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Photonic Lab-on-a-Chip for Rapid Cytokine Detection Ryo Usuba, Masatoshi Yokokawa, Tobias Nils Ackermann, Andreu Llobera, Kiyoshi Fukunaga, Soichiro Murata, Nobuhiro Ohkohchi, and Hiroaki Suzuki ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00193 • Publication Date (Web): 21 Jun 2016 Downloaded from http://pubs.acs.org on June 25, 2016
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Photonic Lab-on-a-Chip for Rapid Cytokine Detection Ryo Usuba†, Masatoshi Yokokawa†, Tobias Nils Ackermann‡, Andreu Llobera‡, Kiyoshi Fukunaga§, Soichiro Murata#, Nobuhiro Ohkohchi§, Hiroaki Suzuki*† †
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan Instituto de Microelectrónica de Barcelona, Centre Nacional de Microelectronica, Cerdanyola del Vallès, Barcelona 08193, Spain § Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan # Graduate School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan ‡
KEYWORDS: photonic lab-on-a-chip, lymphocyte transformation test, microfluidic ELISA, cytokine detection
ABSTRACT: A photonic lab-on-a-chip (PhLoC) was developed for rapid determination of interleukin-2 (IL-2) levels secreted by lymphocytes using measurement of optical absorbance. Optical and microfluidic components, including lenses, mirrors, and flow channels, were monolithically created using poly(dimethylsiloxane) (PDMS). Sample and reagent solutions were processed in the form of liquid columns separated by air. A flow channel structure with an air bypass was used to release air between solution columns and to exchange solutions in the measuring chamber, avoiding the intrusion of air into the measuring chamber and the denaturation of the capture antibodies. The concentration of IL-2 was measured by enzyme-linked immunosorbent assay (ELISA) in the PhLoC. The total time required for the ELISA was less than 30 min. Clear concentration dependence was confirmed for IL-2 concentrations ranging from 50 to 1000 pg/mL. Thus, the concentration of IL-2 secreted by lymphocytes following activation by concanavalin A (Con A) could be measured using the PhLoC. Hypersensitivity drug reactions (HDRs) are caused by immunemediated mechanisms and may lead to diagnostic errors.1 HDRs account for 15–20% of all adverse drug reactions2 and occasionally lead to serious reactions. One representative HDR is the T-cell mediated delayed drug hypersensitivity reaction, which involves sensitized T cells that recognize drugs and induce an immunological response.3 For clinical diagnosis, lymphocyte transformation tests (LTT) are widely used.4 In these tests, peripheral blood mononuclear cells (PBMCs) are separated and cultured in the presence of drugs to allow sufficient proliferation of allergenspecific cells. The production of DNA is then measured as an indicator for the proliferation. The in vitro test is neither harmful nor painful to the patient and is effective for identification of autoimmune disorders5 and drug susceptibility for immunosuppressive drugs.6 However, this method requires 5–7 days for evaluation. For on-site analysis in routine clinical settings and in emergencies, more rapid methods are needed. Cytokines are immunomodulatory molecules that have been shown to have applications as critical biomarkers in the blood.7–9 Cytokine secretion from PBMCs of patients suffering from HDRs can also be used to determine the associations between certain cytokines and specific clinical phenotypes.6,10 Immunoassays are currently the most widely used method for detection of cytokines.11,12 Among representative methods, immunochromatography is suitable for on-site analysis and is widely used. However, this method does not yield highly sensitive, precise measurements. In contrast, enzyme-linked immunosorbent assay (ELISA) is highly sensitive but requires time-consuming antibody immobilization and long incubation periods for the antigen-antibody reaction. Microfluidics provides an effective solution for achieving more rapid, efficient immunoassays.13,14 Microfluidic channels have large surface-to-volume ratios, which accelerate antigen-
antibody reactions with immobilized capture antibodies. Additionally, the microfluidic platform minimizes the consumption of expensive reagents and precious samples, and statistical or multiplexed analyses can easily be implemented by integrating multiple sensors. Many proteins can be used as analytes owing to the commercial availability of antibodies for sandwich immunoassays, and most of these antibodies can be applied for optical detection. Synergistic combinations of optics and microfluidics for analytical applications are becoming popular, leading to the development of the photonic lab-on-a-chip (PhLoC).15,16 In this method, an optical path length in the centimeter scale is created within the PhLoC and facilitates the absorbance measurements using appropriate micro-optical components, such as lenses and mirrors. The components can be created monolithically with the microfluidic components, ensuring perfect alignment between the optical path and the measuring chamber without increasing the cost of fabrication. For the application of PhLoC in immunoassays, researchers must consider two points. First, capture antibodies should be immobilized only on the surface of the measuring chamber to minimize loss during the transport of the analyte solution. Second, intrusion of air during the exchange of solutions should be avoided, because it can result in light scattering and protein denaturation.17,18 Accordingly, in this study, we fabricated a PhLoC with a microfluidic structure with an air bypass for rapid cytokine-based LTT. Our flow channel structure enabled the introduction of only solutions into the measuring chamber and facilitated the selective immobilization of capture antibodies on the measuring chamber surfaces. The photonic and biochemical properties of the PhLoC were characterized. Then, the PhLoC was used to detect cytokine secreted from lymphocytes. As a representative cytokine, which activates the immune system, interleukin-2 (IL-2)19 can be used as
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Figure 1. The PhLoC with integrated optical and microfluidic components. (A) Schematic of the optofluidic device. A magnified view of the major part of the device is shown at the bottom. (B–D) Photographs of critical structures. (B) Lenses and structure to insert and fix the optical fiber. Scale bar: 1 mm. (C) Magnified view of T-junction 1. (D) Magnified view of the lenses. (E) Procedure for the sandwich ELISA conducted using the PhLoC.
a good indicator for LTT. We therefore used the IL-2 concentration readout to demonstrate that diagnostic results can be obtained rapidly. ■ EXPERIMENTAL SECTION Reagents and Materials. Glass wafers (#7740, 3-inch, 500 µm thick) were obtained from Corning Japan (Tokyo, Japan), and the thick-film photoresist (SU-8) was purchased from MicroChem (Newton, MA). The prepolymer solution of PDMS (KE-1300T) and (3-aminopropyl) triethoxysilane (APTES) was obtained from Shin-Etsu Chemical (Tokyo, Japan). Phosphate-buffered saline (PBS, pH 7.4), bovine serum albumin (BSA), horseradish peroxidase (HRP; activity: 5.4 × 106 U/mg), RPMI-1640 medium, and concanavalin A (Con A) were purchased from Wako Pure Chemical Industries (Osaka, Japan), and streptomycin and fetal bovine serum (FBS) were purchased from Life Technologies Japan (Tokyo, Japan). Sodium pyruvate and MEM-Non-essential Amino Acids were purchased from Sigma-Aldrich (St. Louis, MO). HEPES was purchased from ICN Biomedicals (Irvine, CA), and 2-mercaptoethanol was purchased from MP Bio Japan (Tokyo, Japan). Density gradient media for separation of lymphocytes (Lymphoprep) was purchased from Axis-Shield (Dundee, UK). FITC anti-human IL-2 antibody, Cy5-streptavidin, and the standard ELISA kit for human IL-2 (Human IL-2 ELISA MAX Deluxe) were purchased from BioLegend (San Diego, CA). The kit contained recombinant human IL-2, anti-IL-2 antibodies as capture antibodies, biotinylated detection antibodies, HRP conjugated with avidin (avidin-HRP), and tetramethylbenzidine (TMB; enzyme substrate). The concentrations of the reagents in the kit were not disclosed. Protein solutions other than the capture antibody solution were diluted with PBS containing 0.5% BSA. The
capture antibody solution was diluted 200-fold with the buffered solution included in the kit. The detection antibody and avidinHRP solutions were diluted by 200- and 1000-fold, respectively. All solutions were prepared with ultrapure water. Structure and fabrication of the PhLoC. The PhLoC is shown in Figure 1. PDMS structures, including optical components, the measuring chamber, the air bypass, and other flow channels for introduction and flushing of solutions, were formed by replica molding using a SU-8 2050 thick-film photoresist template. The prepolymer solution of PDMS was poured onto the template. After curing, the PDMS was removed from the template and cut according to the appropriate dimensions. The PDMS substrate with the formed structures and a flat cleaned glass substrate were exposed to oxygen plasma to activate the surfaces (20 W, 15 s). Both surfaces were then placed in contact immediately, resulting in strong bonding between the PDMS and glass. In the completed PhLoC, the length, width, and height of the measuring chamber were 5 mm, 250 µm, and 200 µm, respectively. For measurement of samples, the required volume was less than 500 nL, including that of solutions left in the connecting flow channels. For the other flow channels, including the air bypass, the height and width were 200 and 125 µm, respectively. The function of the PDMS optical components was based on the difference in the refractive indices between PDMS and air.15 Collimating lenses were formed at both ends of the measuring chamber. Highly specialized air mirrors were defined on this PhLoC, based on the following two aims: i) the outer interface was defined with semi-random teeth structures so as to prevent stray light from interfering with the sample to be measured, thus lowering both the signal-to-noise ratio and the limit of detection; and ii) the internal interface that faced the measuring chamber
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was flat, facilitating the delineation of a waveguide-like structure, in which light is confined at the region comprising the sample and the small PDMS walls, and boosting the light-analyte interaction. Optical fibers (250 µm in diameter; Mitsubishi Cable Industries, Japan) were inserted from both ends of the PhLoC. The protrusions in the structure facilitated the alignment of the optical axis between the measuring chamber and optical fibers while not causing excessive stress which may lead to the damage or even break of the optical fiber. Photonic simulation. The commercial software TracePro (Lambda Research Corporation) was used to simulate the light behavior in the PhLoC by tracing 40000 rays from the light source to the detector. Ray colors (red, green, and blue) represent the energy that each ray carries. The initially red rays traced from the source lose energy in the various reflections at the interfaces between PDMS (refractive index nPDMS = 1.41) and water (nH2O = 1.33) and are displayed in green when their energy is reduced to between two-thirds and one-third of their initial energy or in blue after having lost more than two-thirds of the initial energy. The dual role of the air mirrors is illustrated in Figure 2. In the flat (internal) region, the flat surface of the air mirrors, displayed at both sides of the fluidic channel, allows light confinement and its interaction with the analyte along the optical path. Here, only rays reaching the detector (end of the output fiber) and thus contributing to the signal are displayed. Conversely, as discussed above, the tooth-shaped external interface of the air mirrors is used for multi-angle light scattering and thus prevents the background light from reaching the readout. Numerical confirmation of this behavior is shown in Figure 2, where a wide-angled light source was positioned outside the opti-
Figure 2. Numerical simulation on the propagation of light beams in the PhLoC. (A) Light confinement and interaction with the analyte in the measuring chamber. (B) Blockage of external background noise by the semi-random tooth-shaped air mirror.
cal path to simulate stray light interacting with the semi-random tooth-like outer shape of the air mirrors. As shown in the figure, light rays are either reflected or refracted, but no significant rays are collected in the output. Exchange of solution in the microfluidic structure. Our PhLoC features processing of solutions using an air bypass formed in parallel with respect to the measuring chamber. When immunoassays are conducted, solutions are exchanged several times. During this process, it is difficult to avoid intrusion of air into the measuring chamber. Denaturation of proteins has been reported to occur at water/air interfaces because of the hydrophobic nature of air.20,21 Additionally, drying by the air accelerates the denaturation and aggregation of proteins. To avoid this, the entrance of the air bypass was constricted to 20 µm at the first junction (T-junction 1 in Figure 1). Although the bottom glass surface was hydrophilic, the air bypass surrounded by hydrophobic PDMS walls was sufficiently hydrophobic overall, which effectively prevented solutions from entering. In addition, a 70µm-wide constriction was also formed in the connecting flow channel at the T-junction to avoid the intrusion of air into the flow channel connected to the measuring chamber. When solution columns separated by air pass through the junction, the air between
the columns is released through the air bypass, and the solutions are merged. By moving the new long column, the first solution (the front part of the column) is replaced with the second solution. Procedures. The inserted optical fibers were connected to a UV-VIS fiber light source (L7893; Hamamatsu Photonics, Japan) with a wavelength range from 200 to 1100 nm and a spectrometer (HR2000+; Ocean Optics, USA). The wavelength resolution of the spectrometer was 1.74 nm. Solutions were transported to the measuring chamber, and the absorbance measurement was then conducted. The solutions were moved manually using a pipette unless otherwise noted. The flow rate was approximately 0.8 µL/s. All measurements were conducted at room temperature. To measure the concentration of the oxidized form of TMB (TMB (Ox)) produced by the enzymatic reaction of HRP, the HRP enzyme was immobilized in the measuring chamber by physical adsorption. The hydrophobic PDMS walls of the measuring chamber were brought into contact with 5 µL of PBS containing 0–10 µg/mL HRP and 0.5% BSA for 1 h. Then, the measuring chamber was washed with 100 µL PBS, and the absorbance measurement was started immediately after introducing the 5 µL TMB (Red) solution into the measuring chamber. TMB changes its color as a result of the following reaction:22 HRP TMB (Red) + H2O2 → TMB (Ox) + 2H2O. The absorbance at 650 nm was measured. Absorbance for the reaction of TMB was measured in a light-shielding box to prevent light from entering the device. IL-2 was detected by ELISA with capture antibodies immobilized in the measuring chamber. The antibodies were immobilized on the hydrophobic PDMS surface of the measuring chamber by physical adsorption or electrostatic interaction with APTES. APTES tends to self-polymerize in an aqueous solution.23,24 This partially polymerized network provides a suitable scaffold by increasing the site of interaction for antibodies. The APTES network also increases the amino groups for electrostatic interaction with antibodies.25,26 To immobilize the capture antibodies, the 5 µL solution in the ELISA kit containing the antibodies and APTES (1% [v/v]) was introduced into the measuring chamber and was allowed to adsorb onto the PDMS walls and the glass bottom for 30 min. The surface was then blocked with PBS containing 0.5% BSA for 30 min. In standard ELISAs, analyte molecules, biotinylated detection antibodies, and avidin-HRP are added, and reactions are allowed to proceed in a step-wise manner. However, in our case, complexes consisting of IL-2, the detection antibody, and avidin-HRP mixed and then introduced into the measuring chamber and was then incubated for 15 min (see Figure S1) to form the complexes with the immobilized capture antibodies (Figure 1E). After flushing the solution, the measuring chamber was washed with 100 µL PBS. Finally, a 5 µL TMB (Red) solution was introduced. Immediately after the introduction of the solution, the absorbance measurement of TMB (Ox) was started. To investigate the influence of air on the activity of the immobilized capture antibodies, air was introduced into the measuring chamber at 1 µL/s using a syringe pump. Then, a mixture of IL-2 (500 pg/mL), detection antibodies, and Cy5-streptavidin (2.5 µg/mL) was introduced into the measuring chamber and incubated for 15 min. Finally, the flow channel was washed with 100 µL PBS, and the fluorescence signal of Cy5 was measured using a fluorescence microscope (IX-73; Olympus, Japan) with a filter unit (Cy5-4040C, Olympus) and CMOS camera (ORCA-Flash 4.0; Hamamatsu Photonics, Japan). To compare the amount of capture antibodies immobilized by the two methods, parallel flow channels were used. The width of
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the flow channels was the same as that of the measuring chamber. FITC-labeled anti-human IL-2 antibodies (2.5 µg/mL) were immobilized instead of capture antibodies, as described above, and the fluorescence was detected using the microscope with a filter unit (U-FBNA, Olympus). Measurement of the activity of lymphocytes separated from whole blood. Lymphocytes were separated from human whole blood samples obtained from healthy volunteers. For this purpose, a blood sample and PBS were mixed in a 1:1 volume ratio in a centrifuge tube. Then, the solution was carefully added to the blood-separating reagent (Lymphoprep) in another centrifuge tube. The merged but still separated solutions were centrifuged (800 × g for 20 min). Lymphocytes were separated as a clearly distinguished layer. Activation of lymphocytes started at the time of their cultivation in RPMI1640 medium in a humidified incubator at 37°C (95% air/5% CO2). The medium contained Con A (50 µg/mL) as an activator in addition to FBS (5%), HEPES (25 mM), MEM-Non-essential Amino Acids (1%), streptomycin (1%), sodium pyruvate (1 mM), and 2-mercaptoethanol (0.1%). The density of cultivated lymphocytes was 5 × 105 cells/mL. Sandwich ELISAs were performed with the supernatants of the medium using the PhLoC and a conventional 96-well plate following the manufacturer’s instructions. ■ RESULTS AND DISCUSSION Movement of solutions in the flow channel structure. Figure 3 shows the movement of solutions in the flow channel structure
Figure 3. Fluorescence photographs showing the movement of solutions in the flow channel structure. The yellow and white arrows indicate the movement of solutions and air, respectively. Flow rate: 5 µL/min. Scale bar: 1 mm. (A–C) The solution was moving toward the measuring chamber but did not enter the air bypass and instead passed through the measuring chamber. (D) The air that followed flowed into the air bypass, and the part of the solution that already passed the Tjunction 2 was separated. The other part of the solution remained in the measuring chamber. (E, F) The next solution flowed into the flow channel and merged with the previous solution. The air between them was released through the air bypass.
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with the air bypass. When the first solution was introduced into the structure (Figure 3A), it did not penetrate into the air bypass and moved only to the measuring chamber (Figure 3B). In other words, the 20-µm-wide constriction of the air bypass at T-junction 1 blocked the solution that passed the junction effectively. The solution moved forward in the measuring chamber and the connecting flow channel on the left (Figure 3C). When the end of the first solution column passed T-junction 1, the column stopped because pressurized air that followed was released through the air bypass. At the same time, the part of the solution column that extended to the lower stream from T-junction 2 was cut and flushed (Figure 3D). The part of the solution left in the measuring chamber was used for the absorbance measurement. After the measurement, the second solution column approached T-junction 1, releasing air between the two solution columns into the air bypass (Figure 3E) and finally merged with the first solution column that stopped in the measuring chamber (Figure 3F). The 70µm-wide constriction formed in the connecting flow channel at Tjunction 1 prevented the intrusion of air and connected the two solution columns smoothly. The merged solution column moved forward, and the first solution in the measuring chamber was replaced with the second solution. For solution columns that followed, the same steps could be repeated. With this structure, the air that separated two solution columns could be removed before entering a measuring chamber. In other words, the measuring chamber was always filled with a solution.
Figure 4. Movement of two solution columns of different colors and a long solution column formed by merging the two columns. The arrows indicate the movement of the front of the resorufin solution. (A) The fluorescein solution was placed in the measuring chamber, and the next resorufin solution was approaching. (B–D) The two solutions were merged. (E) The solution in the measuring chamber was completely replaced with the resorufin solution after 1 µL of the resorufin solution was introduced. (F) The resorufin solution occupied the measuring chamber completely. The flow rate of the solutions was 5 µL/min, and the time to exchange the solution in the measuring chamber was approximately 12 s at this flow rate.
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When conducting ELISAs in the PhLoC, several solutions containing various proteins passed T-junction 1. Although the entrance of the air bypass must have sufficient hydrophobicity to ensure the proper functioning of the air bypass, the surface state may change based on the adsorption of the proteins, and this may affect the transport of solution columns. To examine this, we introduced a row of 1-µL columns of PBS or BSA solution (1 mg/mL) into the measuring chamber. The solution column left in the measuring chamber was replaced with the following solution column one by one, as explained earlier. When only the PBS columns were introduced, penetration of the solutions into the air bypass was not observed for at least 20 exchanges of the columns. On the other hand, exchange of the BSA solution columns was limited to five times. In this case, air could not be released through the air bypass completely and was therefore able to enter the measuring chamber. This indicates that the adsorption of proteins contaminated the surface of the PDMS walls. In our case, a total of three protein solutions, namely the capture antibody solution, the blocking solution, and the sample solution, were introduced into the measuring chamber. Therefore, as long as a single measurement for a protein is conducted based on our procedure, the adsorption of proteins will not cause any serious problems. For the analysis of more than one protein, an array of the flow channel structure should be used. In our PhLoC, the solution in the measuring chamber was replaced with the following solution after they were merged. We were particularly concerned with the mixing of the components around the interface between the solutions. Figure 4 shows changes observed while the long column that formed by merging of two columns was moving. Here, a 1-µL fluorescein solution was first introduced into the measuring chamber, and a 1-µL resorufin solution column was merged with the first solution column. The new long column was moved at a rate of 5 µL/min. In systems with a pressure-driven flow, such as the system presented here, flow velocity is largest at the center of the flow, whereas parts near the flow channel walls are stagnant, as shown in Figure 4B– D. Nevertheless, the dye in the stagnant layer diffused gradually into the central part and was washed out by the flow that followed. As shown in Figure 4D, the front of the thin flow of resorufin solution at the center passed the air bypass, and the color in the measuring chamber indicated that the chamber was almost completely occupied by the resorufin solution. In the case of short liquid plugs, components mix rapidly with each other because of recirculating flows generated in the plug. However, the result shown in Figure 4 indicated that the flows in the column were similar to those in a continuous pressure-driven flow. Moreover, the result indicated that 1 µL of solution was sufficient for replacing the solution in the measuring chamber. Characterization of the PhLoC. UV-VIS light transmitted through the measuring chamber is shown in Figure 5A. The PDMS collimating lenses worked as expected, and the incident light was effectively introduced into the measuring chamber. The ability of the PhLoC to measure absorbance was checked with methyl orange solutions of different concentrations (Figure S2). An excellent linear relationship was observed between absorbance and the concentration of methyl orange (n = 10). The 3σ of the absorbance measurement of a blank PBS solution was 1.74 × 10-2. Based on this, the lower limit of detection was calculated to be 270 nM, which is in agreement with our previous results considering the difference in the optical path.27 Next, the enzymatic reaction of HRP was evaluated to estimate the length of time needed for the absorbance measurement. Figure 5B shows changes in the absorbance of TMB (Ox) produced in the enzymatic reaction. The change was accelerated with the increase in HRP concentration (Figure S3, left axis). The rate of absorbance change should be proportional to the enzyme activity,28 which was confirmed as shown in Figure S3 (right axis).
Figure 5. Characterization of the PhLoC for on-chip ELISA. (A) Propagation of incident UV-VIS light through the measuring chamber and emission of fluorescence from a fluorescein solution. (B) Time courses of absorbance of TMB (Ox) produced by the enzymatic reaction.
Based on the rate analysis, the measurement could be finished within 30 s. TMB (Ox) is unstable and denatures by deprotonation,29,30 and fading of the color begins after several minutes. This complication could be overcome using our protocol and PhLoC. Figure S4 compares changes in the absorbance measured in the flow channel and a cuvette. We hypothesized that the enzymatic reaction or color change would be accelerated in the PhLoC system owing to the high surface-to-volume ratio of its measuring chamber, and our data confirmed this hypothesis. Although the manufacturer recommends 30 min for the enzymatic reaction to proceed, comparable changes could be observed using the PhLoC system within several minutes, thus providing a major advantage as compared to the cuvette in terms of processing time. APTES-based immobilization of capture antibodies. Next, we examined the effects of APTES on the immobilization of capture antibodies. Figure S5 shows the time course of absorbance of TMB (Ox) produced in the reaction of HRP. To obtain sufficiently large changes in absorbance, 30 min was sufficient for the immobilization of the antibodies; this was significantly shorter than the times required in other conventional methods based on APTES and glutaraldehyde31 or 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride and Nhydroxysuccinimide32, which are often used for immobilization. Here, the curve obtained with physically adsorbed antibodies was also added. The rate of absorbance change for antibodies immobilized with APTES was more than double that observed with physical adsorption. This suggests that the electrostatic interaction between the carboxyl groups of the antibodies and the amino groups of APTES immobilized the capture antibodies more effectively. Immobilization of the antibodies was also confirmed by fluorescence measurement. As shown in Figure S6, the antibodies were immobilized, with greater immobilization observed for the APTES-based design. Notably, the solution for immobilization
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Figure 6. Dependence of the rate of absorbance change within 30 s on the concentration of IL-2. Red and black points show values obtained by the APTES-based immobilization (n = 5) and by physical adsorption (n = 4), respectively. Error bar means standard deviation. The lines are guides for the eyes.
remains in the measuring chamber only with the aid of the air bypass. The length of time for the solution to make contact with the walls of flow channels other than the measuring chamber during solution transport is very short compared with the length of time for incubation in the measuring chamber. Therefore, the capture antibodies were immobilized only in the measuring chamber, avoiding unnecessary loss of analyte during the transport of the solution. We then evaluated the influence of the APTES concentration in the capture antibody solution (Figure S7). The observed rate of absorbance change was highest with 1% APTES. Therefore, this concentration was used in the following experiments. Under these conditions, at least 25 min was required to obtain the highest absorbance (Figure S8). However, immobilization for longer times decreased the absorbance. Based on these results, we assumed that thicker layers may be formed with concentrated APTES and/or longer immobilization times;33 these layers may detach easily from the surface and limit the access of IL-2 to the binding site of the antibodies. Once the capture antibodies were immobilized, they were stabilized, and the rate of absorbance change maintained approximately the same level for at least 7 days (Figure S9). Detection of IL-2 by the on-chip ELISA. The detection of IL2 in standard solutions was implemented using the PhLoC by ELISA with capture antibodies immobilized in the measuring chamber. Figure 6 shows the relationship between the rate of absorbance change and the IL-2 concentration. The change within 30 s was sufficient for determining the rate of absorbance change. A linear relationship was observed for concentrations up to 1000 pg/mL. Figure 6 also shows the changes in absorbance for physically adsorbed capture antibodies. With the present simplified protocol, in which the sample and reagent solutions are mixed without sufficient washing after each step of reactions, the mixed solution may contain incomplete complexes, including complexes with IL-2 and the detection antibody and complexes with the detection antibody and the enzyme. The former may bind to the capture antibodies and decrease the number of available binding sites. The latter may adsorb onto the surface nonspecifically and may produce TMB (Ox) upon addition of the TMB (Red) solution. Nevertheless, the IL-2 concentration could be measured down to 50 pg/mL (3.2 pM) within 15 min from the introduction of the IL2 solution to the absorbance measurement. The detection limit was comparable to those achieved previously using an electro-
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Figure 7. Concentration of IL-2 secreted from lymphocytes stimulated with Con A for different times. The data were obtained using the same blood sample using the device and the conventional 96-well plate ELISA.
chemical device34 and nanoplasmonic device.35 On the other hand, the detection limit was higher than that achieved using a silicon photonic sensor and an amplification process comprising an enzymatic reaction product that precipitates on the sensor surface.36 Moreover, the detection limit was higher, by more than one order of magnitude, than that achieved by the conventional ELISA using the same reagents. However, we need to stress that our device is based on simple absorbance measurements that are generally used, where the incubation time is limited to 15 min, though there may still be room to improve the detection limit by further optimization. We also checked the influence of air on immobilized capture antibodies and the effects of the air bypass by fluorescence detection. Air was intentionally introduced into the measuring chamber, and the fluorescence intensity was compared with that without air intrusion (Figure S10). The result clearly showed that air intrusion into the measuring chamber influenced the activity of the immobilized antibodies and/or exerted shear stress and washed out the antibodies, demonstrating the effectiveness of the air bypass. Measurement of the activity of lymphocytes. The on-chip ELISA was implemented to measure the activity of lymphocytes separated from whole blood. Changes in the amount of IL-2 are closely related to lymphocyte activity. In this study, we measured changes in IL-2 concentrations after the activation of lymphocytes by Con A (Figure 7). Before activation, the secretion of IL-2 was not detected. However, a rapid increase in the concentration of IL2 was observed within 5 min due to the mitogen-induced reaction,37 and the concentration decreased over time. The values measured by the on-chip ELISA were consistent with those obtained by conventional ELISAs conducted in 96-well plates. The rapid increase in IL-2 concentration within 5 min suggests that the PhLoC system may be used to facilitate rapid diagnosis in clinical settings, even taking into consideration the length of time for the measurement of IL-2 (15 min). In this study, we separated culturing of the lymphocytes from detection of IL-2. Ideally, the entire procedure should be carried out on the same chip. To this end, our current studies are examining the influence of cell adhesion on the proper functioning of the air bypass and the influence of APTES on the activity of the lymphocytes. To avoid the latter, the culture chamber and measuring chamber may be separated. If the influences are minimized, onchip culturing and IL-2 detection will be realized.
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■ CONCLUSIONS In this study, we fabricated a PhLoC to detect IL-2 produced from lymphocytes. The components, including lenses, mirrors, and flow channels, could be formed monolithically with PDMS. The micro-optical components enabled effective, reproducible measurement of absorbance. The flow channel structure, including an air bypass, facilitated exchange of solutions in the measuring chamber without allowing air to enter. After merging the solution in the measuring chamber with another solution, the solution in the measuring chamber could be exchanged with the second solution. Capture antibodies could be immobilized only within the measuring chamber within 30 min by the APTES-based method. Exchange of solutions using the air bypass enabled selective immobilization of the capture antibodies and minimized the reduction of the capture antibody activity by avoiding intrusion of air into the measuring chamber and the adhesion of the capture antibody onto the surfaces of flow channels other than the measuring chamber. The concentration of IL-2 could be measured within 15 min for concentrations ranging from 50 to 1000 pg/mL. Changes in the concentrations of IL-2 secreted from lymphocytes following activation by Con A could be measured clearly. Therefore, we expect that the PhLoC could be applied as primary tests for drug allergies, autoimmune disorders, and immunosuppressive drugs.
■ ASSOCIATED CONTENT Supporting Information Supporting Information Available: The following file is available free of charge. Additional figures for detailed characterization of the PhLoC; optimization and stability of immobilized antibodies; effect of air intrusion on the microfluidic immunoassay; dependence on incubation time for the immunoassay.
■ AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected]. Tel: +81- 29-853-5598
Notes The authors declare no competing financial interest.
■ ACKNOWLEDGMENT This study was partially supported by Joint Research Projects from the Japan Society for the Promotion of Science (JSPS) and Spanish National Research Council and by Grants-in-Aid for Scientific Research (No. 25461941 and No. 25286034) under JSPS.
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