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Article Cite This: ACS Omega 2019, 4, 7866−7873
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Direct On-Paper Inkjet Printing of Kinase-to-Kinase Phosphorylation Cascade Reactions Jungmi Lee,† Annie Agnes Suganya Samson,† and Joon Myong Song*
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College of Pharmacy, Seoul National University, Seoul 08826, South Korea ABSTRACT: In this study, we described a novel method to detect the kinase-to-kinase phosphorylation cascade reaction using a commercially available inkjet printing machine. It is very difficult to accomplish the kinase phosphorylation cascade reaction with inkjet printing because of the complex optimization of various reaction conditions inherent to transient protein− protein interactions. To demonstrate the proposed approach, the c-Jun Nterminal kinase (JNK1/2) and mitogen-activated protein kinase (MAPK)mediated phosphorylation cascade reaction was chosen as a model. After printing, the resolution and biofunctional activity of the kinase enzyme was determined, and the reproducibility was evaluated. Our results showed a negligible loss in phosphorylation activity, which confirms the success of the phosphorylation cascade reaction on the printed substrate. Considering the success of the cascade reaction on paper, the assay was extended to determine JNK1/2 inhibition activity by SP600125. It reveals that the method was able to assess the percentage of phosphorylation and inhibition activity by utilizing a relatively small amount of the enzyme; data analysis does not require specialized instrumentation. Thus, the inkjet printing technology-based method will be a suitable platform to screen kinase inhibitors. This is the first report to validate the kinase−kinase phosphorylation cascade reaction using an inkjet printing method.
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INTRODUCTION Phosphorylation is an important cellular event that can modulate the nature of protein−protein interactions, thereby leading to the formation of different protein binding complexes and altering signaling pathways.1−3 Therefore, protein phosphorylation has become a crucial focus point for drug discovery as a result of the identification of promising therapeutic targets, such as protein kinases, phosphatases, and phosphoprotein binding sites.4−6 Recently, many investigators have validated the use of inkjet printing technology to deposit DNA,7 RNA,8 and proteins9 or enzymes10,11 on various substrates (glass, nitrocellulose membranes, and parchment paper) and proposed it as an attractive and versatile method, as it is low-cost, simple, fast, and reproducible.12−16 Therefore, inkjet printing techniques are used as a tool to minimize and simplify the standard experiments performed in well-plates and microarrays.17−23 Considering the advent of inkjet printing technology, MacBeath and Schreiber utilized a printing robot as a tool to demonstrate stable protein−protein interactions.24 Although efforts have been shown to verify stable protein−protein interactions using inkjet printing, it is challenging to achieve the transient protein−protein interactions that are essential in many aspects of cellular functions (protein modification, transport, folding, signaling, apoptosis, and cell cycling) with inkjet printing. Therefore, attempts to illustrate complicated phosphorylation cascade reactions (transient interactions) using inkjet printing assay would provide a significant impact to a wide research community. Until now, studies based on protein−protein interactions using inkjet printing are scarcely reported, with the exception of the paper by MacBeath and © 2019 American Chemical Society
Schreiber This reveals the practical difficulty of manipulating protein−protein interactions elaborately on printed substrates. Moreover, when two different proteins are printed on a single substrate, parameters such as temperature, surface tension, and solution viscosity must be precisely controlled. Particularly, complicate phosphorylation cascade reactions have not been demonstrated using inkjet printing assays. Herein, for the first time, we have developed a new approach to analyze direct onpaper phosphorylation cascade reactions using inkjet printing with free protein diffusion. One of our objectives in pursuing this approach was to make the technique easily accessible and compatible with standard inkjet printing machines. This study reports, for the first time, the direct on-paper phosphorylation and activation of the JNK1/2 mitogen-activated protein kinases (MAPKs) under free diffusion using inkjet printing and a fluorescence imaging system.
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RESULTS AND DISCUSSION Figure 1 illustrates the scheme utilized to perform the direct on-paper phosphorylation cascade reaction using inkjet printing under the conditions of free protein diffusion. To validate the proposed approach, reaction components were sequentially printed on the paper so that the reaction could occur under free diffusion conditions. Herein, the Ser/Thr 4 peptide, labeled with two fluorophores (coumarin and fluorescein) comprising a fluorescence resonance energy Received: March 14, 2019 Accepted: April 19, 2019 Published: April 30, 2019 7866
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Figure 1. Schematic illustration of on-paper kinase−kinase phosphorylation cascade reaction using the inkjet printing method. (A) Reaction components present in individual cartridges were sequentially printed on parchment paper. The phosphorylation cascade reaction occurred on the reaction spot, represented inside the dotted rectangular box. (B) Inhibition of the phosphorylation cascade reaction in the presence of SP600125 (JNK1 and JNK2 inhibitors). (C) After the reaction, the developing solution was printed on the paper, and then the fluorescence signal generated on each reaction spot was measured.
Figure 2. (A) Effects of the concentration of Triton X-100 on the surface tension of 1× kinase buffer. (B) Effects of the concentration of Triton X100 on phosphorylation activity of JNK1 (7.5 × 10−7 g/mL) or JNK2 (4.7 × 10−7 g/mL) and MAPKAPK2 (5.0 × 10−6 g/mL) in 1× kinase buffer. (C) Fluorescence signal [coumarin (Ex/Em = 400 nm/445 nm) and fluorescein (Ex/Em = 400 nm/525 nm)] generated on the paper as an effect of the concentration of Triton X-100, measured using confocal microscope (Leica, TCS SP8). Error bars represent the standard deviation of three independent measurements. (D) Effects of different viscosity modifiers on the viscosity of the solutions containing JNK1 (7.5 × 10−7 g/mL) or JNK2 (4.7 × 10−7 g/mL) and MAPKAPK2 (5.0 × 10−6 g/mL) and 0.1 wt %. Triton X-100 in 1× kinase buffer. (E) Effects of different viscosity modifiers on the phosphorylation activity of JNK1 (7.5 × 10−7 g/mL) and MAPKAPK2 (5.0 × 10−6 g/mL) along with 0.1 wt %. Triton X-100 in 1× kinase buffer. (F) Effects of different viscosity modifiers on the phosphorylation activity of JNK2 (4.7 × 10−7 g/mL) and MAPKAPK2 (5.0 × 10−6 g/mL) along with 0.1 wt %. Triton X-100 in 1× kinase buffer.
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Figure 3. (A) Optimization of JNK1-dependent phosphorylation activity using inkjet printing. (B) Optimization of JNK2-dependent phosphorylation activity using inkjet printing. Error bars represent the standard deviation of three independent measurements.
viscosity modifiers used in commercial ink formulations were explored. Figure 2D summarizes the impacts of different viscosity modifiers at room temperature. All solutions displayed exponential values, depending on the concentration and type of the viscosity modifier. Similarly, the activities of the bioinks formulated with JNK1 or JNK2 and MAPKAPK2 in buffer with the 0.1 wt % surfactant containing various concentrations of six types of viscosity modifiers were evaluated at room temperature. Figure 2E,F illustrates the percent change in JNK1-MAPKAPK2 and JNK2-MAPKAPK2 activities, respectively, based on the increased addition of the viscosity modifiers. Negative values indicate a reduction in activity, and positive values indicate the opposite. A significant decrease in JNK1 and JNK2 activities were observed when viscosity modifiers other than carboxymethyl cellulose (CMC) were added. Typically, a decrease in phosphorylation activity due to the presence of poly(ethylene glycol) (PEG) can be related to the lower diffusion coefficients and larger molecular size of the viscosity modifier. Hence, CMC was considered to be the best viscosity modifier for the preparation of all the bioinks. Parchment paper has been reported as a suitable substrate to measure the fluorescence signal because of its insignificant selfabsorption characteristic; the reaction components interact efficiently upon printing, and the low-background signal makes it available to measure the original fluorescence signal generated on paper.19 Hence, parchment paper was used as a substrate in this study. Subsequently, all the bioinks (JNK1/2, MAPKAPK2, ATP, and Ser/Thr phosphor−peptide), containing optimal volumes of the surfactant and viscosity modifier were prepared and printed on parchment paper using a standard inkjet printer. Two groups (control and test) of experiments were performed to confirm the JNK1/2-mediated phosphorylation cascade reaction on paper under free diffusion conditions. The percentage of phosphorylation for each spot on the parchment paper was determined using equations of the emission ratio, and % phosphorylation was illustrated in Figure 3. Control experiments performed in the presence and absence of ATP showed different emission ratios. The maximum (2.69JNK1 and 2.91JNK2) and minimum (0.67JNK1 and 0.57JNK2) emission ratios obtained by 0 and 100% phosphorylation conditions indicate the effectiveness of the phosphorylation cascade reaction under free diffusion conditions on paper. Similarly, test experiments performed with variable amounts of JNK1 or JNK2 with constant amounts of MAPKAPK2, ATP, and Ser/Thr phosphopeptide showed an increase in the emission ratio. The percentage of phosphorylation was calculated to be 85.16% with an emission ratio of
transfer (FRET) pair, was used to determine the kinase−kinase phosphorylation cascade reaction on the paper. As shown in Figure 1A, reaction components, including JNK1 or 2, MAPKAPK2, adenosine triphosphate (ATP), and FRET phosphopeptide substrates, were printed one after another from CMYK cartridges. The phosphorylation cascade reaction occurred on paper within a dotted rectangular box. In brief, the presence of JNK1 or JNK2 activates MAPKAPK2. The activation of MAPKAPK2 catalyzes a proton transfer from the nucleophilic (−OH) group on the Ser/Thr 4 peptide, which attacks the γ-phosphate (γ-PO32−) group on ATP, resulting in the transfer of the phosphate group to the Ser/Thr 4 peptide to form phosphoserine or phosphothreonine, along with adenosine diphosphate. The abovementioned phosphorylation cascade reaction is inhibited in the presence of SP600125 (JNK1 and JNK2 inhibitors) (Figure 1B). After the phosphorylation reaction, a developing reagent was printed onto the paper, which specifically recognizes and cleaves the nonphosphorylated peptide. Peptide cleavage disrupts FRET between the donor fluorophore (coumarin) and acceptor fluorophore (fluorescein) on the peptide, while uncleaved (phosphorylated) peptides sustain FRET. As a result, phosphorylated and nonphosphorylated reactions on paper were distinguished based on the emission fluorescence intensity (Figure 1C). Because we aimed to verify the printing-based kinase-to-kinase phosphorylation cascade reaction on paper, factors such as surface tension and viscosity were optimized for bioink formulation in order to preserve the biological functionality upon printing. For instance, inappropriate ink formulation can negatively affect enzyme or protein activity. In this study, Triton X-100, a nonionic surfactant, was used to modify the bioink surface tension. Figure 2A illustrates the surface tension values obtained at room temperature. The critical micelle concentration (cmc) for the study was found to be at 0.1 wt % surfactant concentration in buffer. In the cases of surfactant concentrations above the cmc, surface tensions stabilize in its lower limit, which is ∼30 mN/m. Similarly, bioink solutions of JNK1 (7.5 × 10−7 g/mL) or JNK2 (4.7 × 10−7 g/mL) and MAPKAPK2 (5.0 × 10−6 g/mL) in buffer containing various concentrations of the surfactant were prepared, and their activities in solution were measured immediately after preparation. Figure 2B,C shows the impact of the surfactant dose on phosphorylation activity. For surfactant doses up to 0.1 wt %, there is a negligible loss in enzyme activity. Therefore, surfactant concentration of the bioink solutions was fixed at 0.1 wt %. This surface tension value was previously reported as an optimal value for printing.25 To modify the bioink viscosity, some common 7868
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Figure 4. (A) Fluorescence signals generated on the reaction spot as an effect of JNK1-dependent 0% phosphorylation, 0% inhibition, and 100% phosphorylation. (B) Fluorescence signal generated on the reaction spot as a function of the inhibitor was measured. (C) Percentage of phosphorylation activity achieved as a result of the inhibitor present on each reaction spot. (D) Emission ratio as a function of the inhibitor present on the reaction spot. Error bars represent the standard deviation of three independent measurements.
Figure 5. (A) Fluorescence signal generated on the reaction spot as an effect of JNK2-dependent 0% phosphorylation, 0% inhibition, and 100% phosphorylation. (B) Fluorescence signal generated on the reaction spot as a function of the inhibitor was measured. (C) Percentage of phosphorylation activity achieved as a result of the inhibitor present on each reaction spot. (D) Emission ratio as a function of the inhibitor present on the reaction spot. Error bars represent the standard deviation of three independent measurements.
0.67 in the presence of 240 ng of JNK1, while a decrease in the level of phosphorylation (15.41%) and an increase in the emission ratio (2.69) were observed with a decrease in the presence of JNK1 (0.12 ng) on the reaction spot. These results confirm that the presence of JNK1/2 on the reaction spot controls the percentage of phosphorylation, thereby validating the existence of the kinase−kinase phosphorylation cascade reaction under free diffusion conditions on parchment paper. Therefore, 3.75 ng of JNK1 and 2.34 ng of JNK2, phosphorylating 39.24 and 35.63% of the Ser/Thr 4 phosphopeptide on paper, were chosen to determine inhibitor activity under free diffusion conditions (Figure 3A,B). These conditions are considered as optimal, as the kinase−kinase
phosphorylation reactions are readily active to phosphorylate 20−40% of peptides and cleave 60−80% of peptides on the reaction spot. After the successful completion of the phosphorylation cascade reaction on paper, the application of the developed method to determine JNK1/2 inhibition activity by SP600125 (JNK1/2 inhibitor) was studied. In the control group, the assay was performed with and without ATP, to confirm 0% phosphorylation or 100% phosphorylation and 0% inhibition under free diffusion conditions on paper. Therefore, the bioinks (JNK1 or JNK2 → MAPKAPK2 → with and without ATP → Ser/Thr 4 peptide) were sequentially printed on the paper. After incubation, developing solution was printed on 7869
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Table 1. Summary of the Solution Densities of the Bioinks and Their Respective Ejection Volumes Printed/Spot Areas density (g/mL) CMYK value ejected volume (×10−8 L)
JNK1
JNK2
MAPKAPK2
Ser/Thr pep
kinase buffer
developing solution
ATP
1.01 100 4.15
1.04 100 4.08
1.03 100 4.13
1.03 100 4.1
1.02 50 3.98
1.05 100 4.07
1.09 50 4.11
each reaction spot, and fluorescence images were acquired. When the reaction spot on the paper surface was excited at 400 nm, the fluorescein and coumarin emission signals were recorded at the same time. As shown in Figures 4A and 5A, the control group includes three arrays of data. A maximum emission ratio of 3.02 (with an increase in coumarin emission and a decrease in fluorescein emission) was attained by 0% phosphorylation conditions, which contained no ATP, and thus the reaction spot exhibited no kinase cascade reaction. As a result, 100% of the peptide cleaved in the presence of the developing solution. In the case of 100% phosphorylation conditions, JNK1/2 kinase and MAPKAPK2 interacted successfully and phosphorylated the peptide at the Ser/Thr site. An increase in fluorescein emission and a decrease in coumarin emission, leading to an emission ratio of 0.48 was recorded, which indicated that a very low peptide cleavage occurred under the 100% phosphorylation conditions. The minimal emission ratio of 1.76 (with an increase in coumarin emission and a decrease in fluorescein emission) was attained by the 0% inhibition conditions, which represents that the kinase is active and able to phosphorylate approximately 20− 40% of the peptide while cleaving 60−80% of the peptide on each reaction spot. The observed difference in the emission ratios under three different conditions confirmed the effective phosphorylation cascade reaction under free diffusion conditions on paper. For the test group, the assay was performed in the presence of all reaction components to confirm the inhibition of the phosphorylation cascade reaction under free diffusion conditions on paper. The bioinks, including JNK1 or JNK2 → MAPKAPK2 → SP600125 → mixture of ATP and Ser/Thr 4 peptide labeled with two fluorophores (coumarin and fluorescein), were sequentially printed on paper. Table 1 details the solution densities of the bioinks and their respective volume printed/spot areas. The number of moles of JNK1 and JNK2 printed per spot was estimated using the volume printed/spot area.19 As mentioned above, 3.1 × 10−11 g of JNK1 or 1.9 × 10−11 g of JNK2 along with 2.1 × 10−10 g of MAPKAPK2 (that phosphorylates 20−40% of the Ser/Thr 4 phosphopeptide on paper) was chosen to determine inhibitor activity under free diffusion conditions. The printer settings were altered by a 10-order difference per spot area to vary the amount of SP600125 to be printed per spot. It was found that 0.5−4.08 × 10−15 mol of SP600125 was printed on the surface when the cartridge setting was fixed in the range of 10−100. After the printing process, the bioink-printed paper was incubated in room temperature for 1 h, and then the developing solution was printed on each reacted spot. Finally, the fluorescence signal generated on each reaction spot was measured using a confocal microscope. When the reaction spot on the paper surface was excited at 400 nm, a gradient decrease in the fluorescein emission signal and an increase in the coumarin emission signal was recorded at the same time (Figure 4BJNK1 and Figure 5BJNK2). This observed result is directly related to the conditions under which the presence of a gradient range of the inhibitor interacts together
with a constant amount of JNK1 or JNK2 and MAPKAPK2 on each reaction spot and affects the phosphorylation cascade reaction on paper. Figures 4C and 5C represents JNK1 and JNK2 dependent phosphorylation achieved in the presence of inhibitor, respectively in each reaction spot. As shown in Figures 4D and 5D, the X-axis represents moles of SP600125 printed per spot area, and the Y-axis represents the emission ratio. The presence of SP600125 (0.5−4.08 × 10−15 mol per spot) led to a significant increase in the coumarin fluorescence intensity and a linear decrease in the fluorescein fluorescence intensity generated by each reaction spot. The maximum emission ratio (3.21JNK1 and 3.48JNK2) attained in the presence of 4.08 × 10−15 mol of SP600125 displayed 0−5% phosphorylation, while the emission ratio (1.68JNK1 and 1.84JNK2) attained in the absence of SP600125 displayed 30−40% phosphorylation. These differences in the emission ratio confirms that the amount of SP600125 present in each reaction spot interacts with the kinases present on the same spot, thereby inhibiting the phosphorylation cascade reaction on paper. Using the proposed method, the inhibitory mole 50 (IM50) of SP600125, which is required to reduce the JNK1 and JNK2 activity by half, was determined to be 2.58 × 10−15 mol and 2.22 × 10−15 mol, respectively.
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CONCLUSIONS In summary, we developed a novel method of direct on-paper kinase−kinase phosphorylation cascade reaction using inkjet printing. For the printing and evaluation of kinase−kinase activity, inkjet printing was found to be most suitable, as it offers many advantages, such as without deteroriating the biofuntional activity of the kinase enzyme and utilization of small reaction volume (nanoliter range) with high accuracy, it occurs in a printer-friendly environment in terms of surface tension and viscosity, and it does not require specialized instrumentation for data analysis. Collectively, all these factors lead to the success of the kinase−kinase phosphorylation cascade reaction on paper under free diffusion conditions. So far, this is the first report to validate the kinase−kinase phosphorylation and inhibition activity using an inkjet printing method, which was easily accessible and compatible with standard inkjet printing machines.
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MATERIALS AND METHODS Materials. All assay components, including JNK1, JNK2, inactive MAPKAPK2, 10 mM of ATP, 1 mM of Ser/Thr 4 peptide, 1 mM of Ser/Thr 4 phosphopeptide, and developing reagent, were purchased from Invitrogen (California, U.S.). JNK1/2 inhibitor (SP600125), sodium CMC, PEGs-200, -400, and -2000 Da, and glycerol were purchased from SigmaAldrich (MO, USA). All materials were used without further purification. Methods. Bioink Preparation. All reagents used in the experiment were freshly prepared, and the experimental procedures were carried out at room temperature (23 °C) under dark conditions. A 5× Kinase buffer (250 mM of pH 7.5 N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid buffer, 7870
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Em = 400 nm/525 nm) was measured using a multiplate reader. Processing of Inkjet Printing Cartridge, Printing Setup, and Bioink Deposition. A commercially available HP Officejet Pro 8100 printer containing four cartridges was used to study the kinase−kinase phosphorylation cascade reaction on paper. Commercial inks present inside the cartridges were completely removed; the cartridges were washed 10 times with deionized water and 100% ethanol using an insulin syringe, and then completely dried in a hot air oven. Finally, bioink solutions were refilled into the respective cartridges. The C cartridge was filled with JNK1 or JNK2, while the M cartridge was filled with inactive MAPKAPK2, and the Y cartridge was filled with a mixture of ATP and Ser/Thr 4 peptide. Adobe Photoshop software was utilized to obtain RGB color codes, and Microsoft Power Point was used to prepare the desired printing patterns (3 rows by 3 columnscontroland 3 rows by 12 columns test). The size of the printing pattern for the reaction was set to be 3 mm diameter per spot. To optimize the JNK1/2dependent phosphorylation cascade reaction on paper, the printer settings were fixed at 100, 50, and 100 to print constant amounts of MAPKAPK2, ATP, and Ser/Thr 4 phosphopeptide, respectively, while the printer settings were altered by from 10 to 100 to vary the amount of JNK1 or JNK2 to be printed. Similarly, to evaluate the inhibitory activity of SP600125 (JNK1/2 inhibitor) on paper, the printer settings were fixed at 100, 100, 50, and 100 to print constant amounts of JNK1 or JNK2, MAPKAPK2, ATP, and Ser/Thr 4 phosphopeptide, respectively, while the different settings between v10 and 100 were used to vary the amount of SP600125 printed. Determination of JNK1/2-Dependent Phosphorylation and Inhibition Activity on Paper under Free Diffusion Conditions (after Printing). To investigate the JNK1/2mediated phosphorylation cascade reaction and inhibition activity on paper, all bioinks were sequentially printed on parchment paper. First, an inkjet printing-based assay was performed with and without ATP, to confirm 0% phosphorylation or 100% phosphorylation and 0% inhibition under free diffusion conditions on paper. The following printing orders were followed to print all assay components: for 0% phosphorylation, JNK1 or JNK2 → inactive MAPKAPK2 → Ser/Thr 4 peptide; for 0% inhibition and 100% phosphorylation, JNK1 or JNK2 → inactive MAPKAPK2 → mixture of ATP and Ser/Thr 4 peptide. Components were sequentially printed on the paper at each reaction point in a grid of 3 rows by 3 columns. Subsequently, the inkjet printing-based assay was performed to determine the optimal concentration of JNK1/2 for the phosphorylation of Ser/Thr peptide on paper. For this purpose, the JNK1 or JNK2 → inactive MAPKAPK2 → mixture of ATP and Ser/Thr 4 peptide was sequentially printed on the paper at each reaction point of a grid with 3 rows and 12 columns. Lastly, inkjet printing-based phosphorylation cascade reactions were performed to determine the JNK1/2 inhibition by SP600125. For this purpose, the bioinks JNK1 or JNK2 → MAPKAPK2 → SP600125 → mixture of ATP and Ser/Thr 4 peptide were sequentially printed on paper at each reaction point of a grid with 3 rows and 10 columns. The assay component-printed papers were incubated for 1 h at room temperature (25 °C). The 1× kinase buffer was printed once in 20 min to enhance the ongoing phosphorylation cascade reaction under free diffusion conditions on paper. After incubation, the developing solution was printed on the
50 mM MgCl2, 5 mM EGTA, 0.05% BRIG-35) was freshly prepared. JNK1 (7.5 × 10−7 g/mL), JNK2 (4.7 × 10−7 g/mL), inactive MAPKAPK2 (5.0 × 10−6 g/mL), ATP (10 mM), Ser/ Thr 4 peptide (4 μM), and Ser/Thr 4 phosphopeptide (4 μM) were prepared using the 1× kinase buffer. JNK1/2 inhibitor (SP600125, 0.1 μM) was prepared using dimethyl sulfoxide. Surface Tension, Viscosity, and Solution Density Measurements. To modify the bioink surface tension, Triton X-100, a nonionic surfactant, was used. Portions of 1× kinase buffer solutions, containing increasing concentrations of the surfactant (10−4 to 100), were prepared, and their surface tensions were measured to determine the cmc. Similarly, bioink solutions, including JNK1 (7.5 × 10−7 g/mL) or JNK2 (4.7 × 10−7 g/mL), MAPKAPK2 (5.0 × 10−6 g/mL), ATP (0.1 mM), and Ser/Thr 4 peptide (4 μM), in buffer containing various concentrations of the surfactant were prepared, and their phosphorylation activity in solution was measured immediately after preparation. The surface tension values of the bioinks were measured at room temperature (23 °C) with a tensiometer (AquaPi, Kibron) based on the Wilhelmy plate principle. To modify the bioink viscosity, 1× kinase buffer solutions containing viscosity modifiers (sodium CMC, PEG-200, -400, and -2000, and glycerol) were prepared, and their dynamic viscosity was measured at room temperature (23 °C) using a capillary viscometer (manufacturer detail). Similarly, bioink solutions of JNK1 (7.5 × 10−7 g/mL) or JNK2 (4.7 × 10−7 g/ mL), MAPKAPK2 (5.0 × 10−6 g/mL), ATP (0.1 mM), and Ser/Thr 4 peptide (4 μM) in buffer containing various viscosity modifiers were prepared, and their phosphorylation activity in solution was measured immediately after preparation. The solution density of the bioink solutions [JNK1 (7.5 × 10−7 g/mL) or JNK2 (4.7 × 10−7 g/mL), MAPKAPK2 (5.0 × 10−6 g/mL), ATP (0.1 mM), and Ser/Thr 4 peptide (4 μM)] containing surfactant and viscosity modifiers was measured at room temperature (23 °C) using a pycnometer, and the values are expressed as mass per unit volume. The density was calculated using the following formula: ds = (Ws − Wb)/(Ww − Wb), where Ws is solution weight, Wb is bottle weight, and Ww is water weight. Thus, the volume of assay components printed on the paper was estimated using the printed spot size, solution density, and the weight loss of the cartridge after printing a single spot. Activity of Solution (before Printing). All bioinks [JNK1 (7.5 × 10−7 g/mL) or JNK2 (4.7 × 10−7 g/mL), MAPKAPK2 (5.0 × 10−6 g/mL), ATP (0.1 mM), and Ser/Thr 4 peptide (4 μM)] were prepared in buffer containing 0.1 wt % Triton X100 and viscosity modifier. Before printing, the phosphorylation activities of the bioinks containing surfactants and viscosity modifiers were evaluated. For this purpose, 5 μL of JNK1/2, along with inactive MAPKAPK2 and Ser/Thr 4 peptide was added to a 96 well-plate. The kinase cascade reaction was initiated by adding 2.5 μL of 0.1 mM ATP solution. The well-plate was placed on a shaker for 30 s to gently mix the reagents. After shaking, the assay plate was incubated at room temperature (25 °C) for 1.5 h. After incubation, 5 μL of development solution was added to the reaction well. The plate was incubated in room temperature (25 °C) for 1 additional hour, and 5 μL of stop solution was added to the reaction well. Finally, the fluorescence intensity of coumarin (Ex/Em = 400 nm/445 nm) and fluorescein (Ex/ 7871
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reaction spot, and then fluorescence images [coumarin (Ex/ Em = 400 nm/445 nm) and fluorescein (Ex/Em = 400 nm/ 525 nm)] were acquired using a confocal microscope (Leica, TCS SP8). The acquired fluorescence images were analyzed using Image J software. The amounts of assay components printed on each spot were evaluated based on the volume printed per spot and their respective solution densities, as suggested by Song et al.15 The following equation was used to calculate the emission ratio for each spot on parchment paper Emission ratio =
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coumarin emission (445 nm) fluorescein emission (525 nm)
The following equation was used to calculate the percentage of phosphorylation for each spot on parchment paper % phosphorylation =1−
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emission ratio × F100% − C100% (C0% − C100%) + [emission ratio × (F100% − F0%)]
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Joon Myong Song: 0000-0002-9896-5892 Author Contributions
Jungmi Lee has performed most of the inkjet printing assay, Annie A.S. Samson has contributed to data analysis, and Joon Myong Song has designed idea and experiment conditions of this paper. Author Contributions †
Jungmi Lee and Annie A.S. Samson contributed equally to this manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (2018M3A7B4071235 and 2019R1A4A2001651). We are grateful to the Research Institute of Pharmaceutical Sciences at Seoul National University for providing experimental equipment and Brain Korea 21 plus (BK 21 plus). The authors declare no competing financial interests.
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DOI: 10.1021/acsomega.9b00697 ACS Omega 2019, 4, 7866−7873
ACS Omega
Article
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DOI: 10.1021/acsomega.9b00697 ACS Omega 2019, 4, 7866−7873
