Rapid and separation-free sandwich immunosensing based on accumulation of microbeads by negative-dielectrophoresis

Biosensors and Bioelectronics 24 (2008) 1000–1005

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Rapid and separation-free sandwich immunosensing based on accumulation of microbeads by negative-dielectrophoresis Hyun Jung Lee a , Tomoyuki Yasukawa b,∗ , Hitoshi Shiku a , Tomokazu Matsue a,∗∗ a b

Graduate School of Environmental Studies, Tohoku University, 6-6-11-604, Aramaki-Aoba, Aoba-Ku, Sendai 980-8579, Japan Graduate School of Material Science, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan

a r t i c l e

i n f o

Article history: Received 15 May 2008 Received in revised form 11 July 2008 Accepted 4 August 2008 Available online 9 August 2008 Keywords: Negative-dielectrophoresis Microbeads Accumulation Re-dispersion Separation-free Immunosensing

a b s t r a c t We report here a rapid and separation-free immunoassay using a dielectrophoresis (DEP) device consisting of an interdigitated microarray (IDA) electrode and a polydimethylsiloxane (PDMS) substrate. On applying an AC voltage to the IDA in a negative-DEP (n-DEP) frequency region, goat anti-mouse immunoglobulin (anti-mouse IgG)-immobilized microbeads moved to the surface of the PDMS substrate placed above the IDA. The microbeads accumulated at designated areas of the PDMS surface that had been precoated with anti-mouse IgG. When the fluorescence microbeads bearing anti-mouse IgG were suspended in an analyte (mouse IgG) solution, the microbeads trapped the analyte to form immunocomplexes on microbeads. The microbeads reacted with mouse IgG accumulated and were captured at the designated areas of the PDMS surface via an antibody–antigen–antibody (sandwich) reaction. The captured microbeads were detected selectively by fluorescence measurements at the focused designated areas, regardless of the presence of uncaptured microbeads suspended in solution. Thus, the separation and washing-out steps usually required for conventional immunoassay are eliminated in the presented procedure. Since the formation of the sandwich structures was accelerated significantly by n-DEP, a period as short as 30 s was sufficient to detect the immunoreaction at the surface. The fluorescence intensity of the captured microbeads at the designated areas increased with analyte concentration in the range 0.01–10 ng/mL. The present procedure therefore yields a rapid, sensitive, and separation-free immunoassay in a simple device. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Currently, in the field of bioanalytical science, enzyme-linked immunosorbant assays (ELISAs) are widely used as a standard method to measure biomaterials such as marker proteins, peptides, hormones, and environmental chemicals. Conventional immunoassay procedures using a labeled antibody require several steps for the detection of immunocomplexes, and a relatively long reaction time for the formation of the complexes due to the limitation of diffusion. The diffusion-limited reaction can be speeded up by using micro- and nano-scale devices that enable the antibody to capture an antigen within a short distance (Aguilar et al., 2002; Dong et al., 2006; Bange et al., 2005; Niwa et al., 1993; Shiku et al., 1996; Kasai et al., 2006; Ogasawara et al., 2006; Yasukawa et al., 2007b). Simplicity is also an important factor when developing a novel ELISA device. In ELISAs using 96-well plates, manual operations are nec-

∗ Corresponding author. Tel.: +81 791 58 0171; fax: +81 791 58 0493. ∗∗ Corresponding author. Tel.: +81 22 795 7209; fax: +81 22 795 7209. E-mail addresses: [email protected] (T. Yasukawa), [email protected] (T. Matsue). 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.08.002

essary in order to separate the captured antigen (or antibody) and secondary antibody on the substrate from the solution containing unreacted materials and interfering species—operations that are often a source of human error. In this respect, microfluidic devices carrying microbeads have been reported to produce rapid and automatic immunosensing systems (Gijs, 2004; Sato et al., 2000; Sato et al., 2002; Yasukawa et al., 2007a). We have studied the immobilization of negative-dielectrophoresis (n-DEP)-patterned microbeads by using immunoreactions and applied these to immunoassay. DEP is one of the electrokinetic phenomena that cause dielectrically polarized materials to move in nonuniform electric fields (Morgan and Green, 2002). The rate and direction of migration depend on the electric field gradient and dielectrophoretic character of the material and its surrounding medium. Under the influence of a positive-DEP (p-DEP) force, materials move toward the strongest electric field region, whereas in response to a negative-DEP they move toward a relatively weak electric field region. p-DEP has been applied as a driving force for the capture, separation, and pattern manipulation of micro- and nanoparticles, cells, microorganisms, and nanomaterials such as DNA and carbon nanotubes (Kretschmer and Fritzsche, 2004; Muller et al., 1999; Chiou et al., 2005; Sung and Burns, 2006; Dong et al.,

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2005). Negative-DEP (n-DEP) has been applied to the fabrication of microstructures on substrates opposed to a template electrode. The microstructures fabricated by n-DEP are usually re-dispersed into suspension media when the applied voltage is turned off (Gijs, 2004; Matsue et al., 1997; Suzuki et al., 2004). It is therefore necessary to pretreat the substrate or to encapsulate it within a matrix in order to maintain the desired structures (Suzuki et al., 2007; Docoslis and Alexandridis, 2002). Recently, p-DEP phenomena have been applied to the fast immunocapture of cells and probe particles (Yang et al., 2006; Suehiro et al., 2006; Auerswald et al., 2005). Generally, the repeated use of electrode device has many advantages from the viewpoint of economy and simplicity; however, patterns formed by p-DEP are irreversible and remain on an electrode device even if the AC voltage is switched off. Thus, the electrode devices cannot be used repeatedly, resulting in an increased cost per single assay. This is a critical disadvantage of p-DEP-based immunoassays. Our group has previously reported an immunoassay based on a competitive reaction in a caged space created by n-DEP (Yasukawa et al., 2007b). However, the previous method required a complicated electrode design and a flow system using syringe pump for stepwise immunoreaction and washing processes. In this paper, we report a novel method for rapid, sensitive, economical and separation-free sandwich immunoassay based on n-DEP using an interdigitated array (IDA). Fig. 1 shows the outline of the present procedure. The probe molecule, an antibody, is immobilized on the surfaces of both polydimethylsiloxane (PDMS) substrate and microbeads. The microbeads immobilized with antibody are introduced together with the analyte into the device, which is simply composed of the IDA electrode and the antibodyimmobilized PDMS substrate. When an AC voltage at an n-DEP frequency is applied to the IDA electrode to form nonuniform electric fields, the microbeads are forced to move toward the PDMS substrate, thereby accelerating the reaction with the antibodies immobilized on the PDMS. This immunoreaction at the surface creates an antibody–analyte–antibody linkage that captures the microbeads at the designated areas, and prevents the re-dispersion of microbeads into suspension from the PDMS substrate after the AC voltage is turned off. The selective measurement of fluorescence from the captured microbeads depends on the concentration of the analyte. Uncaptured microbeads are re-dispersed from the PDMS surface after the AC voltage is turned off. As a result, the uncaptured microbeads become out of focus in the fluorescent microscope detection. Therefore, the present immunosensing method does not require the separation and washing-out of unreacted microbeads since the undesired fluorescence signal obtained from the redispersed microbeads under captured microbeads can be easily subtracted from the fluorescence intensities of the accumulated microbeads. Furthermore, a very wide detection range of analyte can be realized simply by controlling the period for the accumu-

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lation of microbeads with n-DEP. This immunosensing using a simple device has substantial advantages; i.e., rapid immunoreaction accelerated by n-DEP, repeated use of the IDA electrode device, separation-free, washing-free, and controllable wide detection range. 2. Experimental 2.1. Chemicals and materials All chemicals of reagent grade were obtained from Wako Chemicals (Osaka, Japan) and used as received. Horseradish peroxidase (HRP)-labeled polyclonal goat anti-mouse IgG was purchased from Zymed Laboratories (Invitrogen Corp., Carlsbad, CA) as a liquid, and was diluted with phosphate-buffered saline (PBS) to give solutions of 100 ng/mL or 10 ng/mL. Mouse IgG and rabbit IgG were purchased from Vector Laboratories Inc. (Burlingame, CA) and diluted with PBS to give concentrations varying from 1 ␮g/mL to 0.01 ng/mL. The PBS buffer consisted of 8.1 mM Na2 HPO4 , 1.5 mM KH2 PO4 , 2.7 mM KCl, and 137 mM NaCl (pH 7.6, conductivity of 1.54 S/m). The aqueous solutions were prepared by using distilled and deionized water from Milli-Q filtration (Nihon Millipore Ltd., Tokyo, Japan) and Aquarius GS-200 (Advantec, Tokyo, Japan) systems. 2-Methacryloyloxyethyl phosphoryl choline (MPC) polymer (product code: N101), a blocking reagent, was purchased from NOF Corp. (Tokyo, Japan). PDMS elastomer and a curing agent (Silpot 184) were obtained from Dow Corning Toray Silicone Co., Ltd. (Tokyo, Japan). Polystyrene latex microbeads with incorporated yellow–green (YG) fluorescent dye (Fluoresbrite Plain Microspheres, 2 ␮m diameter) were purchased from Polysciences Inc. (Warrington, PA). Indium-tin-oxide (ITO) glass was purchased from Sanyo Vacuum Industries Co., Ltd. (Tokyo, Japan). A positive photoresist (S-1818) and a negative photoresist (SU-8 2002) were obtained from Shipley Corp. (Marlborough, MA) and MicroChem Corp. (Newton, MA), respectively. 2.2. Fabrication of ITO–IDA The DEP microdevice was composed of an ITO–IDA electrode and a PDMS substrate. The ITO–IDA was fabricated by conventional photolithographic techniques and a wet-etching process. The IDA consisted of two comb-type array with 10 microband electrode elements. Each microband was 1.0 mm long and 20 ␮m wide, separated by 50 ␮m from the adjacent bands. After making the IDA patterns using the S-1818 photoresist on a cleaned ITO glass, the ITO was chemically etched with 4:5:5 (v:v:v) HNO3 /HCl/water for 25 min, followed by removing the photoresist with acetone and isopropyl alcohol. A 2 ␮m thick insulating layer was thus formed on the surrounding of the electrode by the SU-8 2002. The IDA exposed to the solution was 750 ␮m × 750 ␮m.

Fig. 1. Schematic procedure for sandwich immunosensing by the accumulation of microbeads on a PDMS substrate based on n-DEP.

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Before use, the IDA electrode was characterized by cyclic voltammetry in a 4.0 mM K4 Fe(CN)6 /100 mM KCl aqueous solution using a Pt wire counter and Ag/AgCl reference electrodes. The scan rate was set to 50 mV/s. The same and ideal voltammograms were obtained for both microband electrodes of the IDA (data not shown). Therefore, each microband electrode was found to act independently and equivalently. 2.3. Immobilization of antibody on the PDMS substrate and microbeads The PDMS substrate was fabricated by mixing a PDMS prepolymer with curing agent in a 10:1 (v:v) ratio. The mixed elastomer was then degassed for 1 h and poured over the silicon wafer pretreated with 3,3,4,4,5,5,6,6,6-nonafluorohexyl trichlorosilane (ShinEtsu Corp., Tokyo, Japan). The PDMS substrate was cured for 1 h at 80 ◦ C in an oven and then peeled off from the wafer. The HRP-goat anti-mouse IgG was immobilized on the PDMS substrate by physical adsorption. A 20 ␮L aliquot of the antibody solution (100 ng/mL or 10 ng/mL) was dropped onto the PDMS and this was then incubated overnight at 4 ◦ C. After incubation, the antibody-immobilized PDMS was washed three times with MilliQ water. The fluorescent microbeads were also modified with the antibody using a similar method as follows. A 5.7 × 109 particles/mL microbead suspension (10 ␮L) was centrifuged at 16,800 × g for 15 min in an Eppendorf tube. After removing the supernatant, a 10 ␮L aliquot of antibody solution was added. The microbeads were dispersed again by pipetteting several times and incubated at 4 ◦ C overnight. The resulting antibody-immobilized microbeads were then washed three times with Milli-Q water. The PDMS substrate and microbeads were immersed in the blocking solution (N-101)/deionized water (1:4, v:v) for 20 min in order to prevent nonspecific binding of proteins (analyte) to the surfaces. The PDMS substrate and fluorescent microbeads thus obtained were treated with 3,3 ,5,5 -tetramethyl benzidine dihydrochloride (TMB) and H2 O2 for 20 min to promote the coloring reaction catalyzed by the labeled HRP, followed by deactivation of the HRP by the addition of 1.0N H2 SO4 . The absorbance measurement of the resulting solution clearly revealed a characteristic color, confirming that the antibody had been immobilized on the surface of the PDMS and microbeads. 2.4. Manipulation of the microbead probe and sensing of the immunoreaction via n-DEP The analyte solution was added into the antibody-immobilized microbeads. A 20 ␮L solution of the final suspension containing 5.7 × 108 –1.2 × 109 particles/mL microbeads and analyte was dropped onto the ITO–IDA electrode immediately after mixing the analyte and antibody-immobilized microbeads. PDMS substrate immobilized with antibody was placed on the ITO–IDA electrode to fabricate the DEP device. The height between IDA electrode and upper PDMS substrate is about 5–10 ␮m. The n-DEP manipulation of the microbeads was performed under a fluorescence microscope (IX71; Olympus Co., Tokyo, Japan) equipped with a CCD camera (C5985; Hamamatsu Photonics KK, Hamamatsu, Japan). For the manipulation, an AC voltage of 10 Vpp (peak-to-peak voltage), at a frequency of 2 MHz, was applied between the two lead parts of the IDA electrodes for 30, 60, and 180 s. The application of the AC voltage forces the microbeads to move to the PDMS surface by nDEP and, as a result, the microbeads are captured at the surface by the immunoreaction. Therefore, the period of AC voltage application was considered as the period of the immunoreaction. The AC voltage was controlled by a wave-form generator (WF1945; NF Co., Yokohama, Japan).

2.5. Analysis of immunosensing performance after n-DEP The capture of microbeads at the PDMS surface by n-DEPinduced immunoreaction was analyzed based on the accumulation and dispersion motion of the microbeads. The microbeads are assumed to accumulate at the designated area of the PDMS surface above the microband electrode of the IDA. If the analyte molecules are present in the microbead suspension solution, the microbeads should be captured at the surface via antibody–antigen–antibody complex formation. This immunocomplex formation prevents the re-dispersion of microbeads by Brownian motion and migration by gravity. These accumulation/re-dispersion phenomena were monitored by fluorescence measurements in order to determine the antigen in the suspension solution. From the fluorescence images obtained before and 60 s after turning off the AC voltage, we measured the signal densities of fluorescence from the accumulated microbeads in the designated areas (E areas in Fig. 2c). The signal from the gap areas (G areas in Fig. 2c) was considered as a background and subtracted from the signal in the E areas. This background-subtracted density was defined as the fluorescence intensity from the accumulated microbeads. Since the accumulated microbeads formed a clear line, we set the width of the E area to 10 ␮m—half the bandwidth of the IDA. The relative fluorescence intensity from the patterned microbeads was calculated based on the intensity before and 60 s after turning off the DEP voltage. Each point of fluorescence intensity was calculated on the average of three data. 3. Results and discussion 3.1. Accumulation of microbeads on the PDMS substrate by n-DEP Fig. 2 shows the FE-SEM, optical and fluorescence microscopic images of the ITO–IDA electrode and the microbeads manipulated in the DEP device with a 10 Vpp AC voltage at a frequency of 2 MHz. Upon switching on the AC voltage to the electrodes, the n-DEP forced the microbeads to move toward the PDMS substrate with the weakest electric field, and to accumulate at the designated areas (E areas) of the PDMS surface above the IDA band electrode (Fig. 2b). Since the microscopic focus was set at the PDMS surface, the microbeads suspended in the solution or on the IDA surface were observed as out of focus spheres (Fig. 2b). The fluorescence images indicate that fluorescence from the microbeads accumulated in the E areas was intense while the fluorescence from the microbeads in the solution exhibited dispersed and vague images (Fig. 2d). Therefore, the microbeads accumulated at the E areas by nDEP can selectively be determined from the fluorescent microscope image focused on the E areas (Fig. 2c). This situation is schematically presented in Fig. 2e. The microbeads patterned by n-DEP were re-dispersed in the suspension solution after turning off the AC voltage when the solution contained no analyte (Fig. 2d). The above results indicate that the microbeads captured at the surface by an immunoreaction can be detected without separation and washing-out steps to remove unreacted microbeads in the suspension solution—steps that are usually required for conventional immunoassays. The use of suspension solutions with relatively high conductivities causes an imperfect alignment of microbeads at the PDMS surface. DEP force increases with increasing permittivity. The permittivity in low-conductivity solution is higher than that in high-conductivity solution due to the hydration effect (Morgan and Green, 2002; Grant et al., 1978; Loginova et al., 2005). Therefore, the efficiency of microbead manipulation by DEP in a high-conductive medium becomes lower as compared with the efficiency in a lowconductivity medium. In the as-prepared PBS with a conductivity

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Fig. 2. (a) FE-SEM image of the ITO–IDA electrode. (b) Microbeads manipulated in the DEP device under 10 Vpp AC voltage at a frequency of 2 MHz, an electrode width of 20 ␮m, an electrode gap of 50 ␮m, and a microbead diameter of 2 ␮m. Fluorescence microscopic images of (c) the manipulated microbeads and (d) the re-dispersed microbeads 60 s after turning off the AC voltage. (e) Illustration of microbeads accumulated at the PDMS surface and positioned on the ITO electrode. Scale bar: 25 ␮m (a–d).

of 1.54 S/m, almost no microbeads were found to be aligned on the PDMS. Therefore, the suspension solution containing analyte, mouse IgG, and the fluorescent microbeads was prepared in a 1/10 diluted PBS [(1/10)-PBS] or 1/5 diluted PBS [(1/5)-PBS] with Milli-Q water for the n-DEP-based manipulations. The conductivities of the (1/10)-PBS and (1/5)-PBS were 154 mS/m and 360 mS/m, respectively. The conventional ELISA in the low-conductivity PBS solutions indicated that there was no negative effect to form the immunocomplex between mouse IgG and anti-mouse IgG in the (1/10)-PBS and (1/5)-PBS. The accumulation of microbeads proceeded smoothly on applying an AC voltage to the IDA electrode. Fig. 3 shows the time course of the accumulation of the bare fluorescent microbeads (2 ␮m diameter) onto the untreated PDMS surface on applying a 10 Vpp AC voltage at a frequency of 2 MHz in (1/5)-PBS. The uniformly dispersed microbeads initially start to move toward the designated areas (E areas in Fig. 2c) of the PDMS with the weakest electric field region due to the n-DEP force to form line patterns. The relative fluorescence intensity at the E areas increased with voltage application time and saturated within 10 s. The accumulation of microbeads was not affected when the PDMS and microbeads were immobilized with antibody. More-

Fig. 3. Time course of the accumulation of the bare microbeads onto the untreated PDMS surface, determined by relative fluorescence intensity.

over, the presence of analytes (e.g., 1.0 ␮g/mL mouse IgG) in the microbead suspensions had almost no influence on the accumulation behavior. In contrast, the re-dispersion process was markedly retarded when the analyte was present in the solution. Therefore, the analyte can be determined from the re-dispersion of microbeads after turning off the AC voltage. In addition, there was no bead–bead aggregation before and after the DEP manipulation under the experimental conditions. 3.2. Re-dispersion of microbeads from the PDMS substrate We investigated the capture efficiency of microbeads at the PDMS surface via the immunocomplex using mouse IgG as an analyte. For this purpose, the surfaces of both PDMS and microbeads were coated with anti-mouse IgG. To confirm the adsorption of the antibody, PDMS surface immobilized with HRP-labeled antimouse IgG was also prepared. Coloring reactions of TMB by the enzyme reaction of HRP were detected from both PDMS and microbeads immobilized HRP-labeled antibody. The results suggest that antibodies are adsorbed on PDMS and microbeads surface by using the presented method above. When the AC voltage for n-DEP (10 Vpp , 2 MHz) was applied to the IDA in a solution containing 1.0 ␮g/mL mouse IgG for 3 min, the microbeads accumulated in the E areas to form line patterns as described above, and then the voltage was turned off. Fig. 4 shows the re-dispersion behavior of patterned microbeads after turning off the AC voltage. When the analyte was not present in the solution, the pattern of accumulated beads became broad and almost disappeared after 60 s (Fig. 4a). Compared to the accumulation step, the dispersion step proceeded slowly; this is because the re-dispersion process is driven by Brownian motion and migration. In contrast, when the analyte, mouse IgG, was present in the solution, some microbeads were irreversibly captured at the PDMS surface and remained at this position even after the AC voltage had been turned off (Fig. 4b). This result indicates that n-DEP forces the microbeads reacted with mouse IgG to move to the PDMS surface. Consequently, the microbeads reacted with mouse IgG were captured at the surface by the formation of an antibody–antigen–antibody structure. As a result, the microbead patterns were fixed on the PDMS substrate. The relative fluorescence intensities of the aligned microbeads decreased by only 14% in 10 s after the n-DEP was turned off, and then remained at a

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Fig. 4. Re-dispersion behavior of patterned microbeads modified with antibody from the modified PDMS substrate after turning off the AC voltage in the DEP device (a) in the absence of and (b) in the presence of 1.0 ␮g/mL mouse IgG. Time for the application of n-DEP was 3 min.

steady-state value (Fig. 4b). This behavior contrasts well with that observed without the analyte. 3.3. Microbead-based immunosensing accelerated by n-DEP We next investigated the influence of the duration of AC voltage application for n-DEP manipulation. We applied an AC voltage (10 Vpp ) for 30, 60, and 180 s in the presence of 1.0 ␮g/mL mouse IgG for accumulation and immunoreaction. As described above, the fluorescence intensity was saturated approximately 10 s after the voltage application and remained constant for the subsequent voltage application. During this immunoreaction time, the capture of microbeads at the surface proceeded. The voltage was then turned off and the fluorescence intensity relative to the intensity immediately before turning off was measured. Fig. 5 shows the relative fluorescence intensity 60 s after the AC voltage was turned off. The application of n-DEP for 30 s was sufficient to ensure the capture of microbeads at the PDMS surface via the immunoreaction. The signal intensity increased with the immunoreaction time but tended to saturate after 60 s. Without application of n-DEP, no detectable capture of the microbeads on the PDMS surface was

Fig. 6. Calibration curve for mouse IgG using the n-DEP-based immunosensing device. Time for the application of n-DEP: () 30 s, () 60 s, and () 180 s.

found even after 180 s. These results clearly indicate that n-DEP accelerates the immunoreaction by guiding the microbeads to the antibody-immobilized PDMS surface. The fluorescence measurements without analyte indicated that the nonspecific adsorption of the microbeads had only a limited influence on the results as shown in Fig. 4a. In addition, when a different antigen, 1.0 ␮g/mL rabbit IgG, was introduced to the device, at most only 18% of the microbeads was adsorbed nonspecifically. The undesired background responses originated from the nonspecific binding and could not be completely eliminated using a blocking agent, N101. Under the present conditions, the relative background fluorescence noise from the nonspecifically adsorbed and/or floating microbeads was found to be less than 15%. Fig. 6 shows the relationship between the relative fluorescence intensity from the captured microbeads and the concentration of the analyte, mouse IgG, over a wide range (0.01–1000 ng/mL). The relative fluorescence intensity was measured 60 s after turning off the AC voltage. The intensity increased with the analyte concentration up to 10 ng/mL and saturated at higher concentrations. The sensitivity depends on the immunoreaction time (n-DEP application period) and a concentration as low as 0.01 ng/mL can be detected when the time is longer than 30 s. The detectable concentration range also depends on the immunoreaction time. It should be noted again that the time required for immunoreaction at the surface can be significantly shortened by the n-DEP process. This method, based on the n-DEP immobilization of antibodyimmobilized microbeads, will be applicable to many immunoassay protocols. 4. Conclusions

Fig. 5. Relative fluorescence intensity 60 s after turning off the AC voltage as a function of the time of n-DEP application (a) in the presence and (b) in the absence of 1.0 ␮g/mL mouse IgG, and (c) in the presence of 1.0 ␮g/mL rabbit IgG.

The microbead-based immunosensing device described here yields high density sensing sites; therefore, it can perform very efficient analysis. In this paper, we propose a novel procedure for rapid, sensitive, and simple immunoassay based on n-DEP. On applying an AC voltage with a frequency in the n-DEP region, antibody-bearing microbeads were forced to move and accumulate at the designated areas on the PDMS substrate surface. When the solution contained an analyte, mouse IgG, the microbeads were captured on the PDMS surface by the rapid formation of a substrate–analyte–microbead linkage. Therefore, the IDA electrode device can be reused several times. This is an important feature of n-DEP-based manipulation. For comparison, the positive-DEP-based manipulation guides the

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beads to the IDA electrode device and frequently forces the beads to stick on the electrode. This immunoreaction at the PDMS surface was accelerated by n-DEP and completed within 30 s, while almost no immobilization of microbeads was found without applying an AC voltage. The captured microbeads on the PDMS substrate were not re-dispersed when the AC voltage was turned off. On the contrary, uncaptured beads moved far from the substrate and became out of focus. As a result, the fluorescence signals were detectable without the separation of unreacted microbeads since the signals were collected from the focused areas at the PDMS surface. The fluorescence intensity from the captured microbeads depended on the concentration of mouse IgG in the solution in the range 0.01–10 ng/mL. The sensitivity and detectable concentration range can be controlled by changing the DEP time. The present procedure yields a rapid and sensitive immunoassay in a simple system without the necessity of measures required to generate complicated structures for accelerating the immunoreaction, such as agitation in a microchamber and reducing the size of the device in order to overcome diffusion problems. Acknowledgement This work has been financially supported by Grants-in-Aid for Scientific Research (Nos. 18101006, 18048001, 19650121, and 19710112) from the Ministry of Education, Science and Culture, Japan. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2008.08.002. References Aguilar, Z.P., Vandaveer IV., W.R., Fritsch, I., 2002. Anal. Chem. 74, 3321–3329.

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