Easy and Fast Western Blotting by Thin-Film Direct Coating with

Jun 2, 2016 - Thin-film direct coating (TDC) has been successfully used in ... ng of purified recombinant glutathione-S-transferase (GST) proteins and...
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Easy and Fast Western Blotting by Thin-Film Direct Coating with Suction Chao-Yuan Liu,†,§ De-Chao Lu,‡,§ Yi-Wei Jiang,‡,§ Yi-Kuang Yen,†,§ Shih-Chung Chang,*,‡ and An-Bang Wang*,† †

Institute of Applied Mechanics and ‡Department of Biochemical Science & Technology, National Taiwan University, Taipei 106, Taiwan S Supporting Information *

ABSTRACT: Thin-film direct coating (TDC) has been successfully used in Western blotting (WB). In this study, the advanced technique of TDC with suction (TDCS) was developed to reduce the consumption amount of antibody by a factor of up to 104 in comparison with the amount consumed by the conventional WB using the capillary tube without any need of special micromachining processes. The operation time for completely finishing a high-quality WB can be reduced from 3 h in conventional WB to about 5 min or even less by TDCS. In addition, the signal-to-noise ratio of the immunoblotting by TDCS can be markedly increased. TDCS WB showed a high linearity within a 6-log2 dynamic range for detecting 90−6000 ng of purified recombinant glutathione-S-transferase (GST) proteins and could particularly detect extrinsic GST proteins added in crude Escherichia coli or 293T cell lysates. Moreover, a protein mixture containing bovine serum albumin, GST, and ubiquitin could be specifically probed in parallel with their corresponding antibodies through multichannel TDCS WB. This simple and innovative TDCS WB offers various potential applications in simultaneously finishing multiple antibody−antigen screenings in a fast and single experiment. microfluidics with a microtiter-plate-based μWB array was developed to simultaneously analyze 10 proteins.8 The μWB array improved the immunoassay, but it may still require complex micromachining techniques. Recently, Hughes and Herr5 took advantage of a high-throughput microfluidic technique (up to 48 channels) to finish protein separation, transfer, and blotting procedures in a single glass microfluidic platform in 10−60 min. These results reveal that the multichannel detection could significantly improve the multiplexing capability of Western blotting method; however, there is still no widely obtainable and inexpensive way for simultaneous multiple detections without the need for special and precision micromachining processes. In addition to the development of the immunodetection method with reduced antibody consumption, several methods using different design concepts have been introduced to increase the efficiency and sensitivity of WB.9,10 Compared with conventional WB, the SNAP i.d. 2.0 Protein Detection System (by EMD Millipore)9 has significantly reduced the operation time from 3 h to 30 min by using an active vacuum mechanism for conducting antibody probing and washing steps. However, in this system, only the durations in staining and washing steps are improved, whereas antibody consumption is not. To enhance the detection sensitivity, quantum-fluores-

W

estern blotting (WB), which is composed of various biochemical techniques, is widely used in life science studies and clinical diagnoses.1 WB can identify specific targets in protein mixtures because of specific antibody−antigen interactions.2−4 However, WB has some drawbacks, such as its high costs of excessive antibody consumption and timeintensive procedures.5 In comparison with conventional WB, microfluidic methods require lower sample volumes and take much shorter operation time. Thus, they have been gradually applied in immunodetection.5−8 For instance, He and Herr6,7 introduced a glassbased microfluidic chip, which was fabricated by the wet etching process, and a photopatterned membrane for antibodybased in-gel blotting. The whole immunoblotting process only consumed ∼1 μg of antibody, which was benefited from the automated microfluidic methods. Although this method has lower antibody consumption, it can detect only one target protein in each assay. In applying this approach to separation of multiple proteins, which are confined in a narrow region, the sufficient protein migration distance would be required for a better resolution. In addition, the technical barrier of microfabrication technology for glass-based microfluidics still exists. For instance, a clean foundry, costly etching processes, complicated chemical modifications for microfluidic surfaces, and other delicate microscopic instruments, among others, are necessarily required to achieve multifunctions of microfluidic chip. Therefore, the wide applications of the microfluidic method remain limited. Recently, a method incorporating © XXXX American Chemical Society

Received: February 22, 2016 Accepted: May 24, 2016

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by adding isopropyl-1-thio-beta-D-galactopyranoside to a final concentration of 1 mM for 4 h.16 GST proteins were purified using a GSTrap FF column (GE Healthcare) pre-equilibrated with a binding buffer (20 mM sodium phosphate, 0.15 M NaCl, pH 7.3), and bound proteins were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0. The recombinant human Ub was purified using a HisTrap HP column (GE Healthcare) pre-equilibrated with a binding buffer (20 mM sodium phosphate, 20 mM imidazole, 0.5 M NaCl, pH 7.3), and bound proteins were eluted with a linear gradient of 20− 250 mM imidazole in 20 mM sodium phosphate and 0.5 M NaCl (pH 7.3). The protein purity was examined using 16% SDS-PAGE, and the concentration was determined by the Bradford dye-binding method.17 Cell Lysate Preparation. HEK-293T cells cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin−streptomycin solution (Gibco, Invitrogen, Carlsbad, CA, U.S.A.) were lysed in a lysis buffer (50 mM Tris-HCI, pH 7.8, 4% glycerol, 50 mM NaCl, 0.5% NP-40, 1% SDS, 1% Protease Inhibitor Cocktail Set III (EMD Millipore), and 0.1% sodium azide) for 2 h at 4 °C. The supernatant was collected by centrifugation at 20 000g for 10 min at 4 °C. To prepare the E. coli BL21(DE3) cell lysate, cells were cultured in LB medium overnight at 37 °C. Cells were collected by centrifugation at 6000g for 10 min. The cell pellet was washed three times with PBS and then incubated with SDS-PAGE sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% beta-mercaptoethanol, and 0.004% bromophenol blue) at 100 °C for 30 min. Antibody. Anti-GST antibody was purchased from AbOmics (New Taipei City, Taiwan) (catalog no. M0006). Anti-Ub monoclonal antibody was purchased from SigmaAldrich (St. Louis, MO, U.S.A.) (product no. U0508). Horseradish peroxidase-labeled goat antimouse IgG secondary antibody (GAM) was purchased from KPL (Gaithersburg, MD, U.S.A.) (catalog no. 04-18-06). Anti-bovine serum albumin (BSA) polyclonal antibody was prepared by immunizing Balb/c mice one time with a mixture of 100 μg BSA and complete Freund’s adjuvant (Sigma-Aldrich, product no. F5881) and three times in a 2-week interval with a mixture of 50 μg BSA and incomplete Freund’s adjuvant (Sigma-Aldrich, product no. F5506) followed by a final boost of 50 μg BSA in phosphatebuffered saline without adjuvant. Antisera were collected 1 week after the final boost. The animals’ care and use protocol have been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University (IACUC Approval Number: NTU-102-EL-93). Gel Electrophoresis. SDS-PAGE was performed according to the method described by Laemmli.18 Tris-boric acid SDS running buffer (90 mM Tris, 2.5 mM EDTA·2Na, 80 mM boric acid, 0.1% SDS, pH 8.4) was used as the electrophoresis buffer. After electrophoresis, proteins were transferred onto the Immobilon-P PVDF membrane (EMD Millipore) at 400 mA for 1 h by using 10 mM CAPS (pH 11) transfer buffer with 15% methanol and then stained with Ponceau S (Catalog number 22001, Biotium, Hayward, CA, U.S.A.) for rapid reversible detection of protein bands before conducting the Western blotting analysis. Conventional WB. Conventional WB was performed on an orbital shaker at room temperature. PVDF membranes were blocked with a blocking buffer (5% skimmed milk in PBS with 0.05% Tween-20 (PBST)) for 1 h and then probed with primary antibodies diluted with Gelatin-NET (0.25% Gelatin,

cence-based WB was introduced to detect target proteins10 by using the high brightness and multiple colors of Qdot secondary antibody conjugates. The results showed high sensitivity at the picogram level and demonstrated a short operational time of 0.6−5 min in the incubation process. However, a general application of this method is expensive by apparatus requirements and thus still very limited in the biochemistry laboratory. Precise wet coating film has been extensively employed in various industrial products due to its good reliability and uniformity in producing mass production.11,12 However, it is very difficult to apply a conventional precision coating technique (e.g., slot-die coater) in a desktop immunoblotting system that is commonly used in the biochemistry laboratory. Nevertheless, an important breakthrough of direct coating technique using disposable, light, compact, and thin coaters13 has been revealed to remarkably reduce the manufacturing cost through standard microelectromechanical system (MEMS) technology. This plane coating technique was successfully employed in the labeling process of WB to substantially reduce antibody consumption, namely, the thin film direct coating (TDC) WB method.14 Conceptually, TDC WB has demonstrated that the antibody consumption can be further significantly reduced by shortening the coater width. This means that the newly developed TDC coater would produce a narrow line, instead of a plane, for the WB probing with much lower material consumption but without losing its sensitivity. It is a pity that the TDC coater has not been mass produced in the real market, similar to most of the μWB methods, and as a result, the TDC WB system is still not generally available to the market and thus limited in its applicability to general laboratories. Theoretically, also from the successful result of TDC used in WB, increasing local antibody concentration could enhance the antibody−antigen interactions and therefore shorten the reaction time. In order to shorten the operation time and to further reduce the material consumption in the WB probing process, the TDC with suction (TDCS) technique was thus first proposed and tested in the study. Because the coating method by the capillary tube has been realized in the line coating concept and also recognized as the most material-saving and the easiest way to achieve parallel coating,15 the commonly available capillary tube would then be introduced as an alternative of TDC coater before the plane coater can be mass produced in the real market. In this study, the enhancement of detection sensitivity by effectively removing residual antibodies on the polyvinylidene fluoride (PVDF) membrane to decrease the background noise and the functionality to simultaneously detect multiple proteins with parallel individual or mixed antibodies by the aid of capillary coating have also been examined and characterized.

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EXPERIMENTAL SECTION Protein Expression and Purification. The expression vector pGEX-4T-3 (GE Healthcare, Pittsburgh, PA, U.S.A.) encoding glutathione S-transferase (GST) or the expression vector pET28a encoding human ubiquitin (Ub) with an Nterminal His-tag was transformed into E. coli BL21(DE3) cells (Novagen, EMD Millipore, Merck KGaA, Darmstadt, Germany). Cells were cultured in Luria−Bertani (LB) medium with ampicillin (50 μg/mL) or kanamycin (50 μg/mL) and incubated at 37 °C on an orbital shaker at 150 rpm. Expression of the recombinant protein was induced at an A600 of 0.6−0.7 B

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Figure 1. Schematic diagram of TDCS Western blotting (WB): (a) The specifications of the TDCS WB system. (b) The photo of the new compact TDCS system used in the study. (c) The operation process of TDCS WB. A video about operation process of TDCS WB with essential notes and captions is provided in Supporting Information.

0.15 M NaCl, 5 mM EDTA·2Na, 0.05% Tween 20, 50 mM Tris) for 1 h. The membranes were subsequently washed three times for 10 min each with PBST and then incubated with GAM secondary antibody for 1 h. The membranes were then washed three times for 10 min each with PBST. VisGlow chemiluminescent substrate (Visual Protein Biotechnology, Taipei, Taiwan) was used to detect the protein targets on the membranes, and signals were visualized by the UVP BioSpectrum Imaging System (Upland, CA, U.S.A.). TDC/TDCS WB. The schema of TDCS WB is illustrated in Figure 1. A video with essential notes is provided in Supporting Information. The coater can be silicon coater13,14 or the capillary tube15 for wet coating films of different coating width. The TDC method is using relative motion of the coating head and substrate to define uniform film of antibody solution on the PVDF substrate. To compare the benefit of the suction effect for TDCS, the super light-and-slim slot die coater,19 which had been demonstrated in the previous TDC study,14 was used first in combination with the suction platform underneath of the PVDF membrane (as shown in Figure 1a). The machine was first built to achieve the suction function by connecting the suction chamber to a vacuum pump (G-735, TOHAMA, Yeong-Shin Co., Hsinchu City, Taiwan), and the coater was made by the standard MEMS technique consisting of a fishtailtype diffuser, a guide fin, and a contraction to ensure a uniform flow velocity across the slit width at the end of the silicon mold. The coater is 150 μm in depth and 20 mm in width at the coater exit and contains 80 μL of total interior volume. Detailed descriptions of manufacturing process of the coater can be found in Yen et al.14

Because the above-mentioned slot die coater is still not generally available in the market and the smaller coating width can also reduce the consumption of the coating material without losing its detecting quality,11 the commonly available capillary tube in the range of 10−1000 μm was thus used as the alternative coater to test its feasibility and performance. The coater with 100 μm in width was utilized for coating specific antibodies on the PVDF membrane in this study. The total volume of this capillary tube coater is approximately 0.39 μL. Furthermore, to achieve automatic parallel immunoblotting, a new desktop coating system equipped with multicapillary coaters (e.g., three capillary tubes are shown in Figure 1a) was developed and the photo of the new compact TDCS system is shown in Figure 1b. In this novel TDCS system, different coating modules (i.e., the slot coating die or capillary tube(s)), can be easily exchanged in the replaceable TDC coating head, and a small vacuum pump (KPV30A-12A, Koge Micro Tech Co., Fujian, China) was integrated inside the system for the suction operation. The linear motorized stage was constructed by two stepping motors (42BYG200A-SASSML, SYNTRON, Beijin, China) and linear sliders (SVRN24−160, MiSUMi, Tokyo, Japan) to move the substrate in the X−Y direction with spatial resolution of 1.25 μm, maximum speed of 15 mm/s and working distance of ±100 mm. For the Z-axis, a manually controlled translational stage (06RTS-0.5, Unice, Taoyuan City, Taiwan) and a rotary stage (06RSP-4, Unice) were built for adjusting the height and perpendicularity of micronozzle to the substrate, respectively. All the motion control of stages was implemented by micro controller unit (Arduino MEGA 2560, Turin, Italy) and motor control cards (ADIO-A4988 and ADIO-RAMPS, Tun-Hwa Electronic Material Co., Taichung, Taiwan). In addition, a liquid pump (MP5, Bartels C

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Figure 2. TDCS WB showed a high linearity within a 6-log2 dynamic range. A series of purified GST proteins (0.09, 0.19, 0.38, 0.75, 1.5, 3, and 6 μg) were analyzed on SDS-PAGE and transferred to PVDF membranes for conducting conventional WB (a), TDC WB (b), and TDCS WB (c). The consumption of the primary anti-GST antibody (1st Ab) for conventional WB, TDC WB, or TDCS WB was 2, 0.2, or 0.02 μg, respectively. The consumption of the secondary GAM antibody (2nd Ab) for each method was kept the same as 0.2 μg. The top panels showed the immunoblotting results, and the middle panels showed the GST proteins stained by Ponceau S. In bottom panels, the measured signal intensity of the top panels by a densitometer was plotted against the amount of GST proteins loaded on SDS-PAGE. The error bar indicates the standard deviations of three independent experiments.

Mikrotechnik GmbH, Dortmund, Germany) could be utilized to deliver the antibody from the reservoir to the slot coater by manual/program control. The open-sourced software, CURA, was adopted as Graphical User Interface (GUI) to easily construct the needed coating patterns. In the coating process, the PVDF membrane (6 cm × 8 cm) was first placed on the stage to air-dry excess liquid for 10 min. The primary antibody solution was fed into the coater (slot die coater or capillary tube) by using a liquid pump (100 μL/min) or just by capillary force,15 respectively. The coater was mounted on the vertical axis of a translational stage to control the coating gap between the coater and the membrane by using a linear encoder. The stage running speed was programmable and was set as 1 mm/s as that used in TDC WB.11 The coating process contained three steps as shown in Figure 1c. The coater first descended to the cover glass surface to form the stable liquid bridge. To start the coating, the coating gap was then controlled as 150 μm with the help of a linear encoder, and the coater was moved from the cover glass to a PVDF membrane without direct contact (to PVDF) along the coating direction. After the coating process has been completed, the coater ascended to its resetting position. In this study, unless otherwise mentioned, the coating duration was less than 1 min, and less than 0.1 mL (for a 20 mm coating width and 60 mm coating length by slot die coater) or less than 0.2 μL (for capillary tube) of the primary antibody (1st Ab) solution was used in each coating stroke. After finishing the antibody coating, the PVDF membrane was incubated at room

temperature for 2−10 min and ready for the subsequent three washing steps (10 s/step) with suction (−30 cmHg). The total suction time was less than 1 min by using the first vacuum pump (G-735, TOHAMA) and was less than 0.5 min in the new compact TDCS system (Figure 1b). The secondary antibody (2nd Ab) was coated by slot die coater with 20 mm coating width and 60 mm coating length. The incubation and washing steps were performed as the conditions applied in the probing process of first Ab.

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RESULTS AND DISCUSSION

Performance Test of TDCS WB. TDC method has been successfully applied for WB and has considerably increased WB efficiency and reduced antibody consumption.14 To further improve the performance of TDC, the TDCS (Figure 1) was studied in the present work for maintaining its high detection quality in addition to reducing the operation time and material consumptions for completing WB. To start the performance test of TDCS WB in comparison with that of conventional WB, the PVDF membrane blotted with a series of purified GST proteins (90−6000 ng) was mounted on a porous plate and coated once with anti-GST antibody (10-μm thick) by using a thin-film coater (Figure 1). The membrane was then incubated for 10 min and washed three times with suction by using an automatic suction pump (Figure 1). The experimental results in Figure 2 showed that the signal intensity of TDCS WB was stronger than that of conventional WB. Furthermore, TDCS WB displayed high linearity within a 6-log2 dynamic range for D

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Analytical Chemistry GST measurement in the bottom panel of Figure 2c. It is noted that the consumption of anti-GST antibody was 2 μg in conventional WB (Figure 2a), 0.2 μg in TDC WB (Figure 2b), and 0.02 μg in TDCS WB (Figure 2c), indicating that the antibody consumption of TDCS WB was reduced 100-fold or 10-fold in comparison with the amount consumed by conventional WB or TDC WB, respectively. The suction mechanism of TDCS WB also reduced the nonspecific blotting background (Figure 2). In addition, the signal intensity for detecting 90 ng of GST by TDCS WB can be easily observed (Figure 2c), implying that the lower limit of detection should be much smaller than 90 ng. Reduction of the Antigen−Antibody Incubation Time through TDCS WB. To examine whether the immunoblotting signal intensity of TDCS WB is higher than that of conventional WB under the same incubation time, purified GST proteins were analyzed through SDS-PAGE and transferred to PVDF membranes for performing WB by incubating the membranes with the anti-GST antibody for various durations (2−60 min). Signal intensity was measured using a densitometer and plotted against the antibody incubation time in Figure 3. The signal intensity of TDCS WB was generally higher than that of the conventional WB, as shown in Figure 3a. The signal intensity of TDCS WB increased rapidly before 10 min and then increased gradually to the maximum intensity after 20 min, whereas conventional WB required 40 min to reach the same value (Figure 3b). The results showed that the intensity of TDCS WB is significantly higher than that of

conventional WB at both 2 and 10 min, indicating that the antibody incubation time in TDCS WB can be dramatically reduced from 60 to 10 min or even 2 min with only 12.5% signal intensity reduction. In fact, by assuming the signal intensity at 60 min as saturation intensity, the incubation time for conventional WB and TDCS WB to reach half of the saturation intensity are 1.96 and 0.55 min, respectively (Figure 3); that is, the characteristic time scale for the latter is shortened to be only 28% in comparison with that of the former. It reveals that the performance of TDCS WB is really superior to that of conventional WB for enhancing antigen− antibody interaction during a very short incubation time. To be more conservative, the suggested value14 of 10 min has been adapted and used in the present study. Actually, the incubation time could be further optimized through the best combination of coating film thickness, first and secondary antibody consumptions, addition of suction, and design of porous size, among others, and the washing time could be also further reduced by adjusting the suction power, the washing flow rate, and so on; however, this is not the focus of the present work. Compared with the 10 mL of antibody solution used in conventional WB, TDCS WB uses a much smaller volume of antibody solution (0.1 mL) in a coating and suction platform, implying that the molecular traveling distance, molecular collision possibilities, and diffusion mechanism between an antigen and an antibody should be tremendously changed. The ability of TDCS WB in reduction of the antigen−antibody incubation time suggests that the suction steps of TDCS WB could be the major factors for reducing the diffusion effect and considerably increasing the molecular collision possibilities of the antigen−antibody interaction. By contrast, the diffusioncontrolled interaction between antigen and its antibody in conventional WB is time intensive because it uses a low concentration of antibody. Thus, an orbital shaker is often used to enhance molecular collisions in conventional WB to obtain a more favorable WB result. Detection of Target Proteins in Cell Lysates by TDCS WB. In addition to the detection of purified GST in a cell-free system, TDCS WB was evaluated for probing extrinsic GST proteins added in Escherichia coli and 239T cell lysates. The cell lysate without adding GST was shown as the negative control. The lower panels of Figure 4 show that the extrinsic GSTs could not be clearly recognized by Ponceau S staining on the PVDF membranes because of the complex protein composition and the small amount of GSTs in the cell lysate. However, GSTs could be specifically probed through TDCS WB with anti-GST antibody (Figure 4, upper panels). Moreover, the nonspecific bindings in the negative control of conventional WB (left photo) could be eliminated in the TDCS WB (right photo) in the upper panel of Figure 4a. It indicates that TDCS WB has better signal quality and specificity compared with conventional WB because the specific primary antibody could be homogeneously coated on the PVDF membrane, and nonspecific bindings could be reduced by the mechanism of suction. Application of Capillary-Tube-Based TDCS WB in Detecting Multiple Antigen−Antibody Interactions. TDCS WB has been demonstrated as a reliable method for quantitative analysis in specific protein detection. It is quite straightforward that the antibody consumption of TDCS WB could be further reduced by simply using a coater with smaller coating width. On this basis and also to overcome the restriction of general availability of the TDC coater in the

Figure 3. Investigation of the antibody incubation time for conventional WB and TDCS WB. (a) Purified GST protein (5 μg) was analyzed on SDS-PAGE and transferred to a PVDF membrane for incubating with anti-GST antibody in the time periods from 2 to 60 min. (b) Signal intensity measured by a densitometer was plotted against the incubation time. Error bars represent standard deviation (n = 3). Statistical significance is marked with an asterisk (*P < 0.05 by one-way ANOVA with post hoc Tukey’s test). E

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Figure 4. TDCS WB specifically detected extrinsic GST proteins in cell lysates. Purified GST protein (1.6 μg) was mixed with 10 μg of E. coli lysate (a) or 293T cell lysate (b) and then analyzed on SDS-PAGE for Ponceau S staining or TDCS WB. The cell lysate without adding GST protein was also analyzed as a reference.

tube-based TDCS WB (0.1 mm coating width) could be further reduced in comparison with that of the TDC WB due to better combination of first Ab and second Ab usage. It is worth mentioning that the antibody consumption in the method of microfluidic polyacrylamide gel electrophoresis in situ immunoblotting6 was about 1 μg, and the corresponding value in TDCS WB was only 0.02 μg, which could be further reduced to 0.0001 μg in the capillary tube-based TDCS WB (Table 1). The reduced consumption of materials can reach up to 10 000-fold. As noted before, the antibody consumption could be still improved through the better combination of optimal operation parameters (e.g., coating film thickness, first and secondary antibody consumptions, addition of suction and design of porous size, etc). Therefore, a further reduction of the antibody consumption in Table 1 should be not a surprise after optimization in the real applications. Table 2 shows the comparisons of operation time durations used in TDC14 and TDCS WB. It is important to point out that the coating speed of 1 mm/s tested in TDC14 and also adapted in this work was very conservative and also really slow (more than 2 orders of magnitude smaller) in comparison with that commonly adapted in the coating procedure; therefore, the duration of coating period should be actually negligible among the total operation time for TDCS in the real applications. Furthermore, if we choose the incubation time to be 2 min instead of 10 min (with slight sacrifice of signal intensity as shown in Figure 3), it suggests a great potential of TDCS WB for screening multiple antigen−antibody interactions by significantly reducing antigen and antibody consumptions and total operation time within 5 min as shown in Table 3. For shortening the processing time, TDCS WB can be finished in 5 min, which is shorter than that in SNAP i.d. 2.0 Protein Detection System9 (30 min) and in the microfluidic Western blot by Pan et al.8 (10−60 min). These results clearly

traditional laboratory, the commonly available capillary tube was thus proposed as the alternative TDC coater for testing its feasibility in antigen or antibody screening and has been experimentally demonstrated its availability. To further investigate its advanced multisample screening ability, three target proteins of different molecular sizes (i.e., BSA, GST, and Ub) were chosen as the detection targets in the present experiments. The basic concept of multisample target protein screening by using capillary-tube based TDCS WB is illustrated in Figure 5a. In lane 1, the parallel coating by three TDC coaters (here three capillary tubes) containing individual antibody of BSA, GST, and Ub, respectively, could be applied instead of the one-by-one series coating. In lanes 2−5, the advanced parallel coating by only a single TDC coater filled with mixed antibodies would be tested for all three targets (lane 2) or for only one individual target (lanes 3−5), respectively. Figure 5b shows that all specific proteins could be correctly detected by TDCS WB in each lane of SDS-PAGE either by parallel coating from three coaters (capillary-tubes) filled with individual antibody (lane 1) or by a single coater with mixed antibodies of three target proteins (lanes 2−5). It is worth noting that the signals are almost similar in lanes 1 and 2 although the concentrations of all three antibodies were 3 times diluted in the latter due to equally used volume (1/3 of 0.15 μL) of αBSA, αGST, and αUb. This indicates that the advanced parallel coating of mixed antibodies could further reduce the antibody consumption to a much lower level, thereby also largely reducing the experimental cost. The comparisons on antibody consumption of conventional WB, TDC WB, and TDCS WB are summarized in Table 1. Recall that the TDC WB could reduce 2000-fold antibody consumption14 in comparison with the conventional WB under the same coating parameters (Table 1). The present data demonstrated that the antibody consumption by the capillaryF

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Analytical Chemistry Table 1. Comparisons of Antibody Consumption in Conventional WB, TDC WB, and TDCS WBa Ab consumption (μg) method

1st Ab

2nd Ab

Conventional WB TDC WB11 TDCS WB

2 0.2/0.001 0.02/0.0001

2 0.2 0.2

a

The values of TDC WB were deduced from our previous study.11 The two values separated by a slash character shown in the column of 1st Ab represent primary antibody consumption of 20 mm or 0.1 mm coating width, respectively. The secondary antibody (2nd Ab) was coated by 20 mm coating width in TDC WB and TDCS WB.

Table 2. Comparisons of Operation Time Durations Used in TDC WB and TDCS WBa operation time duration (min) method

1st Ab

1st wash

2nd Ab

2nd wash

total (min)

TDC WB11 TDCS WB

1/10 1/10

30 0.5

1/10 1/10

30 0.5

82 23

a

The two values separated by a slash character are coating and incubation time, respectively. The values of TDC WB were obtained from our previous study.11

Table 3. Comparisons of Suggested Operation Time Durations in Conventional WB and TDCS WB operation time duration (min) method

1st Ab

1st wash

2nd Ab

2nd wash

total (min)

Conventional WB TDCS WB

60 2

30 0.5

60 2

30 0.5

180 5

availability of TDC coater but also significantly enhance the overall performance and flexibility of the TDC system. This study demonstrated that TDCS technology could provide a 6log2 dynamic detection range, much shorter operation time of about 5 min (1/16 of the total processing time required in TDC WB) or even less (through the system optimization). Furthermore, compared with conventional WB, the consumption of primary antibody and the total operation time in TDCS WB were substantially reduced by a factor of 1/20 000 and 1/ 36, respectively. The TDCS method could further realize the simultaneous probing for multiple antigen−antibody interactions and benefit WB on material and time savings for providing reliable and rapid detections. The TDCS WB also obtained enhanced immunoblotting results with markedly reduced nonspecific background in comparison with that of conventional WB. The highly efficient, user-friendly, and easily adapted TDCS method can therefore be expected to widely spread in general biomedical laboratories for daily WB assays without the special and complex micromachining requirements.

Figure 5. Multiple detections of target proteins by using the capillarytube based TDCS WB. BSA (9 μg), purified GST (6 μg), and purified Ub (6 μg) were analyzed on the SDS-PAGE and then transferred to PVDF membranes for conducting the capillary-tube-based TDCS WB. The corresponding diluted antibody solutions against BSA, GST, and Ub are 13 ng/μL, 2 ng/μL and 1 ng/μL. (a) The schema of the multisample screening concept for target proteins of BSA, GST and Ub. (b) The protein mixture of BSA, GST, and Ub was first analyzed on one lane of SDS-PAGE and then subjected to TDCS WB by three individual capillary-tube coaters filled with 0.15 μL of antibody solution against BSA, GST, or Ub (lane 1) to demonstrate that TDCS WB could be applied in parallel instead of the one-by-one series coating for saving the total operation time. To further verify the specificity in antibody−antigen, the protein mixture of BSA, GST, and Ub was also analyzed on SDS-PAGE and then subjected to TDCS WB by 0.15 μL of mixed anti-BSA, anti-GST, and anti-Ub antibody solution (lane 2), anti-BSA antibody only (lane 3), anti-GST antibody only (lane 4), or anti-Ub antibody only (lane 5), respectively.

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demonstrate that the capillary-tube-based TDCS WB technique not only solve the availability of the TDC coater but also give reliable immunoblotting results and provide parallel detection approaches.

ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00699. Caption and description of SI video (PDF) Video showing the process for conducting TDCS WB (MPG)

CONCLUSIONS Suction has been added in the recently developed TDC WB14, and this newly developed method is denoted as TDCS to further enhance its performance. The capillary-tube based TDCS could not only offer as an excellent alternative before the G

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for A.-B.W.: [email protected]. *E-mail for S.-C.C.: [email protected]. Author Contributions §

C.-Y.L., D.-C.L., Y.-W.J., and Y.-K.Y. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors appreciate the financial support of Ministry of Science and Technology, Taiwan (MOST 104-2221-E-002138-MY3, NSC101-2811-E-002-048, MOST 104-2321-B-002019 and NSC101-2311-B-002-009) and the Industrial Technology Research Institute (ITRI), Taiwan, R.O.C. (contract no. 102-S-C19).

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DOI: 10.1021/acs.analchem.6b00699 Anal. Chem. XXXX, XXX, XXX−XXX