Anal. Chem. 2000, 72, 1144-1147
Integration of an Immunosorbent Assay System: Analysis of Secretory Human Immunoglobulin A on Polystyrene Beads in a Microchip Kiichi Sato,† Manabu Tokeshi,† Tamao Odake,† Hiroko Kimura,‡ Takeshi Ooi,§ Masayuki Nakao,§ and Takehiko Kitamori*,†,|
Integrated Chemistry Project, Kanagawa Academy of Science and Technology, Sakado, Takatsu, Kawasaki, Kanagawa 213-0021, Japan, Department of Forensic Medicine, School of Medicine, Juntendo University, Hongo, Bunkyo, Tokyo 113-8421, Japan, and Department of Engineering Synthesis and Department of Applied Chemistry, School of Engineering, The University of Tokyo, Hongo, Bunkyo, Tokyo 113-8656, Japan
An immunosorbent assay system was integrated into a glass microchip. Polystyrene beads were introduced into a microchannel, and then human secretory immunoglobulin A (s-IgA) adsorbed on the bead surface was reacted with colloidal gold conjugated anti-s-IgA antibody and detected by a thermal lens microscope. The scale merits of liquid microspace on the molecular behavior remarkably contributed to reduced assay time. The integration cut the time necessary for the antigen-antibody reaction by 1/90, thus shortening the overall analysis time from 24 h to less than 1 h. Moreover, troublesome operations required for conventional immunosorbent assays could be replaced by simple operations. Immunoassay is one of the most important analytical methods for clinical diagnoses and biochemical studies because of its extremely high specificity. The conventional immunosorbent assay, however, needs a long time and involves troublesome procedures and many expensive reagents. To overcome these drawbacks, integration of the immunoassay system seems to be effective. There have been some papers about microchip immunoassays, but most were based on separation of the free form and the complex of the antigen and the antibody by microchipbased capillary electrophoresis; there have been very few reports in which the antigen-antibody reaction was performed on a microchip.1-6 A heterogeneous immunosorbent assay is used widely and is superior to liquid-phase separation because of its easy and clear separation of the free form and the complex. * Corresponding author: (fax) +81-3-5841-6039; (e-mail)
[email protected]. † Kanagawa Academy of Science and Technology. ‡ Juntendo University. § Department of Engineering Synthesis, The University of Tokyo. | Department of Applied Chemistry, The University of Tokyo. (1) Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, 18-22. (2) von Heeren, F.; Verpoorte, E.; Manz, A.; Thormann, W. Anal. Chem. 1996, 68, 2044-2053. (3) Chiem, N.; Harrison, D. J. Anal. Chem. 1997, 69, 373-378. (4) Colyer, C. L.; Tang, T.; Chiem, N.; Harrison, D. J. Electrophoresis 1997, 18, 1733-1741. (5) Chiem, N. H.; Harrison, D. J. Electrophoresis 1998, 19, 3040-3044. (6) Chiem, N. H.; Harrison, D. J. Clin. Chem. 1998, 44, 591-598.
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Integration of the heterogeneous immunoassay system, which is based on the same principle as the conventional immunosorbent assay, has not been reported so far, however. For integrated analytical systems, a detection method with high sensitivity in microspace is indispensable. We have reported a new, highly sensitive immunoassay method on a bulk scale using photothermal spectrometry for detection, in which colloidal gold was used as a labeling material.7-10 Among some photothermal spectrometries, the laser-induced thermal lens microscope (TLM) is especially useful for ultrasensitive determination in microspace because of its high space resolution. In this paper, we report the integration of an immunosorbent assay system into a microchip using the TLM as a detector. Human secretory immunoglobulin A (s-IgA), which has an important role in local immunity and is known as a stress indicator, was assayed with this system. EXPERIMENTAL SECTION Principle. Schematic illustrations of our immunosorbent assay in a microchip using colloidal gold are shown in Figure 1. Polystyrene beads, introduced into a microchannel, were selected for the reaction solid phase. s-IgA (antigen) was adsorbed on the bead surface, and then the antibody to s-IgA, conjugated with colloidal gold, was fixed on the solid phase by antigen-antibody binding. After free antigen was washed out, colloidal gold bound to the bead surface via the antigen-antibody complex as detected by TLM. Microchip Fabrication. To dam up the reaction solid phase, i.e., the polystyrene beads in the microchip, a dam was fabricated in the microchannel. The chip layout is shown in Figure 2. The details of the microchip fabrication were described previously11 except for the dam region. The chip was composed of three quartz (7) Matsuzawa, S.; Kimura, H.; Tu, C. Y.; Kitamori, T.; Sawada, T. J. Immunol. Methods 1993, 161, 59-65. (8) Tu, C. Y.; Kitamori, T.; Sawada, T.; Kimura, H.; Matsuzawa, S. Anal. Chem. 1993, 65, 3631-3635. (9) Kimura, H.; Matsuzawa, S.; Tu, C. Y.; Kitamori, T.; Sawada, T. Anal. Chem. 1996, 68, 3063-3067. (10) Kimura, H.; Mukaida, M.; Kitamori, T.; Sawada, T. Anal. Sci. 1997, 13, 729-734. (11) Sato, K.; Tokeshi, M.; Kitamori, T.; Sawada, T. Anal. Sci. 1999, 15, 641645. 10.1021/ac991151r CCC: $19.00
© 2000 American Chemical Society Published on Web 02/10/2000
Figure 2. Layout of the glass microchip for immunosorbent assay. (a) An overview. (b) A cross section.
Figure 1. Schematic illustrations of integrated immunosorbent assay.
glass plates (30 mm × 70 mm), i.e., the cover, middle, and bottom plates with thicknesses of 170 µm, 100 µm, and 1 mm, respectively. Two access holes of 0.5-mm diameter for an inlet and an outlet were mechanically bored on the cover glass. A deep channel was made in the middle plate with a highly focused and intensified CO2 laser beam. The middle plate was attached to the bottom plate by using an optical contact; that is, the plates were polished to an optical smoothness and then laminated together without any adhesive in an oven at 1150 °C. A shallow channel of the dam region was fabricated by the fast atom beam fabrication method on the upper side of the laminated plates as reported previously.12 The fast atom beam fabrication is suitable for microfabrication below 10-µm scale for glass chip. The shallow channel was etched with SF6 beam accelerated at 5 kV, and the etching time was 8 h. The depth of the shallow channel was estimated as 10 µm, and the reaction solid phase, i.e., polystyrene beads (45 µm in (12) Toma, Y.; Hatakeyama, M.; Ichiki, K.; Huang, H.; Yamauchi, K.; Watanabe, K.; Kato, T. Jpn. J. Appl. Phys. 1997, 36, 7655-7659.
diameter), would be retained in the dam region. Finally, the cover plate was laminated on described above. General Reagents. Phosphate buffer (PB; 1/15 M, pH 7.4) was made from phosphate buffer powder (Wako Pure Chemical, Osaka, Japan) and ultrapure water, and the buffer was filtered with a 0.2-µm membrane filter prior to use. Polystyrene bead (45.6 µm in diameter with 14.5% CV) suspension (10% solid) was obtained from Duke Scientific (Palo Alto, CA). The beads were washed with PB prior to use. To prevent nonspecific binding to the polystyrene or glass surface, 0.2% casein (Merck, biochemistry grade) in PB was used as a blocking reagent. Antigen and Antibody-Colloidal Gold Conjugate. Chromatographically purified human s-IgA and goat antiserum to human s-IgA were purchased from Cappel (Durham, NC). The antibody concentration of the antiserum was 6 mg/mL, and the antiserum was used without further purification. Colloidal gold was used as a labeling material for the antibody. Colloidal gold has been used for photothermal immunoasssays,7-10 because it is stable to strong radiation and does not interfere with the immunological activity of the conjugated antibody. The monodispersed colloidal gold suspension was prepared as reported previously.13 Antibody-colloidal gold conjugate (AbCG) was prepared essentially by the reported method.14 In brief, 50 µL of the antiserum was added to 5 mL of the pH-adjusted gold suspension. After 30 min reaction at room temperature, the residual free colloidal gold was blocked by addition of 25 µL of 30% bovine serum albumin (BSA; 4 × crystallized, ICN) in PB, followed by overnight incubation at 4 °C. The conjugate was then centrifuged at 15000g for 15 min, and the supernatant was removed. The pellet obtained was resuspended with 2.5 mL of 0.5% BSA in PB, followed by 5-min centrifugation at 3000g to remove aggregated large particles. The resulting supernatant (2.2 mL) which has an absorbance maximum at 525 nm, can be stored at 4 °C for 1 week without loss of the activity. Apparatus. The TLM used was illustrated elsewhere.11 In brief, the TLM was composed of a microscope with two laser(13) Frens, G. Nature Phys. Sci. 1973, 241, 20-22. (14) Chakraborty, U. R.; Black, N.; Brooks, H. G., Jr.; Campbell, C.; Gluck, K.; Harmon, F.; Hollenbeck, L.; Lawler, S.; Levison, S.; Mochnal, D.; O’Leary, P.; Puzio, E. D.; Tien, W.; Venturini, A. Ann. Biol. Clin. (Paris) 1990, 48, 403-408.
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oscillation apparatuses and other optical devices. The excitation beam was the 514.5-nm emission line of an Ar+ laser (Lexel Laser, Fremont, CA, model 95-SHG) with output power of 200 mW, and its intensity was modulated by a mechanical chopper with a modulation frequency at 1.03 kHz. A He-Ne laser (Melles Griot, Carlsbad, CA, model 05LHP171, 15 mW) with an emission line of 632.8 nm was used for a probe beam. The two beams were made coaxial by a dichroic mirror and tightly focused by an objective lens (Nikon, CF IC EPI Plan 20×, NA 0.46). The transmitted beams were collected by a condenser lens and filtered. Only the probe beam intensity was monitored by a photodiode. The preamplified signal from the photodiode was synchronously amplified with a lock-in amplifier LI-575 (NF Corp, Yokohama, Japan). The liquid flow was controlled by microsyringe pumps (KD Scientific, Boston, MA) and Hamilton gastight syringes with an untreated fused-silica capillary tubing, miniature inert valves, and capillary column connectors (GL Science, Tokyo, Japan). The flow rate of the solution was adjusted to 20 µL/min for washing and solution exchanging or 2 µL/min for the antigen-antibody reaction and the surface blocking. For a conventional immunoassay, Nunc immunoplates (flat bottom) and a microplate reader (Wallac, 1420 ARVOSX) with a detection wavelength at 490 nm were used. For each reaction step, 50 µL of the solution was added to each well. Analytical Procedures. Fused-silica capillaries were connected to the inlet and outlet of the microchannel. The inlet capillary was then connected to the syringe via valves and/or connectors if necessary, and the outlet capillary was connected to the waste reservoir. The solution exchange could be achieved simply by switching the valve. After the inner wall of the capillary and the channel was blocked by the casein solution for 1 h, the solution was replaced by PB. Introduction of the beads into the microchannel was performed as follows. After the inlet capillary was disconnected, 0.5 µL of polystyrene bead suspension was dropped onto the inlet hole of the microchip. The capillary was reconnected to the inlet hole, and PB was pumped in to move the beads to the dam region. After the reactions, the solution in the microchannel was replaced by PB, and then colloidal gold adsorbed on the bead surface was monitored by the TLM. For the measurement, five beads were randomly selected per microchannel and the laser beam was focused on the top of each bead. After the measurement, the beads were removed from the microchip by a reverse flow of PB. Thus, the microchip can be used repeatedly. Adsorption of s-IgA onto the beads was also carried out without the microchip. The polystyrene beads (50 µL of suspension) were washed with PB and suspended with 0.5 mL of the antigen solution in a 1.5-mL microtube. The suspension was stirred gently at room temperature for 1 h, followed by overnight incubation at 4 °C. The beads were washed and blocked with the casein solution for 1 h at room temperature. The resulting beads were washed with PB and stored at 4 °C. RESULTS AND DISCUSSION Antigen-Antibody Reaction. The time course of the antigenantibody reaction was examined. The polystyrene beads preadsorbed with s-IgA (10 µg/mL) were introduced into the microchip. AbCG solution was injected into the microchannel at a flow rate 1146 Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
Figure 3. Time course of antigen-antibody reaction. (a) Conventional microtiter plate assay. (b) Integrated immunoassay.
Figure 4. Schematic illustrations of the antigen-antibody reaction. (a) Microtiter plate. (b) Microchip.
of 2 µL/min for the antigen-antibody reaction. A comparison of the immunoreaction rate between the integrated immunoassay and the conventional assay with a microtiter plate is shown in Figure 3. In the conventional immunoassay using a microtiter plate, the immunoreaction was nearly completed after 15 h. In this assay, the antigen was adsorbed on the solid surface, and the antigenantibody reaction occurred only at the surface. Therefore the reaction efficiency was very poor, and it took a long time to complete the reaction with the conventional assay. On the other hand, the signal intensity became constant after a 10-min reaction in the microchannel. These results indicated that the integration of the immunoassay shortens the reaction time by one-ninetieth. This effect seemed to be brought about by an increase in the specific interface and reduction of the diffusion distance (Figure 4). Specific interface (S/V) is defined as the S-to-V ratio, where S and V are the surface area of the reaction solid and the solution volume, respectively. An increase in S/V means an increase in the reaction field. The S/V of 50-µL solution in the microtiter plate well (0.65 mm in diameter) was estimated to be 13 cm-1, whereas that of the microchannel (11 beads in 100 µm × 100 µm × 200
Figure 5. Time course of the antigen adsorption onto the bead surface.
µm channel space) was 480 cm-1. Therefore the S/V of the microchannel was 37 times larger than that of the microtiter plate, and the reaction rate may be increased by this larger reaction field. In case of the conventional microtiter plate assay, a 1.5-mm movement would be necessary for the most distantly located antibody molecule to react with the antigen fixed on the surface of the well, since the liquid depth was 1.5 mm. On the other hand, the liquid phase of the microchannel filled with polystyrene beads was much smaller. The longest distance from an antibody molecule to the reaction-solid surface may be less than 20 µm. Diffusion time is proportional to the squares of the diffusion distance, so that the diffusion time of the antibody molecule to the antigen in the microchip would be more than 5600 times shorter than the conventional method. From these results, we decided that the reaction time for the antigen-antibody reaction was 10 min. For a liquid-phase immunoassay, the antigen-antibody reaction takes ∼10 min at room temperature. Therefore, these results indicate that the integration of the heterogeneous immunosorbent assay brings a reduction of the reaction time to the same level as a homogeneous liquidphase assay, and a weak point of the heterogeneous immunosorbent assay, i.e., long analysis time, can be overcome. Antigen Adsorption. The time course of the amount of the antigen adsorbed on the bead surface was examined. After introduction of the polystyrene beads, s-IgA solution (10 µg/mL) was introduced into the microchannel, and liquid flow was stopped to allow adsorption of the antigen onto the surface of the beads. After incubation, s-IgA solution was washed out with PB, and then the residue-free surface of the polystyrene beads was blocked by a 10-min flow of casein solution, followed by a 1-min flush of PB. After blocking, AbCG solution was introduced into the microchannel, and then the antigen-antibody reaction was performed for 10 min. The time course of the signal intensity is shown in Figure 5. The antigen adsorption was almost finished in 20 min. For quantitative immunoassay, we concluded that a 20-min adsorption time was sufficient for the antigen adsorption at room temperature. Immunoassay in the Microchip. All of the immunosorbent assay procedures, including adsorption of a sample, blocking,
Figure 6. Calibration curves of human secretory immunoglobulin A. Solid square: the microchip assay (left scale). Open circle: the conventional assay (right scale).
antigen-antibody reaction, washing, and detection. were performed in the microchip. The times for s-IgA adsorption, blocking by casein solution, antigen-antibody reaction, and washing by PB were decided to be 20, 10, 10, and 1 min, respectively. Therefore, the total analysis time was less than 1 h. The analysis time for the conventional method, which was performed with a microtiter plate as a reference experiment, was almost 24 h. Thus, the time necessary was reduced by about one twenty-fourth by integration of the assay. The calibration curve for human s-IgA by microchip immunosorbent assay is shown in Figure 6. The sample containing 1 µg/mL s-IgA showed an obvious TLM signal, whereas this sample showed only the same absorbance as the control by means of the conventional immunoassay. Since the concentration of s-IgA in human saliva is normally around 200 µg/mL, this integrated system is expected to be suitable for practical measurements. These results indicated that the integrated immunosorbent assay in the microchip with a much shorter analytical time has better sensitivity than that of the conventional method. In the integrated assay, simple operations, i.e., pumping with syringe pumps and switching of the valves, take the place of troublesome liquid-handling procedures, i.e., numerous washing and solution removal procedures, which are necessary for the conventional assay. Moreover, consumption of the reagents can be reduced by the integrated assay. We concluded that this integrated immunosorbent assay system will be useful to be put into practical use including an automated system in future, because of its short analysis time, easy procedures, and low cost. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for University and Society Collaboration (11794006) from the Ministry of Education, Science, Sports and Culture of Japan.
Received for review October 5, 1999. Accepted January 3, 2000. AC991151R Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
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