Online Preconcentration by Transient Isotachophoresis in Linear

Apr 17, 2007 - concentration on standard cross-channel microchips made of poly (methyl methacrylate). Sample injection, precon- centration, and separa...
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Anal. Chem. 2007, 79, 3667-3672

Online Preconcentration by Transient Isotachophoresis in Linear Polymer on a Poly(methyl methacrylate) Microchip for Separation of Human Serum Albumin Immunoassay Mixtures Mohamad Reza Mohamadi,*,†,‡ Noritada Kaji,†,§ Manabu Tokeshi,†,§ and Yoshinobu Baba†,§,⊥,|

Department of Applied Chemistry, Graduate School of Engineering, MEXT Innovative Research Center for Preventive Medical Engineering, and Plasma Nanotechnology Research Center, Nagoya University, Nagoya 464-8603, Japan, Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu 761-0395, Japan, and Department of Molecular Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima 770-8505, Japan

Online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody by on-chip transient isotachophoresis is reported. An 800fold signal enhancement was achieved following the preconcentration on standard cross-channel microchips made of poly (methyl methacrylate). Sample injection, preconcentration, and separation were done continuously and controlled solely by a sequential voltage switching program. The preconcentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution. The method was applied to microchip electrophoresis immunoassay of HSA. Baseline separation of HSA and its immunocomplex was achieved in 25 s in the first 1 cm of the microchannel. In a direct immunoassay, the minimum detectable concentration of fluorescent labeled HSA by laser-induced fluorescence detection was 7.5 pM. Microchip electrophoresis-based immunoassay (MCEIA) is a developing technology in which microchip electrophoresis (MCE) is used to separate an immunocomplex from a free antibody or antigen. Compared to conventional immunoassay, MCEIA possesses a number of advantages such as lower sample consumption, simpler procedure, and shorter analysis time. However, some drawbacks have hindered application of MCEIA in practical assays of protein samples. In most reported MCEIAs, free solution electrophoresis has been applied to separate small antigens with molecular weight of less than 6000 from their immunocomplexes.1-9 Generally the difference in charge-to-mass ratio of a protein and * Corresponding author. Phone, fax: 81-52-789-4666. E-mail: rezam@ mail.apchem.nagoya-u.ac.jp. † Department of Applied Chemistry, Graduate School of Engineering, Nagoya University. ‡ The University of Tokushima. § MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University. | Plasma Nanotechnology Research Center, Nagoya University. ⊥ National Institute of Advanced Industrial Science and Technology (AIST). (1) Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, 18-22. 10.1021/ac0623890 CCC: $37.00 Published on Web 04/17/2007

© 2007 American Chemical Society

its immunocomplex is not enough to achieve baseline resolution using free solution electrophoresis in a short microchannel.10 Therefore, the reported applications of free solution electrophoresis in MCEIA of protein samples, unlike for small molecules, suffer from low resolution and low reproducibility.11-18 The low resolution and reproducibility will, in turn, raise the detection limit (DL) in MCEIA.19 To improve the resolution and increase the reproducibility of separation of proteins from their immunocomplexes, different strategies have been tried.20 Gel electrophoresis under a nondenaturing condition, which does not affect the interaction between protein and antibody, has been reported for MCEIA.21 These authors applied photopolymerized cross-linked polyacrylamide gel on a glass microchip for separation of tetanus neurotoxin from (2) Schmalzing, D.; Koutny, L. B.; Taylor, T. A.; Nashabeh, W.; Fuchs, M. J. Chromatogr., B 1997, 697, 175-80. (3) Chiem, N.; Harrison, D. J. Anal. Chem. 1997, 69, 373-8. (4) Chiem, N. H.; Harrison, D. J. Clin. Chem. 1998, 44, 591-598. (5) Cheng, S. B.; Skinner, C. D.; Taylor, J.; Attiya, S.; Lee, W. E.; Picelli, G.; Harrison, D. J. Anal. Chem. 2001, 73, 1472-1479. (6) Tao, L.; Kennedy, R. T. Anal. Chem. 1996, 68, 3899-3906. (7) Roper, M. G.; Shackman, J. G.; Dahlgren, G. M.; Kennedy, R. T. Anal. Chem. 2003, 75, 4711-7. (8) Bromberg, A.; Mathies, R. A. Electrophoresis 2004, 25, 1895-1900. (9) Bromberg, A.; Mathies, R. A. Anal. Chem. 2003, 75, 1188-95. (10) Yang, W. C.; Schmerr, M. J.; Jackman, R.; Bodemer, W.; Yeung, E. S. Anal. Chem. 2005, 77, 4489-4494. (11) Harrison, D. J.; Fluri, K.; Chiem, N.; Tang, T.; Fan, Z. Sens. Actuators, B 1996, 33, 105-109. (12) Chiem, N.; Harrison, D. J. Electrophoresis 1998, 19, 3040-4. (13) Qu, J. P.; Wang, Q. G.; Cheung, T. M.; Chan, S. T. H.; Yeung, W. S. B. J. Chromatogr., B 1999, 727, 63-71. (14) Qiu, C. X.; Harrison, D. J. Electrophoresis 2001, 22, 3949-3958. (15) Lin, C. H.; Lee, G. B.; Lin, Y. H.; Chang, G. L. J. Micromech. Microeng. 2001, 11, 726-732. (16) Chen, S. H.; Lin, Y. H.; Wang, L. Y.; Lin, C. C.; Lee, G. B. Anal. Chem. 2002, 74, 5146-53. (17) Martynova, L.; Locascio, L. E.; Gaitain, M.; Kramer, G. W.; Christensen, R. G.; MacCrehan, W. A. Anal. Chem. 1997, 69, 4783-4789. (18) Jiang, G.; Attiya, S.; Ocvirk, G.; Lee, W. E.; Harrison, D. J. Biosens. Bioelectron. 2000, 14, 861-9. (19) Taylor, J.; Picelli, G.; Harrison, D. J. Electrophoresis 2001, 22, 3699-3708. (20) Kawabata, T.; Watanabe, M.; Nakamura, K.; Satomura, S. Anal. Chem. 2005, 77, 5579-5582.

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its immunocomplex. Although the two peaks for antigen and the immunocomplex were resolved, the peak for the immunocomplex was still broadened. Recently we developed a nondenaturing gel electrophoresis of protein samples using methylcellulose as linear polymer in microchips made of poly(methyl methacrylate) (PMMA).22 We applied the nondenaturing gel electrophoresis for separation of human serum albumin (HSA) from its immunocomplex. Although the separation efficiency was comparable with the previous report using cross-linked polyacrylamide gel,21 the detection sensitivity was not exceptional and the peak for the immunocomplex was much broadened. Therefore, to improve the sensitivity of the method, we decided to develop a preconcentration and stacking method that is appropriate for application in the MCEIA. Many methods have been developed for protein preconcentration on microchips; some of these methods have required special nanostructures or long times for preconcentration and others needed special chip designs.23,24 These approaches are either not applicable on a PMMA microchip because of the limits in fabrication or are not amenable to the interactions between antigen and antibody in the immunoassay due to the long time for preconcentration. Electromigration-based sample stacking processes such as electrokinetic suppercharging and isotachophoresis (ITP) also have been applied for electrophoresis of DNA, amino acids, and SDS-denatured protein samples.25-30 But the previous reported electrokinetic supercharging for preconcentration of SDS-denatured protein samples in a single channel on a quartz microchip required the working voltage be stopped and the sample reservoir be cleaned, which is time-consuming and complicated.25 Fully automated ITP stacking and gel electrophoresis of SDS-denatured protein samples was reported26 in which the preconcentration was done in a special coupled channel on a glass microchip, and 40-fold signal enhancement was achieved. More recently, a new on-chip transient ITP method for preconcentration of fluorescent tracer on a standard cross-channel glass microchip has been reported.31 Despite the high efficiency, the method was not fully automated and it required vacuum application for sample injection and a pause between the sample injection and ITP. These requirements will lower the reproducibility, which is not favored for applications in clinical analysis and immunoassays. In the current work, we report a fully automated preconcentration and stacking method based on transient ITP on a standard cross-channel microchip made of PMMA. Sample injection, (21) Herr, A. E.; Throckmorton, D. J.; Davenport, A. A.; Singh, A. K. Anal. Chem. 2005, 77, 585-590. (22) Mohamadi, M. R.; Mahmoudian, L.; Kaji, N.; Tokeshi, M.; Baba, Y. Electrophoresis 2007, 28, 830-836. (23) Foote, R. S.; Khandurina, J.; Jacobson, S. C.; Ramsey, J. M Anal. Chem. 2005, 77, 57-63. (24) Wang, Y. C.; Stevens, A. L.; Han, J. Anal. Chem. 2005, 77, 4293-4299. (25) Xu, Z.; Ando, T.; Nishine, T.; Arai, A.; Hirokawa, T. Electrophoresis 2003, 24, 3821-3827. (26) Huang, H.; Dai, Z.; Xu, F.; Lin, B. Electrophoresis 2005, 26, 2254-2260. (27) Xu, Z. Q.; Hirokawa, T. Electrophoresis 2004, 25, 2357-2362. (28) Xu, Z. Q.; Hirokawa, T.; Nishine, T.; Arai, A. J. Chromatogr., A 2003, 990, 53-61. (29) Grass, B.; Hergenroeder, R.; Neyer, A.; Siepe, D. J. Sep. Sci. 2002, 25, 135-140. (30) Gong, M.; Wehmeyer, K. R.; Limbach, P. A.; Arias, F.; Heineman, W. R. Anal. Chem. 2006, 78, 3730-3737. (31) Jung, B.; Bharadwaj, R.; Santiago, J. G. Anal. Chem. 2006, 78, 2319-2327.

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preconcentration, and separation are done continuously and are controlled solely by a sequential voltage switching program. The method was applied to preconcentration of HSA and its immunocomplex with a monoclonal antibody. The ITP stacking was followed by on-chip nondenaturing gel electrophoresis, which successfully resolved the free HSA from its immunocomplex. We also applied the method for direct and competitive immunoassay of HSA and a monoclonal antibody anti-HSA (mAb anti-HSA) on the PMMA microchip. EXPERIMENTAL SECTION Reagents, Polymer, and Sample Preparation. HSA and methylcellulose (MC-4000) were purchased from Sigma Chemical Co. (St. Louis, MO). The methylcellulose solutions were prepared in Tris-HCl (20 mM pH 8.3) according to the method suggested by the manufacturer. HSA was labeled by Alexa Fluor-647 (Molecular Probes, Carlsbad, CA) according to the manufacturer’s instructions. Protein concentration and dye ratio were calculated using a NanoDrop ND-1000 UV-visible spectrophotometer from NanoDrop Technologies Inc. (Wilmington, DE). Fluorescein isocyanate (FITC)-labeled human serum albumin was purchased from Sigma (St. Louis, MO). FITC-HSA was used for microscopy imaging and online monitoring of separation and detection at any desired distance. HSA labeled with Alexa Fluor 647 was used for immunoassays and reproducibility tests with the microchip electrophoresis system Hitachi SV1210 (Tokyo, Japan). Microchip Electrophoresis. A plastic microchip made of PMMA, (i-chip 3 DNA, Hitachi Co.) was used in this research. The channel cross section was 100 µm (width) by 30 µm (depth), and effective separation length was set at 30 mm. The distances from the intersection of microchannels to the buffer reservoir (BR), buffer waste reservoir (BW), sample reservoir (SR), and sample waste reservoir (SW) were 5.7, 37.5, 5.2, and 5.2 mm, respectively (Figure 1-a). An inverted fluorescence microscope, Axivert 135TV, equipped with a 10/0.3 NA objective lens (both from Carl Zeiss, Tokyo, Japan) was used to view the protein separations. Illumination for viewing was done by a 100-W mercury arc lamp (Carl Zeiss). A HVS448 3000V (LabSmith, Livermore, CA), power supply, which was controlled by a PC, has been used for MCE. A CCD camera, (EB-CCD, Hamamatsu Photonics), was used to capture the separation process. The captured photos were analyzed by image processing software (Cosmos32, Library Inc., Tokyo, Japan). The electropherogram was simulated for each separation from acquired data. After conditioning the microchip, electrophoresis was done on the Hitachi microchip electrophoresis system SV 1210 equipped with laser-induced fluorescence (LIF) detection. Control MCE. For the control MCE, the immunoassay mixture of HSA and its immunocomplex was separated in 1% methylcellulose solution in Tris-HCl 20 mM (pH 8.3) plus 0.01% Tween-20. During injection for 45 s, the SW reservoir was maintained at 450 V and the other reservoirs were grounded. During separation, the SW reservoir and SR were set at 350 V, the BR was grounded, and the BW reservoir was set at 1450 V. Online Preconcentration and ITP. For the sample preconcentration, the BW, SW, and all the microchannels were filled with a desired concentration of methylcellulose in the leading buffer (LE) (Figure 1a). The BR was filled with the same

Figure 1. Schematic of the preconcentration followed by ITP in the separation channel. Part a shows the chip layout. The BW, SW, and all the channels were filled with a desired concentration of methylcellulose in the LE. The BR was filled with the same concentration of methylcellulose as used in the microchannels prepared in the TE. The sample was loaded into the SR. Parts b-f show the channel intersection during the sample injection and preconcentration. Part b shows the sample flow before arrival of the TE zone at the intersection. Part c shows the intersection when the TE zone faced the sample flow. Part d shows the intersection after switching the voltage to the separation step. Part e shows formation of the sample plug at the beginning of the separation channel. Part f shows the stacked protein sample zones, which resulted from the transient ITP. Parts d-f show autonomous phenomena, which happened sequentially without any stop in less than 1.5 s. E1, E2, and E3 show the electric field strength in TE, sample, and LE zone, respectively. E1 in part b has increased in part c due to replacement of LE with TE in this zone. The applied voltages are the optimized voltages used for all the following experiments.

concentration of methylcellulose as used in the microchannels prepared in the terminating buffer (TE). The sample was loaded into the SR. The sample was electrokinetically injected by applying +450 V at the SW for 45 s; all the other reservoirs were kept grounded during the injection. During the separation, the SW and SR were set at +350 V, the BR was grounded, and BW was set at +1450 V (Figure 1a). LE and TE Buffer. Methyl cellulose solution with different concentrations was prepared in the LE and TE buffer. In a single experiment, a similar concentration of methylcellulose was used in both LE and TE zones. The LE buffer in all ITP experiments (except for those mentioned in the figure’s caption) was Tris 20 mM and pH was adjusted by HCl (pH 8.3). In some experiments, which have been mentioned in the related figure’s caption, the LE buffer included 5 or 10 mM NaCl. The TE in all ITP experiments was 192 mM Gly, Tris 25 mM, (pH 8.3). All the polymer solutions included 0.01% Tween-20 to decrease protein adsorption onto the microchannel walls. Immunoassays. All the immunoassays were done off-chip in the same buffer (Tris-HCl 20 mM pH 8.3 including 0.01% Tween20). For the direct quantitation of labeled HSA, a constant concentration of mAb anti-HSA (20 nM) was added to serial dilutions of Alexa Fluor-647-labeled HSA (Alexa-HSA), and the mixtures for direct immunoassay were incubated for 1 h at room temperature while shielded from light. For affinity tests of mAb anti-HSA, a constant concentration of Alex-HSA (15 nM) was added to serial dilutions of the antibody, and the mixtures were incubated for 1 h at room temperature while shielded from light.

For the competitive immunoassay of unlabeled HSA, a constant concentration of Alexa-HSA was added to serial dilutions of unlabeled HSA sample. A constant concentration of mAb anti-HSA was added to the all competitive solutions. Samples were mixed and incubated at room temperature for at least 20 min while shielded from light. Different concentrations of labeled HSA (500, 250, 125, 62.5 ng/mL) and different molar ratios (16, 8, 4, 2, 1) of monoclonal antibody to HSA were tried in a range of incubation times (20, 40, 60, 90 min). RESULTS AND DISCUSSION Principle of the Preconcentration and Stacking Method. Figure 1 shows the principle of preconcentration and stacking we used in the current work. When the injection voltage was applied (+450 V at SW), the sample was electrokinetically moved from the SR toward the SW and it arrived at the intersection of the channels after 10 s (Figure 1b). A few seconds (∼3 s) later, the TE zone, which included low mobility ion (Gly-), arrived from the BR at the intersection of the channels (Figure 1c). The sample plug was forced into the separation channel, which, during the rest of the sample injection, led to the accumulation of the sample at the beginning of the separation channel (cf. Figure 2c). This force may originate from a repulsion force by low mobility co-ion or be caused by different electric field strength in the separation and injection channels. After 45 s, when the injection step was switched to the separation step, the TE zone moved toward the separation channel and swept the sample plug into the separation channel (Figure 1d). Due to the low mobility of Gly- compared to the protein sample, a short time lag happened between steps Analytical Chemistry, Vol. 79, No. 10, May 15, 2007

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Figure 2. Fluorescent images of the intersections of microchannels during the sample injection and sample stacking during first 3 s of separation step. Images a and b show the sample flow before and after arrival of the high ionic zone to the intersection of the microchannels. By switching the voltage to the separation step, images c-i were taken sequentially with a time interval of 240 ms. The arrow in part c indicates the accumulation of the sample at the beginning of the separation channel. The arrows in image g show two protein zones, which are separating and stacking. The sample was FITCHSA and its immunocomplex and the chip layout, buffer positions, and applied voltages were similar to those shown in Figure 1. The sample was immunoassay mixture of FITC-HSA 2.5 µg/mL and mAbanti HSA 5 µg/mL. The immune reaction was done by incubation at romm temperature for 25 min. The ITP on microchip was done in 1% of methylcellulose and the LE included 5 mM NaCl.

d and e (Figure 1). During this lag, the sample continuously entered the separation channel and a big sample plug was formed (Figure 1e). At the beginning of the separation step, the sample plug, which was captured between TE and LE, was stacked because of the transient ITP effect (Figure 1f). Figure 2 shows a series of images from sample injection and the ITP afterward. The last seven images (Figures 2c-i) were taken sequentially with a time interval of 240 ms, and they showed that the stacking process happened in less than 1.5 s. During the preconcentration, the immunocomplex and HSA were stacked (Figure 2g). The two sample zones were further resolved by sizebased separation in the methylcellulose solution. Figure 3 shows the electropherograms of separation of HSA from its immunocomplex in normal MCE (Figure 3a) compared to the presented method. The results in Figure 3b showed that the intensities of the peaks for HSA and the immunocomplex were respectively enhanced by 40- and 270-fold. The analysis of peak areas showed that the peak area of both peaks were similarly increased by 30 times, which means the injected amount of both HSA and the immunocomplex after the ITP was similarly increased; but as is obvious, the peak efficiency for two peaks before and after the ITP were different. The immunocomplex (peak 2) is adjacent to the TE zone, which includes a high concentration of Gly (192 mM) and therefore showed higher 3670 Analytical Chemistry, Vol. 79, No. 10, May 15, 2007

Figure 3. Comparison between two electropherograms of separation of HSA (peak 1) from its immunocomplex (peak 2). The sample was HSA 2 µg/mL and mAb-anti HSA 5 µg/mL (molar ratio of mAb/ HSA 1:1). The immune reaction was done by incubation at room temperature for 25 min. Part a shows the electropherogram in the control MCE in 1% methylcellulose solution in Tris-HCl 20 mM (pH 8.3) including 0.01% Tween-20. Part b shows the result after applying preconcentration by ITP to the same sample as in part a. The ITP was done in 1% of methylcellulose solution. The LE did not include NaCl. In both MCE and on-chip ITP during injection for 45 s, the SW reservoir was maintained at 450 V and the other reservoirs were grounded. During separation, the SW reservoir and SR were set at 350 V, the BR was grounded, and the BW reservoir was set at 1450 V.

stacking efficiency compared to HSA (peak 1), which is adjacent to the LE zone (Tris-HCl 20 mM). By increasing the concentration of Cl- in the LE the peak area of both immunocomplex and HSA was increased (see the Supporting Information), which led to further enhancement in the signal intensity (Figure 4a). By adding 5 mM NaCl in the LE, the signals for HSA and the immunocomplex were respectively enhanced by 70- and 675-fold. And finally, by adding 10 mM NaCl, the signals for HSA and the immunocomplex were respectively enhanced by nearly 100- and 800-fold. The signal intensity enhancement by increasing chloride ion in the LE buffer was due to the field-amplified sample stacking effect in which increasing the ion concentration in background electrolyte enhanced the peak intensity of samples in capillary electrophoresis. Adding Cl- ion also changed the migration time of HSA and the immunocomplex. To explain the changes in the migration times of the two peaks, we must return to the theory of ITP in a constant electric field mode. Increasing the concentration of Cl- in the leading zone decreases the electric field in this zone. According to eq 1, velocity (V) or migration time (t) of an ion species in an applied electric field (E) is correlated with electrophoretic mobility (µ) of the ion.

Figure 5. Separation of a FITC-HSA sample (peak 1) from its immunocomplex (peak 2) at 1 cm from the intersection of the microchannels. The sample was FITC-HSA 3 µg/mL and mAb-anti HSA 5 µg/mL. The immune reactions were done by incubation at room temperature for 25 min. The on-chip ITP was done in 1% methylcellulose solution and 5 mM NaCl was added to the LE buffer.

means lower resistance in this zone, hence

RLE+NaCl < RLE

therefore I 2 > I1

Figure 4. (a) Effect of concentration of chloride ion in the leading buffer on peak intensities and migration times of HSA (peak 1) and its immunocomplex (peak 2). The on-chip ITP was done in 1% methylcellulose. The quantitative analysis of peak area is shown in Supporting Infromation. (b) shows the preconcentration in different concentrations of methylcellulose. The LE did not include NaCl. The sample was Alexa-HSA 3 µg/mL and mAb-anti HSA 5 µg/mL. The immune reactions were done by incubation at room temperature for 25 min.

V ) µE

or

µ)

l l wt) tE µE and E in leading zone ∝ 1/[Cl-] (1)

From eq 1 we concluded that, by increasing Cl- in the LE, the LE zone moved slower and so did the HSA peak, which was moving adjacent to the LE zone. Since the total electric field did not change during the electrophoresis, the lower electric field in the LE zone was balanced with a higher electric field in the TE zone. To explain this balance in the electric field, we should refer to the Ohm’s law for a resistor in series. If we consider each zone in ITP as a resistor so before adding NaCl to the LE buffer

V ) I1(RTE + RSample + RLE)

and

VTE-1 ) I1RTE

and after adding NaCl to the LE buffer by applying the same voltage

V ) I2(RTE + RSample + RLE+NaCl)

and

VTE-2 ) I2RTE

Adding NaCl increases the conductivity in the LE zone, which

and

VTE-2 > VTE-1

In other words, the local potential (or the electric field strength) in the TE zone in the presence of NaCl in the LE zone (VTE-2) is higher than the local potential when the LE does not include NaCl (VTE-1). Therefore, after adding NaCl to the LE buffer, the TE zone and the immunocomplex peak that moved at the front of the TE zone moved faster. The overall result of this phenomenon was that, by increasing the concentration of Cl- in the LE, the two peaks of HSA and its immunocomplex were less resolved. One of the key points to achieve the above-mentioned transient ITP and preconcentration was to suppress the electroosmotic flow (EOF). If the EOF was not suppressed, when the voltage was switched to the separation step in Figure 1d, a strong EOF opposed the mobility of the TE zone and prevented the formation of the transient ITP in Figure 1e. This would cause a continuous leaking of the sample flow to the separation channel. Therefore, the EOF in the microchannels was suppressed by using methylcellulose solutions at concentrations higher than the entanglement point (0.2%). Using a too low concentration of methylcellulose, which did not properly suppress the EOF, caused leakage of the sample to the separation channel (Figure 4). Increasing the concentration of the methylcellulose also led to further resolution of the peaks for HSA and the immunocomplex (Figure 4). Monitoring the separation channel by fluorescence microscopy showed that the separation of the HSA from its immunocomplex was completed in the first few millimeters of the microchannels. Figure 5 shows the separation of FITC-HSA from its immunocomplex at 1 cm from the intersection of the microchannels in 25 s. However, for ease of handling and to get higher reproducibility in the later immunoassays, we did the detection using the Hitachi MCE 1210 system, in which the detection point was fixed at 3 cm from the intersection of the microchannels. Direct MCEIA of Labeled HSA and a mAb-Anti HSA. To evaluate the performance of the preconcentration in biological assays, we applied the method to a direct immunoassay of a HSA Analytical Chemistry, Vol. 79, No. 10, May 15, 2007

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Figure 6. (a) Direct immunoassay of Alexa-HSA. mAb anti-HSA, 15 nM, was added to solutions of Alexa-HSA of varying concentration. The peak area for the highest concentration of Alexa-HSA has been considered as 1, and the other peak areas were normalized by dividing to this peak area. The mixtures were incubated for 60 min while shielded from light. (b) Direct immunoassay of a mAb anti-HSA and the simulated dose-response curve. Peak areas of Alexa-HSA and the immunocomplex was used to calculate the ratio of bounded antigen to the total antigen. The error bars show the standard deviations (n ) 4).

sample labeled with Alexa Fluor-647. A constant concentration of mAb anti-HSA was added to serial dilutions of an Alexa-HSA sample. Using the peak areas of HSA and the immunocomplex, we reproduced a saturation curve. Figure 6a shows a linear correlation between concentration of Alexa-HSA and peak area of the immunocomplex from 7.5 pM up to 250 pM. We also characterized binding affinity of an anti-HSA monoclonal antibody by using the current method (Figure 6b). Quantitation of peak area was used to produce a dose-response curve for the monoclonal antibody anti-HSA. Due to the baseline separation of the two peaks and the highly stacked peak for the immunocomplex the ratio of bound-to-total antigen in the immunoassays was easily calculated using the peak areas of HSA and the immunocomplex. Our result showed that, compared to the previous report in which the immunocomplex peak was normalized by peak height of a free dye,21 the current way of normalization led to a smaller relative standard deviation in the normalized peak areas (data not shown) and it was also unnecessary to use an internal standard.

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The data in Figure 6b were fitted using a four-parameter logistic model, and from this, the Kd of the monoclonal antibody and the labeled HSA was calculated as 211 ( 33 nM. Therefore, the affinity constant of the antibody (Ka) for Alexa-HSA was ∼5 × 106 M-1. Competitive Immunoassay of Unlabeled HSA. To conduct a competitive immunoassay of an unlabeled HSA sample, we studied factors such as concentration of the reporter Alexa-HSA and the antibody, molar ratio of antibody/antigen, and incubation time. The standard curves for molar ratios of antibody/antigen of 16, 8, 4, 2, and 1 for concentrations of 500, 250, 125, and 62.5 ng/mL Alexa-HSA were prepared. Also, we investigated the incubation times of 20, 40, 60, and 90 min for the reaction. At the optimized condition for the minimum time (20 min), the molar ratio of antibody/antigen of 8 for concentrations of 250 and 125 ng/mL produced a standard curve with DL of 12 and 35 nM for unlabeled HSA. Considering the affinity of the antibody (5 × 106 M-1), this was 10 times less than the minimum theoretical DL. The minimum theoretical DL for competitive immunoassay by an antibody with the same affinity and by LIF detection is limited to 150-450 nM.19,32 To the best of our knowledge, without applying a preconcentration method and by normal competitive MCEIA, the reported DL for protein samples have been 1 or 2 orders of magnitude higher than the expected minimum theoretical DL.21,33 CONCLUSION A novel online preconcentration and stacking method based on transient ITP on a PMMA microchip was introduced. The preconcentration did not require any special chip design or structure, and the method was applicable to standard crosschannel microchips. The method was applied to direct immunoassay of mAb anti-HSA and competitive immunoassay of an unlabeled HSA. The preconcentration enhanced the signal for the peak of the immunocomplex by 800-fold. The preconcentration was followed by nondenaturing gel electrophoresis in methylcellulose solution for size-based separation of HSA from its immunocomplex. The separation was completed at a distance 1 cm from the intersection of the cross-channels in less than 25 s. Picomolar concentration of labeled HSA was detected. The method is promising for developing highly sensitive immunoassays of protein samples in inexpensive plastic microchips. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 18, 2006. Accepted March 19, 2007. AC0623890 (32) Jackson, T. M.; Ekins, R. P. J. Immunol. Methods 1986, 87, 13-20. (33) Tsukagoshi, K.; Jinno, N.; Nakajima, R. Anal. Chem. 2005, 77, 1684-1688.