KR101959612B1 - Quantitative evaluation method for drug efficacy based on inkjet printing - Google Patents

Abstract

The present invention relates to a method for quantitatively evaluating the effect of a compound which inhibits or activates its activity by acting on a target protein enzyme or the like based on inkjet printing at a molecular level. Specifically, the method according to the present invention quantitatively measures the volume of reactants produced by printing substrates, dye materials, drug candidates, and enzymes contained in an inkjet printer cartridge, thereby determining the number of moles of the substance to be printed It is possible to perform an accurate and rapid quantitative analysis while using a smaller amount of the test substance than in the conventional method, and therefore, it can be usefully used for drug efficacy evaluation analysis.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a quantitative evaluation method for drug efficacy based on inkjet printing,

The present invention relates to a method for quantitatively evaluating the efficacy of a drug based on inkjet printing.

In order to develop excellent new drugs, it is essential to establish systematic new drug development system through establishment and accumulation of technologies that can test synthetic, active screening, drug efficacy, pharmacokinetics, pharmacokinetic and safety. Enzymes are biological protein catalysts that mediate a number of biochemical reactions involved in various metabolic processes in biological systems. Methods for analyzing these enzymes to identify biological activities of metabolism, metabolic defects in organs and organ function are used. ELISA and absorbance analysis are most commonly used for enzyme analysis, but the accuracy and reproducibility of the assay varies depending on the ability of the researcher to perform the experiment, which is costly and takes a long time. In addition, proteins containing enzymes should be low in stability to maintain the stability of the material during analysis. To solve this problem, various automation technologies with high precision and reproducibility such as robot well-plate have been developed, but they are very expensive. Therefore, it is necessary to develop an easy, fast and cheap enzyme analysis method.

XOD (xanthine oxidase) inhibition assay is a widely used method for studying the antioxidant capacity of drugs. As shown in Figure 1 below, XOD is a catalyst used to convert xanthine into hydrogen peroxide and uric acid, in which oxygen is converted to the active oxygen, superoxide anion (O ₂ ^- ). The resulting superoxide anion reduces the yellow NBT (nitro blue tetrazolium) to produce a purple NBT formazan. At this time, the amount of the candidate substance can be calculated by measuring the absorbance of NBT formazan produced at a wavelength of 560 nm. Therefore, antioxidant capacity can be analyzed through the XOD inhibitory effect of added drug candidates.

[Figure 1]

However, these analytical methods are economically problematic because the analytical results vary depending on the ability of the researcher to perform the experiment by injecting the analytical solution directly by the researcher, and a large amount of analytical solution is required.

Inkjet printing is a technique for printing by ejecting ink through a nozzle in a microdroplet shape. These include continuous jet (CJ), which continuously injects liquid droplets, and drop-on-demand (DOD), in which liquid droplets are ejected through application conditions. Among the drop-on-demand methods, there are a heating method using a bubble spray by heat treatment and a piezoelectric method applying an electric signal to a piezoelectric body to apply pressure. The inkjet printing process is easy to operate and is suitable for commercialization because of its high speed. The electric actuator can uniformly eject the substance to be analyzed, and the test substance can be analyzed with a small amount of the test substance. Also, unlike vapor deposition or photolithography, the native form of the test material can be maintained by depositing the biomaterial solution in a noncontact manner.

Due to these advantages, studies have been made actively on the production of cell engineering devices for cell and biomaterial patterning using ink-jet printing or the manufacture of drug delivery vehicles. In this regard, U.S. Patent Application Publication No. 2009/0208577 discloses the patterning of living cells into a scaffold using an ink-jet printing apparatus, and Korean Patent Registration No. 10-1429477 discloses a method of manufacturing a polymer drug delivery system using an inkjet printing method A method is disclosed.

Accordingly, the inventors of the present invention have developed a method for quantitatively analyzing the efficacy of drugs using inkjet printing, and have found that when inkjet printer cartridges are filled with xanthan, NBT, drug candidates, and xanthine oxidase, An analytical method capable of quantitatively measuring the volume of the reactant produced was developed. The above analytical method can quantitatively evaluate the drug efficacy of the drug candidate substance by calculating the number of moles of the substance to be printed depending on the kind or concentration of the compound, which is similar to the result obtained by the conventional method. Thus, the method of the present invention can be used to accurately and rapidly quantitatively analyze the efficacy of a drug, while using a smaller amount of test substance than conventional methods, by using an inkjet printing technique.

An object of the present invention is to provide a method of analyzing the efficacy of a drug using an ink jet printer equipped with a cartridge filled with bio-ink necessary for analysis.

It is another object of the present invention to provide an ink-jet printing apparatus for analyzing the efficacy of a drug containing a cartridge filled with bio-ink necessary for analysis.

In order to accomplish the above object, the present invention provides a method of manufacturing an ink jet printer, comprising: mounting a first cartridge including a substrate, a second cartridge including a dye material, a third cartridge including a drug candidate material, step; And a method of sequentially analyzing the efficacy of a drug using an inkjet printer including the steps of sequentially inkjet-printing the cartridges.

The present invention also provides a method of manufacturing an inkjet printer, comprising the steps of: mounting a first cartridge containing a substrate and a dye material, a second cartridge containing a drug candidate material, and a third cartridge containing an enzyme into an inkjet printer; And a method of sequentially analyzing the efficacy of a drug using an inkjet printer including the steps of sequentially inkjet-printing the cartridges.

The present invention also relates to a bio ink storage cartridge comprising a jetting device, a bio ink storage cartridge, and a drive device, wherein the bio ink storage cartridge comprises a first cartridge including a substrate, a second cartridge including a dye material, And an inkjet printing apparatus for analyzing the efficacy of a drug, the fourth cartridge comprising a cartridge and an enzyme.

In addition, the present invention provides a method for producing a bio ink, comprising: a jetting device; a bio ink storage cartridge; and a drive device, wherein the bio ink storage cartridge comprises a first cartridge including a substrate and a dye material, a second cartridge including a drug candidate material, And an inkjet printing apparatus for analyzing the efficacy of a drug, which is composed of a third cartridge.

The method according to the present invention can quantitatively measure the volume of the substance to be printed depending on the kind and concentration of the compound by measuring the volume of the substrate, the dye substance, the drug candidate substance and the reactant produced by printing the enzyme contained in the inkjet printer cartridge Therefore, it is possible to perform accurate and rapid quantitative analysis while using a smaller amount of test substance than the conventional method, and therefore, it can be usefully used for the evaluation of the efficacy evaluation.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an overall schematic view (A) of an XOD inhibition assay using inkjet printing and a schematic diagram (B) for measuring the volume of bio-ink ejected from a cartridge. Fig.
FIG. 2 is a graph showing the maximum absorbance of NBT formazan using an XOD inhibition assay in a well plate according to a conventional method.
3 is a graph showing the absorbance of SOD (A) and naringenin (B) at a wavelength of 560 nm using an XOD inhibition assay in a well plate in a conventional manner. The x-axis represents the concentration of the compound and the y-axis represents the absorbance of NBT formazan used as an indicator of the antioxidant properties.
4 is a graph showing a correlation between the K value and the amount of bio ink ejected from the cartridge. FIG. 4A shows the results obtained by plotting the values of the buffer solution (1), SOD (2), aminoglutethimide (3), ditranol (4), naringenin (5) , A mixture of xanthine (⑥), NBT (⑦), XOD (⑧), NBT and xanthine (⑨) or a mixture of xanthine and XOD (⑩) . 4B is a graph showing the amount of bio-ink ejected from the cartridge containing a buffer solution, SOD, aminoglutethimide, dithranol, or naringenin against the K value in a reaction spot having a diameter of 0.3 cm.
FIG. 5 is a graph showing the antioxidative activity of a test drug (A: SOD; and B: naringenin) by charging xanthan, NBT, test drug, and XOD into each cartridge of an inkjet printer. Purple spots are photographs showing NBT formazan formed in the reaction spot by inkjet printing according to the printing order.
FIG. 6 is a graph (A: SOD; and B: naringenin) of a test drug by charging a mixture of xanthine and NBT, a test drug and XOD to each cartridge of an inkjet printer. Purple spots are photographs showing NBT formazan formed in the reaction spot by inkjet printing according to the printing order.
7 is a graph showing the antioxidative activity of a test drug by charging a mixture of xanthine and XOD, a test drug and NBT to each cartridge of an ink jet printer (A: SOD; and B: naringenin). Purple spots are photographs showing NBT formazan formed in the reaction spot by inkjet printing according to the printing order.

Hereinafter, the present invention will be described in detail.

The present invention provides a method of manufacturing an inkjet printer, comprising the steps of: 1) mounting a first cartridge containing a substrate, a second cartridge containing a dye material, a third cartridge containing a drug candidate material and a fourth cartridge containing an enzyme into an inkjet printer; And 2) sequentially inkjet printing the cartridges of the step 1). The present invention also provides a method of analyzing the efficacy of a drug using an inkjet printer.

As used herein, the term "bio-ink" refers to an analytical reagent that is charged to a printer cartridge when analyzing the efficacy of a drug using an ink-jet printing method in accordance with the present invention. Specifically, the bio-ink may include a substrate, a dye material, a drug candidate substance and an enzyme capable of being charged into the cartridge, and a solution capable of dissolving the enzyme.

The inkjet printing may include a technique of spraying and printing a dye.

The method according to the present invention may further comprise the step of dissolving the substrate, the dye substance, the drug candidate substance or the enzyme in a solution to prepare a bio-ink, and charging each cartridge with the bio-ink. The solution may generally be any substrate, dye material, drug candidate material, and any material that can be used to dissolve the enzyme. For example, the solution may contain at least one solvent selected from the group consisting of polyethylene glycol, tetrahydrofuran, dimethylacetamide, dimethylformamide, chloroform, dimethylsulfoxide, butanol, isopropanol, isobutyl alcohol, tetrabutyl alcohol, Toluene, oso-xylene, dimethylformamide, and the like, which may be used alone or in combination. In one embodiment of the invention, the solution may be Tris buffer, PEG-400 (polyethylene glycol-400) or tert-butanol.

The substrate may be xanthine, and the dye material may be NBT. The drug candidate substance is not limited and may be a solid drug or a liquid drug. The enzyme may be a xanthine oxidase.

The cartridges may be sequentially inkjet printed onto a substrate. The substrate may be a plate or a film made of a polymer such as a slide glass, a paper, a silicon wafer, polyimide (PI), polyethylene terephthalate (PET) or polystyrene (PS) Any substrate capable of printing substrates, dye materials, drug candidates and enzymes can be used without limitation. In one embodiment of the invention, the substrate may be paper.

The amount of bio ink ejected from the cartridge can be controlled by adjusting the K value, which is an ejection amount parameter that can be controlled by software. The K value may vary depending on the substrate, the dye substance, the drug candidate substance and the physical and chemical properties of the enzyme contained in the cartridge. In one embodiment of the present invention, the K value may be between 10 and 100.

The method according to the present invention may further comprise calculating the number of moles of the printed material. The number of moles of the printed material can be calculated by multiplying the amount of bio-ink consumed by the concentration of the solution. The amount of bio-ink consumed can be calculated by dividing the average weight of the bio-ink consumed in the cartridge by the density of the bio-ink charged in the cartridge. The bio-ink may be a material to be charged into the first to fourth cartridges as described above. Specifically, the bio-ink may be a substrate, a dye substance, a drug candidate substance, or an enzyme.

The diameter of the droplets jetted by the inkjet printing may be 0.1 cm to 0.5 cm. Specifically, it may be 0.2 cm to 0.4 cm. In one embodiment of the invention, the diameter of the droplets jetted by inkjet printing may be 0.3 cm. The droplet diameter If it is too small, there is a problem in a certain amount of printing, and if the diameter of the droplet is too large, there is a problem that the sample consumption is too much.

In a specific embodiment of the present invention, the inventors analyzed the degree of XOD inhibition by charging and printing bio-ink on a cartridge of an ink-jet printer under three different conditions to calculate the amount of bio ink in the cartridge consumed during printing, (Naringenin) was measured. As a first condition, cartridges filled with xanthine, NBT, test drug and XOD were used for C, M, Y and K cartridges respectively. C, M and Y cartridges were used as a second condition for a mixture of xanthine and NBT, And XOD were used. In the third condition, C, M, and Y cartridges were packed with a mixture of xanthine and XOD, test drug, and NBT, respectively. The amount of bio-ink ejected from the cartridge was calculated by dividing the average weight of the bio-ink lost in the cartridge by the density of the bio-ink filled in the cartridge using the correlation between the amount of bio-ink ejected from the cartridge and the K value (see FIG. 4). Meanwhile, the number of moles of bio-ink ejected was calculated by multiplying the volume (volume) of ejected bio-ink by the molar concentration of bio-ink (see Fig. 1B). Also, a gray-scale value indicating the intensity of the NBT formazan color precipitated on paper is calculated using a function of the number of moles of ejected bio ink / unit of compound / SOD, and a test required to inhibit the gray-scale value by 50% to define the amount of drug in IM ₅₀ value was compared with the antioxidant activity of the test drug over the IM ₅₀ value.

The measured results were similar to those obtained by measuring the antioxidative activity using a XOD inhibition assay in a well plate (see FIGS. 3 and 5 to 7). In addition, the first and second conditions were more reproducible than the third condition, and the gray-scale intensity graph of NBT Formazan versus the number of moles of ejected bioinfine was more linear (see FIGS. 5 to 7).

Therefore, the above method can be used for analyzing the efficacy of the drug, because accurate and rapid quantitative analysis can be performed using a smaller amount of the test substance than the conventional method.

Further, the present invention provides a method of manufacturing a color printer, comprising the steps of: 1) mounting a first cartridge containing a substrate and a dye material, a second cartridge containing a drug candidate material, and a third cartridge containing an enzyme to an inkjet printer; And 2) sequentially inkjet printing the cartridges of the step 1). The present invention also provides a method of analyzing the efficacy of a drug using an inkjet printer.

The method according to the present invention may further comprise the steps of preparing the bio-ink by dissolving the substrate and the dye material in a solution, and filling the bio-ink into the first cartridge. The substrate and the dye material may be mixed in a molar ratio of 1: 0.1 to 5. In one embodiment of the present invention, the substrate and the dye material may be mixed in a molar concentration ratio of 1: 1. The substrate and the dye material may have the characteristics as described above. The solution can generally be any material that can be used to dissolve the substrate and dye material. The solution may have the characteristics as described above. In one embodiment of the invention, the solution may be Tris buffer, PEG-400 (polyethylene glycol-400) or tert-butanol. In addition, the substrate may be xanthine, and the dye material may be NBT.

The method according to the present invention may further comprise the steps of preparing the bio-ink by dissolving the drug candidate substance or enzyme in a solution, and charging each cartridge with the bio-ink. The drug candidate substance is not limited and may be a solid drug or a liquid drug. The enzyme may have the characteristics as described above. The solution may be any substance that can be generally used to dissolve drug candidates and enzymes. The solution may have the characteristics as described above. In one embodiment of the invention, the solution may be Tris buffer, PEG-400 (polyethylene glycol-400) or tert-butanol. In addition, the enzyme may be a xanthine oxidase.

The cartridges may be sequentially inkjet printed onto a substrate. The substrate may have the characteristics described above. In one embodiment of the invention, the substrate may be paper.

The method according to the present invention may further comprise calculating the number of moles of the printed material. The number of moles of the printed material can be calculated by multiplying the amount of bio-ink consumed by the concentration of the solution. The amount of bio-ink consumed can be calculated by dividing the average weight of the bio-ink consumed in the cartridge by the density of the bio-ink charged in the cartridge. The bio-ink may be a material to be charged into the first to fourth cartridges as described above. Specifically, the bio-ink may be a substrate, a dye substance, a drug candidate substance, or an enzyme.

The diameter of the droplets jetted by the inkjet printing may be 0.1 cm to 0.5 cm. Specifically, it may be 0.2 cm to 0.4 cm. In one embodiment of the invention, the diameter of the droplets jetted by inkjet printing may be 0.3 cm.

In a specific embodiment of the present invention, the inventors analyzed the degree of XOD inhibition by charging and printing bio-ink on a cartridge of an ink-jet printer under three different conditions to calculate the amount of bio ink in the cartridge consumed during printing, And the measured results were similar to those obtained by measuring the antioxidative activity using the XOD inhibition assay in the well plate by the conventional method.

Therefore, the above method can be used for analyzing the efficacy of the drug, because accurate and rapid quantitative analysis can be performed using a smaller amount of the test substance than the conventional method.

In addition, the present invention relates to a bioinjection cartridge comprising a jetting device, a solution storage cartridge and a drive device, wherein the bio ink storage cartridge comprises a first cartridge including a substrate, a second cartridge including a dye material, , And a fourth cartridge containing an enzyme. The present invention also provides an ink-jet printing apparatus for analyzing the efficacy of a drug.

Each of the substrate, the dye substance, the drug candidate substance and the enzyme may have the characteristics as described above. The cartridge may have the features described above.

In a specific embodiment of the present invention, the inventors analyzed the degree of XOD inhibition by charging and printing bio-ink on a cartridge of an ink-jet printer under three different conditions to calculate the amount of bio ink in the cartridge consumed during printing, (Naringenin), and the results were similar to those obtained by measuring the antioxidative activity of XOD inhibitory assay in a well plate by a conventional method.

Therefore, the above-described method can perform accurate and quick quantitative analysis while using less amount of test substance than the conventional method, so that the ink-jet printing apparatus can be usefully used for the evaluation of the efficacy of the drug.

In addition, the present invention provides a method for producing a bio ink, which comprises a jetting device, a solution storage cartridge, and a drive device, wherein the bio ink storage cartridge comprises a first cartridge including a substrate and a dye material, a second cartridge including a drug candidate material, The present invention provides an inkjet printing apparatus for analyzing the efficacy of a drug.

Each of the substrate, the dye substance, the drug candidate substance and the enzyme may have the characteristics as described above. The cartridge may have the features described above.

In a specific embodiment of the present invention, the inventors analyzed the degree of XOD inhibition by charging and printing bio-ink on a cartridge of an ink-jet printer under three different conditions to calculate the amount of bio ink in the cartridge consumed during printing, (Naringenin), and the results were similar to those obtained by measuring the antioxidative activity of XOD inhibitory assay in a well plate by a conventional method.

Therefore, the above-described method can perform accurate and quick quantitative analysis while using less amount of test substance than the conventional method, so that the ink-jet printing apparatus can be usefully used for the evaluation of the efficacy of the drug.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited thereto.

96 well-plate XOD inhibition assay

Antioxidant activity of naringinin, an anthracene derivative, was measured using an XOD inhibition assay on a well plate as a conventional method. SOD, known as an antioxidant, was used as a control. The IC ₅₀ values of the compounds for inhibiting the enzyme activity of XOD were quantitatively evaluated.

Specifically, using 0.5 M Tris buffer, xanthine containing 5 mM DTPA (diethylentriamine), 0.1 mM naringenin, 5 mM NBT (nitro blue tetrazolium) and 500 U / ml SOD (superoxide dismutase) Solution. NBT was dissolved in Tris buffer by the addition of 2 N NaOH. First, 10 μl of 5 mM xanthine and 20 μl of 5 mM NBT were mixed well in a well-plate and allowed to react for 3 minutes. To measure the antioxidative activity of SOD or the test drug at various concentrations, add 10 [mu] l to 100 [mu] l of a storage solution of the SOD or test drug prepared by the above method to each well, and add 0.5 ml of 0.5 M Tris buffer to the final volume 230 < / RTI > The final concentrations of SOD and test drug were 5 U to 50 U for SOD and 1 nmole to 10 nmole for naringenin. Then, 20 μl of 0.1 U / ml XOD was added to all wells, and the reaction mixture was left at room temperature for 3 minutes, after which 50 μl of 3 N HCl solution was added to stop the reaction. The absorbance of the reduced NBT formazan was then measured in the reaction using a multiple plate reader (Molecular Devices, Spectra MAX M5). The concentration of the solution finally used for absorbance measurement is shown in Table 1 below.

The concentration of the solution used in the assay solution Xanthine
(5 mM) NBT
(5 mM) XOD
(0.1 U / ml) The concentration of the solution used in the assay 5.0 x 10 < ^-8 > mol 1.0 x 10 < ^-7 > mol 2.0 x 10 ^-3 U

As a result of the absorbance measurement, the maximum absorption of NBT formazan was observed at about 560 nm, and the XOD enzyme inhibition by the test drug was analyzed by absorbance at the wavelength of 560 nm (FIG. 2). In addition, the IC ₅₀ value of the test drug was calculated based on the absorbance measurement result of the test drug.

As a result, as shown in FIG. 3, the NBT formazan absorbance of the test drug decreased linearly in inverse proportion to the amount of the test drug added at a wavelength of 560 nm, which was similar to that of SOD. As a result, the amount of NBT decreased due to the inhibition of the activity of XOD enzyme by the test drug, and the formation of NBT formazan was decreased, and it was found that the test drug had an antioxidative activity similar to that of SOD (FIG. 3) . Further, the result of calculating the IC ₅₀ value for the test drug on the basis of the absorbance measurement result, IC ₅₀ values of SOD and Naryn angiogenin was respectively 5.2 U / ㎖ and 24.0 μM.

Inkjet  Using printing XOD  Inhibition Assay

The XOD inhibitory activity was analyzed by charging and printing the bioinfective ink on a commercially available HP Desktop Jetjet 8100e cartridge and calculating the amount of bio ink in the cartridge consumed during printing. The experiment was performed by selecting the optimum reaction spot size in the following manner and measuring the surface covering strength of the reaction spot per printed volume.

<2-1> Optimum reaction Spot  Select size

First, on A4 paper having an overall reaction spot area of 45 pi cm &lt; ² &gt; to determine the optimum reaction spot size, the diameter of the inkjet printing reaction spot was set to 0.2 cm, 0.3 cm, 0.4 cm, or 0.5 cm, The weight and the function of the reaction spot diameter when the solution or bio-ink solution containing the test drug was ejected from the cartridge to fill the reaction spot were calculated. Inkjet printing was repeated 20 times for each spot size and the standard deviation of the lost cartridge weight was obtained at every spot size.

As a result, 0.3 cm was selected as the optimal spot size since the dispersion value of the lost cartridge weight was the smallest standard deviation value when the spot diameter was 0.3 cm.

&Lt; 2-2 > Method for measuring the amount of bio ink ejected from the cartridge

The amount of bio-ink consumed during printing was calculated by dividing the average weight of the bio-ink consumed in the cartridge obtained by repeating the printing 20 times by the density of the solution filled in the cartridge (see FIG. 1B). The density of bio-ink filled in the cartridge was measured by comparing the density of the bio-ink and water at the same temperature condition using a pycnometer. The density was calculated by substituting the measured value into the following formula (1).

In the above Equation 1, W is the weight of the bio-ink filled in the cartridge, W _e is the weight of the grapevine, W ₀ is the weight of water, and d ₀ is the density of water. The amount of ink ejected from the inkjet printer cartridge was adjusted according to the cyan, magenta, yellow, and black (CMYK) values set using a graphics program (Adobe Photoshop CC) Respectively.

<2-3> Quantity of bio ink ejected from cartridge K value  correlation

The amount of bio-ink ejected from the cartridge is affected by viscosity, surface tension and density, so the amount of ejected ink differs depending on the material charged into the cartridge. In order to measure the ejection amount of the bio-ink in the cartridge filled with the bio-ink containing the substance used in the assay, the reaction spot diameter was set to 0.3 cm according to the result of Example <2-1>, and 0.5 M Tris buffer, A solution containing 500 U / ml SOD, 0.1 mM naringenin, 5 mM xanthine, 5 mM NBT or 0.1 U / ml XOD was prepared. 180 [mu] l of PEG-400 and 100 [mu] l of tert-butanol were added to each solution, and a final volume of 1 ml was added to prepare a bio-ink. The cartridge was charged with the bio-ink, and the K value, which is a parameter of the bio-ink ejection amount, was set to 100 using software, and the amount of ejected bio-ink was calculated in the same manner as in Example <2-2>. All cartridges and printheads were thoroughly rinsed thoroughly with 100% ethanol and distilled water several times and dried in air before printing.

The K value is a software adjustable ejection amount parameter, which can control the amount of bio ink ejected from the cartridge by adjusting the K value, thereby freely controlling the number of moles of the printed material in a specific reaction spot. In order to confirm the correlation between the K value and the amount of bio ink ejected from the cartridge, a bio-ink containing 0.5 M of Tris buffer, 500 U / ml of SOD or 0.1 mM of naringenin prepared by the above method in the cartridge was applied to the cartridge And the K value was set to 10, 20, 30, 40, 50, 60, 70, 80.90 or 100, and the ejection amount of the bio-ink was calculated in the same manner as in Example <2-2>.

As a result, as shown in FIG. 4A, when the K value was 100, the Tris buffer solution had a higher ejection amount than the other solutions and test drugs. Also, as shown in FIG. 4B, when the K value was set to 100, the ejection amount of the reagent solution was less than 45 nL, but when the K value was set to 10, the ejection amount of the reagent solution was less than 5 nL. On the other hand, the ejected amount of SOD, aminoglutethimide, dithranol and naringenin was almost the same at the same K value although the solution characteristics were different. The ejection amount of all the bioinf ink solution was increased about 10 times when the K value was increased from 10 to 100 in proportion to the K value (FIG. 4).

<2-4> Inkjet  Measurement of antioxidant activity using printing

The antioxidant activity of naringenin was measured by analyzing XOD inhibition level by inkjet printing. Antioxidative activity of the materials was investigated by filling the ink cartridge with three different conditions. Antioxidant activity was measured by defining the value of IM ₅₀ (inhibitory mole 50) as a quantitative index to evaluate the antioxidant properties of the molecule.

Specifically, a solution containing 5 mM xanthine, 5 mM NBT, 0.1 U / mL XOD, 0.1 mM naringenin, or 500 U / mL SOD (superoxide dismutase) was prepared using 0.5 M Tris buffer Respectively. 180 [mu] l of PEG-400 and 100 [mu] l of tert-butanol were added to each of the prepared solutions, and a final volume of 1 ml was used to prepare a bio-ink. The bio-ink was injected into the cartridge of the ink jet printer under three different conditions . The concentration of the solution finally injected into the cartridge is shown in Table 2 below.

The concentration of solution injected into the cartridge solution Xanthine
(5 mM) NBT
(5 mM) XOD
(0.1 U / ml) The concentration of solution injected into the cartridge 2.3 x 10 &lt; -10 ^{& gt;} mol 2.2 x 10 &lt; -10 ^{& gt;} mol 4.6 × 10 ^-6 U

First, the C, M, Y and K cartridges of an inkjet printer were charged with 5 mM xanthine, 5 mM NBT, test drug (0.1 mM naringenin or 500 U SOD), and 0.1 U XOD, respectively, Were measured. The K value for the test drug was set to 10-100, and the K value for xanthine, NBT and XOD was set to 100 to achieve as normalized in Example 1. The C cartridge was printed 5 times on the reaction spot of A4 paper, the M cartridge was printed 10 times in the same spot, and the mixture was left for 3 minutes. The Y cartridge was then printed 10 times in the same spot and left for 3 minutes. Thereafter, the K cartridge was printed 10 times in the same spot and left for 3 minutes to dry the paper, and the image was scanned using an HP scanner. The intensity of the NBT formazan color precipitated on the paper was measured by Image J software and expressed as a gray-scale value. The amount of bio-ink ejected to the K value was measured using the correlation between the amount of bio-ink ejected from the cartridge obtained as the result of Example <2-3> and the K value, and the amount (volume) of ejected bio- The molar concentration of the bio-ink multiplied by the molar concentration of the bio-ink was calculated (see Fig. 1B). Using the function of the unit / SOD of the mole number / compound of the ejection bio ink gray NBT formazan-calculating scale values, and gray - the amount (mole) of a scale value necessary for 50% inhibition test drug IM ₅₀ Respectively.

When the number of cartridges used in the analysis process is reduced, the cost and time are not consumed. Therefore, the second condition is to measure the antioxidant activity using three cartridges. C, M, and Y cartridges of inkjet printers were charged with a mixture of 5 mM xanthine and 5 mM NBT, test drug (0.1 mM naringenin or 500 U SOD), and 0.1 U XOD, respectively, The antioxidant activity of the test drug was measured in the same manner as described above, except that it was not used.

Antioxidant activity was measured by varying the timing of XOD enzyme eradication as a final condition. C, M, and Y cartridges of inkjet printers were charged with a mixture of 5 mM xanthine and 0.1 U XOD, test drug (0.1 mM naringenin or 500 U SOD), and 5 mM NBT, respectively, The antioxidant activity of the test drug was measured in the same manner as described above, except that it was not used.

As a result, as shown in Figs. 5 to 7, the IM ₅₀ values of SOD and naringenin were 183.9 mU and 3.7 x 10 ^-11 mol, respectively, under the first condition (Fig. 5) and 198.3 mU and 3.6 x 10 ^-11 mol (Fig. 6), and 224.1 mU and 4.2 x 10 ^-11 mol, respectively, under the third condition (Fig. 7).

This tendency was similar to the results of Example 1 in which antioxidative activity was measured using a XOD inhibition assay in a well plate in a conventional method. In addition, the first and second conditions were more reproducible than the third condition, and the gray-scale intensity graph of NBT Formazan versus the number of moles of ejected bioinfine was more linear.

Claims (12)

1) attaching a first cartridge containing xanthine, a second cartridge containing NBT (nitro blue ttrazolium), a third cartridge containing a drug candidate substance, and a fourth cartridge containing xanthine oxidase to an inkjet printer step; And
2) successively inkjet printing the cartridges of step 1), wherein the droplets jetted by the inkjet printing have a diameter of 0.2 cm to 0.4 cm; How to analyze.
1) attaching a first cartridge containing xanthine and NBT, a second cartridge containing a drug candidate substance and a third cartridge containing xanthine oxidase to an inkjet printer; And
2) successively inkjet printing the cartridges of step 1), wherein the droplets jetted by the inkjet printing have a diameter of 0.2 cm to 0.4 cm; How to analyze.
delete delete delete 3. The method of claim 1 or 2, further comprising calculating the number of moles of the printed material.
7. The method of claim 6, wherein the number of moles of the printed material is calculated by multiplying the consumed amount of the material by the molar concentration of the material.
8. The method of claim 7, wherein the amount of consumed bio-ink is calculated by dividing the average weight of bio-ink consumed in the cartridge by the density of bio-ink charged in the cartridge.
delete delete delete delete

Images