Inkjet-Printing Enzyme Inhibitory Assay Based on Determination of

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Inkjet-printing enzyme inhibitory assay based on determination of ejection volume Jungmi Lee, Annie Agnes Suganya Samson, and Joon Myong Song Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04585 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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Analytical Chemistry

Inkjet-printing enzyme inhibitory assay based on determination of ejection volume Jungmi Lee,† Annie Agnes Suganya Samson,† and Joon Myong Song†* †College of Pharmacy, Seoul National University, Seoul 151-742, South Korea ABSTRACT: An accurate, rapid, and cost-effective methodology for enzyme inhibitor assays is highly needed for large-scale screening to evaluate the efficacy of drugs at the molecular level. For the first time, we have developed an inkjet printing-based enzyme inhibition assay for the assessment of drug activity using a conventional inkjet printer composed of four cartridges. The methodology is based on the determination of the number of mole of the drug on the printed surface. The number of mole was quantified through the volume of substance ejected onto the printed surface. The volume ejected on the reaction spot was determined from the density of reagent ink solution and its weight loss after printing. A xanthine oxidase (XOD) inhibition assay was executed to quantitatively evaluate antioxidant activities of the drug based on the determination of the number of mole of the drug ejected by inkjet printing. The assay components of xanthine, NBT, superoxide dismutase (SOD)/drug, and XOD were printed systematically on A4 paper. A gradient range of number of mole of SOD/drug printed on A4 paper could be successfully obtained. Due to the effect of enzyme activity inhibition, incrementally reduced NBT formazan colors appeared on the paper in a number of mole -dependent manner. The observed inhibitory mole (IM50) values of tested compounds exhibited a similar tendency in their activity order, compared to the IC 50 values observed through absorption assay in well-plates. Inkjet printing-based IM50 assessment consumed a significantly smaller reaction volume of two- to three-orders and more rapid reaction time compared to the well plate-based absorption assay.

Inkjet printing has been widely utilized to directly write biomaterials, such as DNA,1 protein,2-3 and cells4-5 on various substrates. In addition, microarrays of biomaterials6-8 can be easily achieved on substrates through direct-writing of various patterns supported by computer programs. Another attractive feature of inkjet printing is its capability to eject biomaterials of pico-liter volume uniformly via electrical actuators.9 Highly precise manipulations up to the pico-liter level cannot be accomplished by conventional pipetting-delivery of the micro-liter unit. The consumption of pico-liter to nano-liter units is quite advantageous for the handling of biomaterials, which are generally expensive. Working with purified proteins presents difficulties in numerous cases due to their limited availability. Inkjet printing possesses a characteristic that makes it quite suitable for handling biologically active materials with limited stability, such as enzymes. Unlike vapor deposition or photolithography utilized by the bioMEMS technique, inkjet printing employs non-contact deposition of biomaterial solution in a highly reproducible manner.10-11 As a result, native confirmation of biomaterials can be expected to be maintained during inkjet printing. Enzyme assays are a widely used tool to ascertain the biological activity of various metabolic processes in biological research, and also used to assess the metabolic defects, functional ability, and deformities of organs in the biological system. Up to now, inkjet-printing technologies have been developed for enzymatic assays on substrates. Earlier reports have verified that the glucose oxidase (GOD)-mediated glucose reaction could be monitored based on amperometric detection. 12-13, inkjet printing for biocompatible enzyme powered silk microrockets,14 quantum dot/enzyme microarrays,15-19 enzymatic test

strips,20-21 and ELISA22-23 have been successfully accomplished. However, considering the various biological processes that occur due to enzymes, numerous research applications that are capable of being driven by inkjet printing of enzymes remain unexplored. In this work, for the first time, activities of enzyme inhibitors as an antioxidant agent are quantitatively assessed using inkjet printing technique. It was evaluated based on the determination of surface coverage of inkjet-printed volume. The purpose of the present study is to develop an inkjet printing-based enzyme inhibition assay using a conventional inkjet printer composed of four cartridges. This approach can be universally utilized by laboratories of any size. Inhibitory mole 50 (IM50) is newly suggested as a quantifying index to evaluate antioxidant properties of molecules tested in inkjet printing-based bioassays. Until now, drug efficacy has been represented in units, such as inhibitory concentration 50 (IC50) or effective concentration 50 (EC50). In the drug discovery process, the amount of tested molecules is not sufficient to cover in vitro assays, including molecular assays. This is mainly due to the limited production yield by synthesis or isolation from natural products. In addition, molecular assays, such as enzyme inhibition, consist of many reaction steps. Compared to manual operation, automation of a whole molecular assay can drive more accurate results, as well as removal of unnecessary reagent consumption. The inkjet printing-based molecular assay constitutes a very suitable tool to meet these needs. This is because inkjet printing enables pico-liter to nano-liter printing reproducibly, in addition to automatic completion of bioassays.

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Figure 1 (a) A schematic representation of inkjet printing-assisted xanthine oxidase inhibitory assay. (b) A schematic diagram to determine the volume of reagent ink solution ejected from ink cartridge.

However, quantitative determination of enzyme inhibitory concentration still presents an unsolved problem in a conventional inkjet printer equipped with four cartridges (cyan, magenta, yellow, and black). At a minimum, a few to several concentrations of test molecules have to be examined to determine the enzyme inhibitory concentration. Although an expensive custom-built inkjet printer can possess a possible number of cartridges for drug solutions of a few to several concentrations, this screening system cannot be expected in a conventional inkjet printer consisting of four cartridges where only one or two cartridges are available for drug solution. To solve this problem, a method to determine the surface coverage of the inkjet-printed volume was developed and introduced in this study. Different concentrations of inkjet-printed drug solution could be prepared without limitation by only a cartridge using the determined surface coverage of inkjet-printed volume. This approach is expected to open the door to the next generation of drug screening tools based on inkjet printing.

■ MATERIALS AND METHODS Materials Superoxide dismutase (SOD), xanthine, xanthine oxidase (XOD), nitro blue tetrazolium (NBT), diethylentriamine penta acetic acid (DTPA), aminoglutethimide, dithranol, naringenin, 2-propanol, and PEG-400 were purchased from Sigma (St. Louis, MO., U.S.A.). 1.5 M tris-buffer (p.H 8.8) was purchased from Bio-Rad. Measurement of antioxidant activity by XOD inhibition assay in 96 well-plates Xanthine oxidase inhibition assay is widely used to study the antioxidant ability of drugs. To perform an inkjet printer-based XOD inhibition assay, the reaction conditions were optimized by colorimetric estimation of xanthine oxidase activity in 96 well-plates. As shown in Fig. 1 (a), Xanthine oxidase is utilized as a catalyst for the conversion of xanthine to hydrogen peroxide and uric acid. During this reaction, oxygen is reduced to superoxide anion (O2-). Then, the superoxide anion reduces NBT,

so that NBT formazan is produced. Accordingly, xanthine oxidase leads to the conversion of NBT (yellow) to NBT diformazan (purple). The detection of NBT diformazan was achieved by measuring the absorbance of NBT formazan at 560 nm. All of the reagents used in the experiment were freshly prepared, and the experimental procedures were carried out at room temperature. Xanthine solution containing DTPA (5 mM), aminoglutethimide (1 mM), naringenin (0.1 mM), NBT (5 mM), dithranol (0.1 mM), and superoxide dismutase (500 U/mL) were prepared using 0.5 M tris-buffer aqueous solution. 2N NaOH was added to dissolve NBT and dithranol in the trisbuffer. Initially, 10 μL of xanthine (5 mM) and 20 μL of NBT (5 mM) solutions were mixed well in a well-plate and incubated for 3 min. Afterwards, to prepare superoxide dismutase or drug solutions at different concentration ranges, their stock solutions were added to each well from 10 μL to 100 µL. Reaction volume was adjusted up to 230 μL using 0.5 M tris-buffer. Finally, 20 μL of 0.1 U xanthine oxidase was added to all of the wells, and the reaction mixture was incubated for 3 min at room temperature. 50 μl of 3N HCl solution was added to stop the reaction, and the absorbance of reduced NBT was measured at 560 nm using a multi-plate reader (Molecular Devices, Spectra MAX M5). Determination of surface coverage per printed volume for the standardization of inkjet printing A commercially available HP desktop inkjet printer (HP Office Jet Pro 8100e) was used to print all of the compounds on A4 paper. Commercially available A4 paper has been utilized as a substrate of inkjet printing for the first time in this work. All of the ink cartridges and printer heads were washed thoroughly several times with 100% ethanol and distilled water, and air-dried prior to printing. To improve the printability of the reagents used in the assay, a dissolving buffer was prepared with sufficient viscosity and surface tension. First, buffer ink formulation was optimized as 1 mL of 0.5 M tris-buffer containing 180 μL PEG-400 and 100 μL tert-butanol. To execute the inkjet printing-based xanthine oxidase inhibition assay on A4 paper, the optimal spot size was determined using the buffer ink. The


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printing condition to adjust the volume ejected from the cartridge of the inkjet printer was regulated according to the cyan, magenta, yellow, and black (CMYK) values which are set using a graphics program (Adobe Photoshop CC). The printing pattern was designed using Microsoft PowerPoint software (Microsoft Inc., Redmond, WA, U.S.A.). The diameter of the inkjet printing reaction spot was tested from 0.2 cm to 0.5 cm at 0.1 cm intervals with respect to A4 paper with an entire reaction spot area of 45π cm2. Buffer ink was printed on all of the reaction spots of A4 paper, and this process was repeated 20 times with respect to A4 paper with reaction spots of identical diameters. Cartridges were filled with reaction components and their weight was measured before and after printing. Thus, volume of ink printed per spot area was determined from the cartridge weight loss. As shown in Fig. 1 (b), the amount of buffer ink consumed during each printing was measured from the weight loss of the cartridge. Its average weight loss was obtained after the process was repeated 20 times. The density of the buffer ink solution contained in the cartridge was measured using a pycnometer and determined by a comparison with water density. The densities of the buffer ink solution and water were measured under the same temperature. The following equation was used to calculate the density of the buffer ink contained in the cartridge.

while NBT ink was printed on the identical spot 10 times continuously. The mixture was incubated for 3 min. Then, drug (or superoxide dismutase) ink was printed 10 times on the same spot and incubated for 3 min. Finally, xanthine oxidase ink was printed 10 times and incubated 3 min to initiate the reaction. In the second printing condition, the C cartridge was filled with both xanthine and NBT. The remaining cartridges were filled with individual ink solution. Xanthine solution along with NBT was printed five times, and then drug/ superoxide dismutase ink was printed 10 times. Finally, xanthine oxidase was printed 10 times and incubated for 3 min. In the third printing condition, the C cartridge was filled with both xanthine and xanthine oxidase, and the remaining components were filled into individual cartridges. Xanthine solution along with xanthine oxidase was printed five times, followed by either drug or superoxide dismutase ink being printed for 10 times. Finally, NBT was printed 10 times and allowed to incubate for 3 min. After printing, papers were air-dried, and the images were scanned using an HP scanner. The gray-scale intensities were measured using ImageJ software.

■ RESULT

d (W-W ) Density =

0

e

W -W 0

e

where W is the weight of the buffer ink solution; We is the weight of the pycnometer; W0 is the weight of water; and d0 is the density of water. Finally, the volume of buffer ink ejected from the cartridge was attained through dividing the cartridge weight loss by its respective density obtained with the above equation. Xanthine oxidase inhibitory assay using inkjet printing The ink solutions of xanthine, NBT, xanthine oxidase, and drug (or superoxide dismutase) were prepared using 0.5 M trisbuffer. 180 μL PEG-400 and 100 μL tert-butanol were added into each reagent solution, and its total volume was adjusted to 1 mL. Four cartridges were refilled with the prepared ink solutions. Three different printing conditions were designed to optimize the inkjet printing- based xanthine oxidase inhibitory assay. In the first printing condition, C, M, Y, and K cartridges were filled with xanthine of 5 mM, NBT of 5 mM, drug (or superoxide dismutase of 500 U), and xanthine oxidase of 0.1 U, respectively. Concentrations of aminoglutethimide, naringenin, and dithranol in the Y cartridge were 1 mM, 0.1 mM, and 0.1 mM, respectively. The value of the Y cartridge (drug or superoxide dismutase) corresponding to the ejected volume was set as 10 to 100 to change the dose of the test drug using Adobe Photoshop software. As this value increases, the ejected volume from the Y cartridge increases. The printing condition of the remaining cartridges was set so that the reaction condition of xanthine, NBT, and xanthine oxidase was achieved as standardized through the colorimetric assay in well-plates. The values of cartridges containing xanthine, NBT, and xanthine oxidase were set as 100. The number of mole of the reagent solution ejected from each cartridge was adjusted via the repeated printing number under the condition of its above-mentioned fixed condition. Xanthine was printed on the reaction spot of A4 paper five times,

Figure 2 Evaluation of XOD inhibitory activity of superoxide dismutase /drugs using absorption detection in wellplates. (a) Absorption spectrum of NBT formazan produced as a result of XOD inhibition assay. (b) XOD inhibition activity of SOD obtained as a function of dose of SOD. (c) naringenin.

Measurement of antioxidant activity by xanthine assay Colorimetric estimations of NBT formazan were performed in a dose-dependent manner to evaluate the xanthine oxidase inhibitory activity of aminoglutethimide (anti-steroid), dithranol (anthracene derivatives), and naringenin (a kind of flavonoid). Superoxide dismutase was used as a control. IC50 values of the test compounds to inhibit enzymatic activity of xanthine oxidase were evaluated quantitatively. The concentration ranges of drug/ superoxide dismutase used in the well-plate assay was as follows: 5 U to 50 U of superoxide dismutase, 0.01 μmole to 0.1 μmole of aminoglutethimide, and 1 nmole to 10 nmole of dithranol and naringenin. The antioxidant activities of compounds against xanthine oxidase were represented as a



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function of their dose, as shown in Fig. 2. The x-axis denotes the dose of the test compounds, while the y axis shows the absorbance of NBT formazan which is used as an index of antioxidant property. Their antioxidant efficacies were expressed as IC50 values. Fig. 2(a) represents the measured absorption spectrum of NBT formazan produced through the executed assay. The maximal absorption is observed around 560 nm. According to this spectrum, the degree of xanthine oxidase enzyme inhibition by test compounds was monitored at 560 nm. As revealed in Fig. 2(b) and 2(c), the absorbance of NBT formazan at 560 nm decreased linearly as the dose of test compounds, including superoxide dismutase enzyme, increased. This represents the reduction of NBT formazan formation produced as a result of the reduction of NBT, due to inhibition of xanthine oxidase enzymatic activity by test compounds. These results demonstrate that the test compounds have antioxidant activities that are identical to superoxide dismutase. Based on the plots of NBT formazan absorbance versus dose, IC50 values of compounds were calculated. Their values are summarized in Table 1. The IC50 values of superoxide dismutase, and naringenin were 5.17 U/mL Fig.2 (b), and 23.97 µM Fig.2 (c), respectively. Similarly, xanthine oxidase inhibitory activity of different compounds like aminoglutethimide Fig. S1 (a) and dithranol Fig. S1 (b) were tested and their IC50 values were determined as 350.1 µM and 23.97 µM, respectively (Table S-1). Naringenin exhibited the lowest IC50 value compared to other test drugs. The assay conditions to acquire the results of Fig. 2 were utilized for the successful completion of the inkjet printing-based xanthine oxidase inhibition assay. Determination of surface coverage per printed volume The weight loss that occurred in the cartridge was measured as a function of the diameter of the reaction spot when the buffer ink solution was ejected from the cartridge and filled the reaction spots. The entire area of reaction spots was identical with respect to five different diameters of 0.2 to 0.6 cm. The inkjet printing was executed repeatedly 20 times for each spot size, and a standard deviation of the weight loss was obtained in all of the spot sizes. The reaction spot of 0.3 cm diameter was selected because it offered reliable performance with the smallest standard deviation in variance of the weight loss corresponding to the ejection of fixed volume (data not shown). Fig. 3(a) shows the change of ejection volume printed by the reagent solution contained in the cartridge. The ejection volume of inkjet printing has been reported to be affected by viscosity, surface tension, and density. CMYK cartridges in this assay were refilled with reaction solutions, such as drug, NBT, xanthine, and xanthine oxidase. Since they have different properties regarding viscosity, surface tension and density, it was expected that the ejection volume of each solution could be variable. The ejection volumes of reagent solutions contained in each cartridge were determined under the condition of their concentrations used for the inkjetprinting antioxidant assay. K value, as a parameter of ejection volume which is controlled in the software, was set as 10. Compared to the buffer ink solution, all of the other reagents showed smaller ejection volumes. Although the K values were set as 10 for the reagent solutions, those of drug solution have to be variable to produce an incremental ejection volume for the determination of IM50 values of individual drugs. As shown in Fig. 3(b), ejection volumes of drug solutions, including superoxide dismutase, were investigated as a function

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Figure 3 Determination of printed volume on the reaction spot. (a) Ejection volumes of reagent ink solutions printed on the reaction spot of 0.3 cm diameter. K value to define the volume of reagent ink solution ejected from the cartridge was set as 100. Solution component are follows; ①Buffer ②superoxide dismutase ③Aminoglutethimide ④Dithranol ⑤Naringenin ⑥Xanthine ⑦NBT ⑧ XOD ⑨Xanthine with NBT ⑩Xanthine with XOD. (b) Determination of ejection volume printed on the reaction spot of 0.3 cm diameter as a function of K value. Each reagent ink solution used in the antioxidant XOD assay was printed as a function of K value. When K values change from 100 to 10 at 10 intervals, the ejection volumes of all the ink solutions decreased proportionally to K values.

Figure 4 (a) A schematic diagram that represents printing order of ink solutions to complete the xanthine oxidase inhibition assay. Evaluation of the XOD inhibitory activity of (b) superoxide dismutase, and (c) naringenin using the inkjet-printing technique. The upper figure is a photo that shows that NBT formazan formed on the reaction spot by inkjet printing according to the printing order (a).

of K values from 10 to 100 at 10 intervals. When the K value was set as 100, ejection volumes of reagent solutions were less than 45 nL. On the other hand, ejection volumes of reagent solutions were less than 5 nL at a K value of 10. The ejection volumes of superoxide dismutase and drug solutions were observed to be almost identical at the same K values, in spite of different solution properties. The ejection volumes of all of the tested ink solutions increased proportionally to the K value. When the K value increased from 10 to 100, the ejection volumes of buffer and superoxide dismutase of three drugs were found to increase almost 10-fold, as shown in Fig. 3(b). This


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Figure 5 (a) A schematic diagram that represents printing order and intra-cartridge mixing of ink solutions to complete the xanthine oxidase inhibition assay. Evaluation of xanthine oxidase inhibitory activity of (b) superoxide dismutase, and (c) naringenin using the inkjet-printing technique. The upper figure is a photo that shows that NBT formazan formed on the reaction spot by inkjet printing according to printing order (a).

Figure 6 (a) A schematic diagram that represents printing order and intra-cartridge mixing of ink solutions to complete the XOD inhibition assay. Evaluation of XOD inhibitory activity of (b) superoxide dismutase, and (c) naringenin using the inkjet-printing technique. The upper figure is a photo that shows that NBT formazan formed on the reaction spot by inkjet printing according to printing order (a).

quantitative trend of ejection volume as a function of K value was utilized for the determination of IM50 values for each drug. The number of mole printed per reaction spot on A4 paper was estimated based on the ejection volumes obtained in Fig. 3. Inkjet printing-based xanthine assay Results of Fig. 4 to Fig. 6 show how the xanthine oxidase inhibition assay was achieved as a function of spotting order and intra-cartridge mixing of reagents, and as a result, how IM50 values of test compounds changed. Fig 4(a) is a schematic diagram that represents the inkjet-printing order of reagent ink solutions for the antioxidant assay. The upper figures of Fig. 4(b) to Fig. 4(c) show that NBT formazan formed on A4 paper as a result of xanthine oxidase inhibition assay using inkjet printing. The lower figures of Fig. 4(b) to Fig. 4(c) plot gray-scale intensities of NBT formazan obtained as a function of ejected number of mole/unit of compounds/ superoxide dismutase. As shown in Fig. 4(b) and Fig. 4(c), in the case of the control without the treatment of superoxide dismutase or drugs, the blue intensities of NBT formazan were the strongest. Their gray-scale intensities of superoxide dismutase and drugs were almost equal, in the range of 23,000 to 25000. On the other hand, gray-scale intensities at each number of mole of compounds proportionally decreased as the number of mole of compounds increased. Superoxide dismutase exhibited the same tendency as the amount increased. IM50 values of superoxide dismutase and compounds were calculated using gray-scale intensities of Fig. 4(b) to Fig. 4(c). IM50 values of superoxide dismutase, and naringenin were determined to be 18.39 x 10mU and 3.7 x10-11mole, respectively. IM50 values of aminoglutethimide and dithranol were determined to be 3.7 x 10-10 and 3.4 x10-11 mole, respectively (Fig. S-2 and Table S-2). The results shown in Fig. 4 revealed that the xanthine oxidase inhibitory activity of naringenin was significantly higher than aminoglutethimide. In addition, the figure shows that the antioxidant activities of naringenin and dithranol were quite similar. This tendency agreed well with that acquired via xanthine oxidase inhibition assay in well-plates. In Fig. 5, intra-cartridge mixing of reagent inks was changed. Xanthine and NBT were contained in an identical cartridge. This approach can reduce the number of used cartridges to complete the antioxidant assay. As long as the inkjet- printing performance does not deteriorate,

this leads to a smaller number of required cartridges. This is especially advantageous for cases in which a larger number of reagent inks are needed to complete an inkjet-printing molecular assay. In addition, this inkjet-printing condition provided a shorter reaction time to complete the assay. In this inkjet-printing mode, IM50 values of superoxide dismutase, and naringenin, and dithranol were found to be 19.8 x 10mU and 3.6 x10-11mole, respectively. IM50 values of aminoglutethimide and dithranol were determined to be 3.5 x 10-10 and 2.9 x10-11 mole, respectively (Fig. S-3 and Table S-2). In Fig. 6, both spotting order and intra-cartridge mixing of reagents were changed. Xanthine and xanthine oxidase existed in the same cartridge, and inkjet printing of this mixed solution was attempted first. IM50 values of superoxide dismutase, aminoglutethimide, naringenin, and dithranol were determined to be 22.4 x 10 mU and 4.1 x10-11mole, respectively. IM50 values of aminoglutethimide and dithranol were determined to be 3.6 x 10-10 and 3.1 x10-11 mole, respectively (Fig. S-4 and Table S2). Among the three printing conditions of Fig. 4 to Fig. 6, the printing conditions of Fig. 4 and Fig. 5 exhibited more linear and reproducible results compared to that of Fig. 6. This can be verified by comparison of three R2 values. R2 values of Fig. 6 were found to be smaller than those of Fig. 4 and Fig. 5, which signified a larger deviation from the linearity of gray-scale intensity of NBT formazan versus the number of mole. In the inkjet-printing condition of Fig. 6, xanthine oxidase enzyme was spotted on the reaction spot. This may affect the deactivation of xanthine oxidase enzyme during the inkjetprinting assay and contribute to the poorer performance of xanthine oxidase. It is normally recommended that the enzyme is used at the end of the assay, rather than at the start. Finally, the antioxidant efficacy of test compounds was expressed in the form of IM50 values in three different printing modes, and their xanthine oxidase inhibitory property could be compared. This demonstrates the successful achievement of the inkjet printingbased molecular assay for drug screening to find hit compounds with antioxidant property. In addition, it must be noted that the inkjet printing-based assay permits a smaller reaction volume, leading to a smaller amount of reagents. Briefly, the amount of superoxide dismutase, aminoglutethimide, dithranol, and naringenin was consumed with a 237, 240, 242 and 242 times smaller volume compared to the assay performed in well- plates. Table 1 and 2 clearly show the difference in the number of mole



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of drugs/enzyme printed on the paper and those of drugs/enzyme consumed while performing colorimetric estimation.

■ DISCUSSION The enzyme inhibition assay is a highly useful tool to validate the efficacy of enzyme inhibitors. Several methods are currently being employed to analyze enzyme inhibition. Absorption detection is one of the most widely used techniques for this purpose. Even though it is highly sensitive techniques, it possesses certain major limitations. Specifically, the accuracy and reproducibility of manually performed absorption assays mostly depend upon the skills of individuals, and pipetting error is considered to be responsible for the high level inter- and intra-assay variability26. In addition, automated techniques which offer assay results with high accuracy and reproducibility are generally very expensive. Apart from manual error, classic analytical assays based on well-plates for drug screening involve the preparation of a vast range of concentrated solutions of assay components, and thus are highly expensive and time- consuming. To overcome these kinds of challenges, various techniques, including robotic well-plates, have been developed26, which permit a better understanding of drug-driven enzyme inhibition and drug screening. In past years, inkjet printing-based approaches have been applied to produce enzyme arrays,26 micro-contact printing, photo lithography, and electrochemical patterning.27 Hence, printing techniques have gradually become efficient in fabrication processes, which permits the organized use of smaller sample volumes (nano to picoliter) spotted on the paper surface. 2831 However, extant literature is scarce with reference to the use of conventional printing systems for enzyme printing for the purpose of drug screening, since inhibition assays are performed in a dose-dependent manner. To achieve efficient observation of drug activity against enzyme in a dose-dependent manner, printing spots with a gradient concentration range of drug is required. Inkjet printers with multiple cartridges containing drug of different doses is critical to satisfy this condition. Although a custom-built inkjet printer adopting a number of cartridges can overcome this hurdle, it is highly expensive and presents a major challenge in the generalized use of inkjet printers for the purpose of drug screening. Hence, a need has arisen for improved methodology for drug screening using conventional low-cost inkjet printers. To solve this existing problem, for the first time, this study proposed the concept of IM50, which enables the quantitative assessment of inhibitor efficacy using a conventional inkjet printer equipped with just four cartridges. The ejection volume needed to fill the surface area of the reaction spot could be obtained by measuring the weight loss of the cartridge occurring after ejection of the reagent solution, in addition to the density of the reagent solution in the cartridge. Furthermore, the ejection volume to be printed on the reaction spot could be controlled by adjusting the printer setup using software (K value). This enables us to freely adjust the number of mole of the substance printed at a particular reaction spot. Quantitative increase of printed volume as a function of K value was successfully verified in this work. This approach successfully demonstrated inkjet-printing molecular assay based on IM50 as a quantifying index for evaluating the inhibitory efficacy of drug against enzyme as drug target, such as xanthine oxidase. The quantitative determination of the ejection volume required to fill the reaction spot on paper enabled the use of a conventional inkjet printer with four cartridges for drug screening. This is because a drug solution contained in a cartridge can fill different reaction spots in a number of mole -dependent manner. Xanthine oxidase is considered as an important biological

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source of uric acid and superoxide radicals. Xanthine oxidase catalyzes the oxidation of xanthine and hypoxanthine to uric acid. During re-oxidation of xanthine oxidase, oxygen acts as an acceptor of electrons and produces superoxide radical (O 2-) and hydrogen peroxide (H2O2). Hyperuricemia and associated free- radical generation induces oxidative stress and causes a number of vascular diseases 32-33. Thus, scavenging xanthine oxidase induced free-radical generation will constitute a beneficial therapeutic approach against cardiovascular disease 34-35. In this work, we newly evaluated the xanthine oxidase activity inhibitory potential of aminoglutethimide, dithranol, and naringenin via assessing their antioxidant ability using an inkjet printer. Determination of volume used to print the reaction spot allows calculation of the number of mole of aminoglutethimide, dithranol, and naringenin printed per surface coverage area of the printed spot. As shown in the experimental results, lesser deviation was observed in the experiment with 20 repetitions, which confirms that the inkjet printer is highly accurate in ejecting liquid from the cartridge. Moreover, we observed a gradient increase in volume used to print per spot according to the gradient increase of K value (Fig. 2b). Hence, by setting the K value, the gradient number of mole of aminoglutethimide, dithranol, and naringenin was printed on the A4 paper. Based on the observed results, it is evident that the printing setup works like an automated pipetting system, which ensures the accuracy and rapidity of the assay. Free radicals generated during the reaction between xanthine oxidase and xanthine oxidize NBT into NBT formazan, which generates a purple color at the reaction spot on the A4 paper. The result in the present study shows that the intensity of the color precipitated on the paper is inversely proportional to the number of mole of the antioxidant drugs printed at the reaction spots. Intensity of the color was measured using ImageJ software and expressed in gray-scale value. Observed results reveal that naringenin exhibits comparatively higher antioxidant property, as compared to aminoglutethimide and dithranol. The observed results were comparable to the data obtained from absorption estimations in well-plates. However, the striking difference between the two methodologies is the volume of assay components consumed in the experiment. Specifically, a two- to three-order smaller volume and number of mole could be observed through the inkjet- printing assay. Therefore, the methodology of inkjet printing is shown to be very cost-effective as compared to the conventional absorption assay performed in well-plates using pipettes.

■ CONCLUSION In conclusion, the present study successfully demonstrated that newly introduced IM50 quantifying index enables inkjet printing-based high-content drug screening to quantitatively evaluate the enzyme inhibitory efficacy of drugs. Determination of the number of mole of the substance needed to cover the reaction spot made it possible to print an array of assay components with different concentration ranges on the paper substrate. This concept was proven by performing a xanthine oxidase inhibitor assay using four cartridges with a conventional printer. The assay components of xanthine, NBT, superoxide dismutase, xanthine oxidase, and xanthine oxidase inhibitors could be printed at different concentrations on A4 paper. As a result, gradient reduction in NBT formazan formation was successfully observed. The obtained results were quantitatively evaluated and compared with a parallel assay executed in well-plates. In


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comparison with standard absorbance estimation, inkjet printing-based colorimetric detection successfully assessed antioxidant properties of tested xanthine oxidase inhibitors with a twoto three-order smaller reagent consumption. Compared to IM50 of 5.1 x10-9 mol obtained via the assay performed in the microplate, inkjet printing assay provided IM50 of 3.7 x10-11 mol for naringenin to inhibit xanthine oxidase under the identical condition.

ASSOCIATED CONTENT SUPPORTING INFORMATION Supporting information is available: [Xanthine oxidase inhibitory assay of aminogluthethimide and dithranol in well plates (Figure. S-1), Inkjet printing-based xanthine oxidase inhibitory assay of aminoglutethimide and dithranol executed based on different printing order of reagents (Figure. S-2 – S-4), Inhibitory concentration 50 (IC50) values of aminoglutethimide and dithranol (Table S-1), and Inhibitory mole 50 (IM50) values of aminoglutethimide and dithranol (Table S-2)] (PDF). This material is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail : [email protected] Author Contributions Jungmi Lee has written the manuscript and done most of the inkjet printing assay, Annie A.S. Samson has contributed to well-plate and inkjet printing assay, and Joon Myong Song has designed idea and experiment condition of this paper.

ACKNOWLEDGMENT This work was supported by National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (2015R1A2A1A05001842 and 2016R1A4A1010796). We are grateful to the Research Institute of Pharmaceutical Sciences at Seoul National University for providing experimental equipment. The authors declare no competing financial interests.

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Table 1. Inhibitory concentration 50 (IC50) values of superoxide dismutase and test compounds obtained using absorption detection in wellplates and their number of mole of reagents used in the assay. Solution

Xanthine (5 mM)

NBT (5 mM)

XOD (0.1 U/mL)

Amount of used mole in 96 well-plates

5.0 x 10-8 mol

1.0 x 10-7 mol

2.0 x 10-3 U

Sample

SOD (500 U/mL)

Naringenin (0.1 mM)

IC50

5.2 U/mL

24.0 µM

Table 2. Inhibitory mole 50 (IM50) of superoxide dismutase and test compounds acquired using the inkjet rinting-based assay and their number of mole of reagents ejected on the reaction spot of A4 paper. Solution

Xanthine (5 mM)

NBT (5 mM)

XOD (0.1 U/mL)

Number of mole ejected in inkjet printing

2.3 x 10-10 mol

2.2 x 10-10 mol

4.6 x 10-6 U

Printing condition I

SOD (500 U/mL) 183.9 mU

Naringenin (0.1 mM) 3.7 x 10-11 mol

Printing condition II

198.3 mU

3.6 x 10-11 mol

Printing condition III

224.1 mU

4.2 x 10-11 mol

Sample

IM50


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Table of Contents graphic


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Inkjet-Printing Enzyme Inhibitory Assay Based on Determination of

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