIG. 1, each reaction spot is coated with a different reagent. Each reagent is, for example, a pH indicator, various ionophores that change color by reacting with various ions such as lead ions, copper ions, nitrite ions, reagents that change color by reacting with various agricultural chemicals, antibodies against pathogenic bacteria such as Salmonella and Escherichia coli, etc. is there. The sample solution spotted on the sample spotting portion 10a reaches each reaction spot 12 through each flow path 10, where it reacts with each reagent and presents according to various components contained in the sample solution. Color or discolor. Based on the color of each reagent after the reaction, various ions, pesticides, pathogens, etc. contained in the sample solution can be detected.

Of course, the shape of the flow path or the like printed on the paper is not limited to that shown in FIG. 1, but may be any one according to the purpose of measurement or reaction. The width of the flow path is not limited at all, and is, for example, about 0.8 to 1.5 mm as set on a computer. Various microchannel chips are known, and any known microchannel can be formed, and a shape suitable for a target measurement or reaction can be designed. In addition, the reaction spot does not need to be located at the end of the flow path as in the example shown in FIG. 1, and a desired reagent can be coated on a part or all of the flow path to form a reaction spot. . Therefore, for example, it is also possible to perform lateral flow type immunochromatography in a single flow path by coating different parts of the single flow path with necessary reagents (antibodies, etc.). Further, the flow path is not necessarily required, and for example, a shape in which a plurality of simple circles serving as reaction spots are arranged may be used. In this case, each reaction spot can be used in the same manner as a well of a microplate widely used in the measurement field. An ink jet printer is used as a peripheral device of a personal computer, and can accurately print an image created with drawing software. Accordingly, the outer edge of a desired flow path or the like can be accurately drawn using a drawing software of a personal computer, and this can be advantageously printed with an ink jet printer.

Since the reaction performed using the reaction chip is often performed using an aqueous liquid such as an aqueous solution, the liquid may permeate the paper and leak to the opposite side. In order to prevent such leakage to the opposite side, it is desirable to coat ultraviolet curable ink at least on the back surface of the flow path 1000 printed on the paper 50 as shown in FIG. In this case, the ultraviolet curable ink may be printed only on the portion 10000 where the liquid comes into contact when the chip is used, that is, only on the back surface of the flow path or the like, or circumscribes the wider area, for example, the entire flow path. It may be printed in a rectangular shape. Since many of the inkjet printers currently on the market support double-sided printing, printing on the back surface 200 can be performed simultaneously with the front surface 100. However, it is also possible to print the back surface after first printing the front surface and irradiating ultraviolet rays to cure the ultraviolet curable ink. Also, the shape to be printed on the back surface can be accurately drawn using the drawing software of a personal computer as with the front surface.

Furthermore, at least a part of the flow path may be covered with ultraviolet curable ink from the paper surface. For example, a cover can be formed with ultraviolet curable ink in a flow path having the pattern shown in FIG. An example of this is shown in FIG. In the example shown in FIG. 6, a cover 20000 is formed by coating a ring-shaped region including eight flow paths 10 communicating with the sample adhering portion 10 a and the reaction spot 12 with ultraviolet curable ink. In this way, the surface of the flow path is coated with the ultraviolet curable ink and cured to form the cover 20000, so that the sample spotted on the sample sticking portion 10a reaches the reaction spot 12 before the sample reaches the reaction spot 12. It is possible to prevent foreign matter (other than the sample) from being mixed in from the external environment, and the measurement accuracy can be improved. As shown in FIG. 6, the coating from the surface is preferably all or part of the surface of the flow path 10 that communicates the sample spotting portion 10 a and the reaction spot 12. % Or more, preferably 90% or more, and most preferably, the entire flow path is covered. In this case, as described above, it is preferable to coat the back surface of the flow path with the ultraviolet curable ink, whereby the flow path is a flow path that is isolated from the outside and has only the inside of the paper. It becomes possible. As a result, it is possible to more strictly prevent foreign matter from entering the sample. In this case, since the sample passes through only the inside of the paper and reaches the reaction spot, it is preferable to use paper that is well infiltrated with liquid, such as filter paper.

The step of coating the surface of the flow path with the ultraviolet curable ink is also preferably performed by printing using an ink jet printer. This coating step may be performed by first printing and curing the flow path, and then printing it on the print pattern using an ink jet printer, or simultaneously covering the outer edge of the flow path or the like when printing. You may print. In the latter case, there is an advantage that the number of steps can be reduced. Even in the latter case, the outer edge of the flow path 10 is drawn with a dark solid line, and the cover 20000 is drawn with a thin solid line, so that the ultraviolet curable ink that draws the outer edge of the flow path 10 and the ultraviolet curing that draws the cover 20000. The density of the ink (the amount of ink per unit area) can be made different, and therefore the depth of the outer edge 10b of the flow path 10 (since the UV curable ink is a solution, it penetrates from the paper surface to a certain depth). A cover 20000 can be formed (see FIG. 7). Note that the thin solid line here can be drawn by adjusting the color transmittance, the gray level, and the RGB value. If UV curable ink is coated (300) also from the back surface of the flow path, the flow path 1000 can pass only through the inside of the paper (preferably filter paper) and can be blocked from the outside as shown in FIG. However, if desired, the cover 20000 may be printed without printing the outer edge of the flow path 10. In this case, only the outer edge of the sample sticking portion 10 a, the outer edge of the reaction spot 12, and the cover 20000 are included. Printed. In this case, the sample spotted on the sample sticking part 10a diffuses radially inside the paper and reaches the reaction spot, but when there is a sufficient amount of sample as in the case of water quality inspection. Such an embodiment is also possible.

After printing the outer edge of the channel or the like, and possibly the back surface of the channel or the cover on the front surface, the ultraviolet curable ink is cured by irradiating the ultraviolet curable ink with ultraviolet rays. Ultraviolet irradiation can be performed using a commercially available ultraviolet irradiation apparatus. Irradiation conditions are not particularly limited as long as the ultraviolet curable ink is cured to become a hydrophobic line, and can be appropriately set according to the used amount of the ultraviolet curable ink and the ink used. Irradiation at an energy density of 300 to 1200 mW / cm ² may be performed for 15 seconds to 2 minutes. Further, the wavelength of the ultraviolet rays is not particularly limited as long as it is a wavelength capable of curing the ultraviolet curable ink, but usually, a safe near ultraviolet ray of about 260 nm to 380 nm can sufficiently achieve the object. When printing on both sides, each side is irradiated with ultraviolet rays to cure each ultraviolet curable ink.

Next, coat the reaction spot with the reagent. The reagent to be coated is a reagent necessary for a target measurement or reaction, and is arbitrarily selected. Examples of such reagents include pH indicators such as ionophores and simazines that react with various ions such as lead ions, calcium ions, copper ions, phosphate ions, nitrite ions, borate ions, and fluorine ions. It is used in various antigens and antibodies for immunoassay, DNA chips and peptide chips, such as reagents that react with various agricultural chemicals such as and atrazine, color bacteria such as Salmonella and antibodies that react with E. coli. Although various DNA, a peptide, etc. can be mentioned, it is not limited to these. Depending on the type of reagent, it may be necessary to react with the second reagent after the reaction with the sample solution (for example, a labeled antibody in immunoassay by the sandwich method). In such a case, The second reagent can be dropped with a dropper or the like as in the prior art, and in some cases, the second reagent can also be printed by an ink jet printer. In recent years, microchannel chips have been used not only for measurement but also for production of desired substances using chemical reactions. The chip of the present invention can be used not only for measurement but also for a chemical reaction for substance production, similarly to the known microchannel chip.

Reagent coating can be performed manually using a dropper or the like, but a liquid containing a reagent and having a viscosity that can be printed by an ink jet printer is prepared and printed by the ink jet printer. Is convenient and preferred. As described above, if the viscosity is in the range of about 0.4 to 20 mPa · s, it can be printed by an ink jet printer. When the viscosity of the reagent solution is larger than this range, the viscosity can be lowered by diluting with an appropriate diluent. On the other hand, when the reagent solution is smaller than this range, there is a thickening effect that does not hinder the reaction. Substances can be added as thickeners. Examples of preferred thickeners include glycerin, ethylene glycol, diethylene glycol and the like. The reagent solution may be in the form of a solution, an emulsion, or a suspension as long as the nozzle of the printer does not clog. In the case of a suspension, printing is usually possible without problems if the size of the suspended particles is about 1 μm or less.

Printing of the reagent solution by the inkjet printer may be performed after the ultraviolet curable ink is cured, or may be performed simultaneously with the printing of the ultraviolet curable ink. In this case, for example, when drawing the pattern to be printed, the outer edges of the flow path and the like are drawn in black, the part where the reagent is coated is drawn in color, the ultraviolet curable ink is applied to the black ink cartridge, and the reagent liquid is applied. The printing may be carried out in a color ink cartridge. However, in order to prevent ink clogging, some commercially available ink jet printers are set to eject a small amount of color ink even in black drawing. In such ink jet printers, a small amount of reagent is contained in ultraviolet curable ink. It will be mixed. If a printer that is not set as described above is used, simultaneous printing is possible by the above method, and if there is no practical problem even if a small amount of reagent is mixed in the UV curable ink, Simultaneous printing is possible even with printers that are set up like this. However, when it is desired to completely prevent the contamination, it is preferable to print the reagent after first curing the outer edge of the flow path and the like as described in the following examples. In addition, when it is necessary to stack multiple types of reagents in the same reaction spot (for example, an antibody and a blocking agent for sandwich type immunoassay), different reagent solutions are printed multiple times in the same reaction spot. It is also possible to do.

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1
1. Preparation of UV curable ink 7: 3 (weight ratio) mixture of photo-radically polymerizable monomer octadecyl acrylate and photo-radically polymerizable oligomer 1,10-bis (acryloyloxy) decane (DDA) did. Benzyl dimethyl ketal (BDK), a photopolymerization initiator, was dissolved in this to a final concentration of 15% by weight to obtain an ultraviolet curable ink. The viscosity of this ink was 8.7 mPa · s.

2. Printing of the flow path, etc. Using a piezo inkjet printer (PX-101) commercially available from Epson, the ink cartridge was filled with the UV curable ink prepared as described above and the flow path was printed. . The printed flow path had a shape shown in FIG. 2 in which two squares each having a side of 3 mm were connected by a flow path having a length of 3 mm and a width of 1.5 mm. One square is a reaction spot, and the other square is a sample sticking part of the flow path. This ink jet printer is equipped with two black ink cartridges and three color ink cartridges (magenta, cyan, yellow), the maximum resolution is 5760 x 1440 dpi, and the minimum droplet amount is 3 pL. The pattern shown in FIG. 2 was drawn with the drawing software of the personal computer, and this was printed with the said inkjet printer. All the ink cartridges were filled with ultraviolet curable ink, and printing was performed by setting to monochrome printing. The paper used was filter paper.

At the same time, the back side was printed with ultraviolet curable ink. On the back side, a solid rectangle that encloses the entire pattern shown in FIG. 2 was printed.

3. Ultraviolet curing Using a commercially available ultraviolet irradiation device, ultraviolet rays having a wavelength of 365 nm were irradiated for 60 seconds at an energy density of 600 mW / cm ² to cure the ink. When the edible red colorant solution was added to one square of the drawn pattern, the colorant solution passed through the channel and reached the other square, and the entire pattern turned red. Moreover, no liquid leakage was observed. After 4 weeks, the same test was conducted and the same result was obtained. Therefore, it was confirmed that the drawn pattern was stable over time.

4). Reagent coating One square serving as a reaction spot was coated with a pH indicator. The coated pH indicator is a mixture of bromothymol blue (BTB), thymol blue (TB) and methyl red (MR). In BTB and TB, long-chain alkyl groups were introduced in order to increase fat solubility. Each indicator was encapsulated in emulsion nanoparticles composed of poly (1-vinylpyrrolidone-co-styrene) (P (VP-St)), and the emulsion was used as a reagent solution. Hereinafter, these operations will be described in detail.

(1) Introduction of alkyl chain into BTB According to the following scheme, an alkyl chain was introduced into BTB.

Specifically, it was performed as follows.
(i) BTB-Na (147.85 mg, 0.229 mmol, 1 eq) and trimethylstearyl ammonium chloride (79.51 mg, 0.228 mmol, 1 eq) were dissolved in Milli-Q water (trade name).
(ii) The solution of (i) was extracted into the chloroform layer overnight using a continuous extraction apparatus.
(iii) On the next day, the obtained chloroform layer was dried over anhydrous sodium sulfate, and the desiccant was filtered off. Chloroform was removed under reduced pressure and further dried with a vacuum pump. Compound 1 was obtained with a yield of 136.6 mg and a yield of 64.0%.

(2) Preparation of nanoparticles containing BTB with alkyl chains
(i) Compound (i) 17.7 mg was dissolved in ethanol 20 mL.
(ii) 38% (w / w) poly (1-vinylpyrrolidone-co-styrene) (P (VP-St)) emulsion in H ₂ O, (64% (w / w) styrene (dry basis)), (Average particle size 245 nm)) (Aldrich) P (VP-ST) emulsion 1.05 g, Milli-Q water (trade name) 40 mL, ethanol 80 mL were placed in a 300 mL eggplant flask and stirred, (i) Was gradually added dropwise.
(iii) Ethanol and water were removed under reduced pressure, and Milli-Q water (trade name) was added to the resulting concentrated solution to make 10 g.
(iv) 100 μL of the dispersion obtained in (iii) was placed in a microtube and centrifuged with a filter using a microtube centrifuge (RCF = 10,000, Time = 30 min). As a result, a transparent filtrate was obtained, and it was confirmed that BTB did not leak from the nanoparticles.

(3) Introduction of alkyl chain into TB According to the following scheme, an alkyl chain was introduced into TB.

Specifically, it was performed as follows.
(i) TB-Na (49.12 mg, 0.101 mmol, 1 eq) and tridodecylmethylammonium chloride (58.24 mg, 0.102 mmol, 1 eq) were dissolved in Milli-Q water (trade name).
(ii) The solution of (i) was extracted three times using a separatory funnel, and the chloroform layer was taken out.
(iii) A saturated aqueous sodium chloride solution was added to the resulting chloroform layer and extracted once more.
(iv) It was dried over anhydrous sodium sulfate, and the desiccant was filtered off. Chloroform was removed under reduced pressure and further dried with a vacuum pump. Compound 2 was obtained in a yield of 92.5 mg and a yield of 67.6%.

(4) Fabrication of nanoparticles containing TB with alkyl chain
(i) Compound (2) 18.63 mg was dissolved in ethanol 20 mL.
(ii) Add 1.05 g of 38% (w / w) P (VP-ST) emulsion, 40 mL of Milli-Q water (trade name) and 80 mL of ethanol to a 300 mL eggplant flask and stir. It was dripped.
(iii) Ethanol and water were removed under reduced pressure, and Milli-Q was added to the resulting concentrated solution to make 10 g.
(iv) 100 μL of the dispersion obtained in (iii) was placed in a microtube and centrifuged with a filter using a microtube centrifuge (RCF = 10,000, Time = 30 min). As a result, a transparent filtrate was obtained, and it was confirmed that TB did not leak from the nanoparticles.

(5) Inclusion of MR in nanoparticles MR is more lipophilic than the former two, and MR sodium salt dissolves in the organic layer, so it was encapsulated in nanoparticles without an alkyl chain. Specifically, it was performed as follows.
(i) MR 8.1 mg was dissolved in ethanol 20 mL.
(ii) 38% (w / w) P (VP-ST) emulsion 1.05g, Milli-Q water (trade name) 40mL, ethanol 80mL are placed in a 300 mL eggplant flask and stirred, and (i) is gradually added dropwise. did.
(iii) Ethanol and water were removed under reduced pressure, and Milli-Q water (trade name) was added to the resulting concentrated solution to make 10 g.
(iv) In the process of removing under reduced pressure, a precipitate of MR that did not fit into the nanoparticles was generated from (iii). These were removed with a 1.2 μm syringe filter.
(v) (iv) 100 μL was taken in a microtube and centrifuged with a filter using a microtube centrifuge (RCF = 10,000, Time = 30 min). As a result, it was colored red, so MR leakage from the nanoparticles was confirmed.

(6) Printing reagent solution
(i) The alkyl group-introduced BTB-containing emulsion, the alkyl group-introduced TB-containing emulsion and the MR-containing emulsion prepared as described above were mixed at a ratio of 220: 330: 660 (μL).
(ii) In order to clarify the pH responsiveness of the ink, the dispersion of (i): 0.1M NaOH solution = 110: 36.3 (μL) was mixed, and it was confirmed that the color of the solution became green.
(iii) The ink of (ii): glycerin: 2-propanol: Milli-Q = 1246.3: 500: 1500: 1753.7 (μL) is mixed, and the solids content of the particles becomes 0.484% (w / w) Adjusted as follows. Glycerin was added for adjusting the viscosity (thickening agent) and preventing the print head from drying, and 2-propanol was added for adjusting the surface tension.
(iv) The prepared pH indicator solution was ultrafiltered to remove solids, filled in the ink cartridge of the same inkjet printer as described above, printed on one square portion of the pattern shown in FIG. 2, and dried. .

5. pH measurement Buffers of pH 4.0, 5.0, 6.0, 7.0, and 8.0 were prepared using citric acid and disodium hydrogen phosphate dodecahydrate (Na ₂ HPO ₄ · 12H ₂ O). In addition, a pH 9.0 buffer was prepared using sodium carbonate Na ₂ CO ₃ and sodium bicarbonate NaHCO ₃ .

4 μL of each standard sample solution having each pH was dropped onto the square of the pattern shown in FIG. 2 and not coated with a pH indicator. The dropped standard sample solution reached the square (reaction spot) coated with the pH indicator through the channel and reacted with the pH indicator. Thereby, the pH indicator reacted with each standard sample solution and changed its color. After capturing as an image using a scanner, quantitative data was obtained by digital color analysis.

The results are shown in FIG. In FIG. 3, a * on the vertical axis represents a * in the L * a * b * color system. As is clear from FIG. 3, the a * value decreases as the pH increases, and it has become clear that the pH can be measured using this pH measuring chip.

Example 2
1. Preparation of lead ion detection chip A lead ion detection chip was prepared in the same manner as in Example 1 except that xylenol orange (Alfa Aesar), which is an indicator of lead ions, was used as a reagent for coating the reaction spot. .

Prepare 0.75 mM xylenol orange -10 (% (w / w) glycerin-30% (w / w) 2-propanol aqueous solution, and after ultrafiltration, fill the ink cartridge of the same inkjet printer used in Example 1 In the same manner as in Example 1, printing was performed on the square portion of the reaction spot.

2. Measurement of

lead ions

10 ^-2 , 10 ^-3 , 10 ^-4 , and 10 ^-5 M lead (II) chloride aqueous solutions are prepared as standard sample solutions, respectively, and 4 μL each is coated with a lead ion indicator as in Example 1. It was dripped at the square which is not. The dropped standard sample solution reached the square (reaction spot) coated with the lead ion indicator through the channel and reacted with the lead ion indicator. Thereby, the lead ion indicator reacted with each standard sample solution, and discolored. After capturing as an image using a scanner, quantitative data was obtained by digital color analysis.

The results are shown in FIG. As shown in Fig. 4, since the a * value suddenly rises when the lead ion concentration reaches about 10 ^-3 M, this lead ion detection chip can detect lead ions with a detection limit of about 10 ^-3 M. It was revealed.

Example 3
The patterns such as the flow paths shown in FIGS. 6 and 7 were printed. Using a piezo ink jet printer (PX-101) commercially available from Epson, the ink cartridge was filled with the UV curable ink prepared as described above, and the surface covered channels and the cover were printed. . The flow path has a circular sample spotting portion 10a at the center, from which a flow path 10 having eight surfaces radially covered with a cover 20000 extends, and a circular reaction spot at the end of each flow path. 6 is the shape shown in FIG. Actually printed flow path etc., the sample spot 10a is a circle with a radius of 2.5mm, the flow path 10 is a rectangle with a width of 1.0mm and a length of 2.5mm, and the cover 20000 is a large circle with a radius of 5mm to a small circle with a radius of 2.5mm The reaction spots 12 were eight circles with a radius of 2 mm. This ink jet printer is equipped with two black ink cartridges and three color ink cartridges (magenta, cyan, yellow), the maximum resolution is 5760 x 1440 dpi, and the minimum droplet amount is 3 pL. The pattern shown in FIG. 6 was drawn with the drawing software of the personal computer, and this was printed with the said inkjet printer. All ink cartridges were filled with ultraviolet curable ink, and printing was performed with color printing set. The paper used was filter paper.

Examples 4 to 16
Experiments using various photoinitiators were conducted. That is, the same operation as Example 1 was performed except having used the photoinitiator as what was shown in Table 1 (a density | concentration is also shown in Table 1). The same results as in Example 1 were obtained when any photopolymerization initiator was used.

DESCRIPTION OF SYMBOLS 10 Flow path 10a Sample sticking part 10b Outer edge cross section of flow path 12 Reaction spot 50 Paper 100 Paper surface 200 Paper back surface 1000 Cross section of flow path etc. 10000 Portion where liquid contacts when using chip 10000a UV curable ink from back surface Cross section of coating 20000 Cover

Claims

A step of printing an outer edge of a desired flow path and / or reaction spot on paper with an ultraviolet curable ink, and a step of irradiating the ultraviolet curable ink discharged on the paper with ultraviolet rays to cure the ink. A method for producing a paper-based reaction chip, comprising a step of coating the reaction spot with a reagent necessary for the reaction.
The method according to claim 1, wherein the coating step is performed by printing a reagent solution on the reaction spot with an ink jet printer.
The method according to claim 1 or 2, further comprising a step of coating an ultraviolet curable ink on at least the back surface of the flow path and / or the reaction spot.
4. The method according to claim 3, wherein the coating of the ultraviolet curable ink on the back surface is also performed by printing the ultraviolet curable ink with an inkjet printer.
The method according to any one of claims 1 to 4, further comprising a step of coating at least a part of the flow path and / or the reaction spot with an ultraviolet curable ink from the surface of paper and curing.
6. The method according to claim 5, wherein the region covered with the ultraviolet curable ink from the surface of the paper is all or a part of the surface of the flow path communicating the sample spotting portion and the reaction spot.
The method according to claim 6, wherein the covering step is also performed by printing ultraviolet curable ink with an ink jet printer.
The method according to claim 7, wherein the covering step is performed simultaneously with the step of printing the outer edge of the flow path and / or the reaction spot with an ink jet printer.
A paper-based reaction chip produced by the method according to any one of claims 1 to 8.