Detection of Surface Antigens on Living Cells through Incorporation of

Aug 19, 2011 - Therefore, the cells accumulated by p-DEP were easily directed to another site by controlling the DEP forces by frequency. The formatio...
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Detection of Surface Antigens on Living Cells through Incorporation of Immunorecognition into the Distinct Positioning of Cells with Positive and Negative Dielectrophoresis Hironobu Hatanaka,† Tomoyuki Yasukawa,*,†,‡ and Fumio Mizutani*,† † ‡

Graduate School of Material Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako, Hyogo 678-1297, Japan JST-CREST, 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan

bS Supporting Information ABSTRACT: Rapid determination of surface antigens on cells is possible by immobilization of cells accumulated by positive dielectrophoresis (p-DEP) via effective surface immunoreactions and removal of unbound cells by negative DEP (n-DEP). The DEP device for cell manipulation comprises a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower ITO microband array electrode (band electrode) modified with an antibody. Cells with the surface antigen introduced into the channel immediately accumulated on the surface of the band electrode during p-DEP generated by the application of ac voltage between the ITO electrode and the band electrode to immobilize by the specific antibody. The removal of accumulated cells to the gap region during n-DEP was used for rapid estimation of the residual cells with a specific surface antigen. We demonstrate here that human promyelocytic leukemia cells with the surface antigen CD33 can be captured on a band electrode modified with anti-CD33 antibody. The time required for the determination of the surface antigen using this compelled accumulation of cells by p-DEP and the separation of unbound cells by n-DEP is decreased to 60 s compared to that required by a cell binding assay using microtiter plates (30 min). Furthermore, the present method for a novel cell binding assay does not require pretreatment such as target labeling or washing of unbound cells and thereby enhancing throughput in the clinic and in cytobiology studies.

S

urface antigen patterns expressed on living cells are influenced by lineage, differentiation, and maturation. Immunophenotyping, a method in which the presence and proportion of pathogenic cell populations can be identified, is useful for early medical diagnosis and prognosis.1 A common approach is to use fluorescent labeling to reveal specific surface antigens on cells; however, these methods are often qualitative, have low throughput, and involve several complex steps of modification and washing. Recently, an antibody microarray, a protein chip that is a powerful tool for investigating the interactions of thousands of proteins in a single assay,2,3 has been employed to detect surface antigens by direct binding of cells to the antibody spot arrays.4 10 Cell populations with distinct phenotypes can be classified according to antibody array patterns and captured cell densities at each spot because immobilization of cells expresses target antigens at spots with corresponding antibodies. Thus, parallel and collective determination of cells bound to the specific array improves the throughput of immunophenotyping. The profiling of surface antigens on leukemia cells5,11,12 and neural stem cells13,14 has been demonstrated using antibody microarrays. Furthermore, high-throughput quantitative numbering of the cells captured at each spot was achieved by holographic and surface plasmon resonance imaging techniques.15,16 However, a relatively long incubation is still required for antibody arrays because of cell integration at spots without active force. r 2011 American Chemical Society

Dielectrophoresis (DEP) is attractive for the manipulation of micro- and nano-objects including biological living cells and bacteria in a microfluidic device because of its noncontact nature.17 19 It has been used in a wide range of applications, such as separation and sorting,20 23 trapping,24 28 and patterning of cells or particles. The classical quadrupole polynomial electrode array was vigorously applied to analyze DEP properties of cells by monitoring the crossover frequencies.29 33 Applications of the DEP manipulation and analysis for cells have been summarized in a recent review.34 36 The particles placed in a spatially inhomogeneous electric field undergo DEP due to polarization induced in the particles. Cell arrays can be fabricated using a positive dielectrophoretic force, a force that directs particles toward regions of the electric field maxima. 37 40 Generally, positive DEP (p-DEP) patterning is used in arrays that have a pair of electrodes at each element modified with cell adhesive layers to produce cell patterns. The registration of cells in an ordered array provides a simple method to study cellular functions in vitro, including cell cell interactions for cell-based biosensor applications. Received: July 13, 2011 Accepted: August 19, 2011 Published: August 19, 2011 7207

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principle of the present method for the detection of the surface antigen. The suspension of cells was introduced into the device comprising an upper indium tin oxide (ITO) electrode and a lower ITO microband array electrode (band electrode) modified with an antibody. When an ac voltage in the p-DEP frequency region is applied between the ITO electrode and the band electrode to form nonuniform electric fields, the cells are directed toward the band electrode and accumulate on it, thereby accelerating the capture of cells with the antibodies via immunoreactions. Uncaptured cells were removed from the band electrode to the gap region between the bands by n-DEP. Thus, the ratio of captured cell density could be easily calculated from the optical images of the line pattern. The uniform removal of unbound cells by careful washing is difficult in a large number of antibody arrays; however, n-DEP provides uniform removal of unbound cells. Therefore, the present method does not require pretreatment such as target labeling or washing of unbound cells. Furthermore, a rapid detection of surface antigens can be realized by incorporating immunorecognition events into the distinct integration of cells with p- and n-DEP. Here we demonstrate that human promyelocytic leukemia (HL60) cells with the surface antigen CD33 can be captured on a band electrode modified with anti-CD33 antibody.

Figure 1. Cross-sectional view of the DEP device and the principle of the present method for detection of surface antigens using a combination of p- and n-DEP.

’ EXPERIMENTAL SECTION

The negative dielectrophoretic force, a force that repels particles from regions of high electric field, has also been used to pattern cells.41 45 The strategy for n-DEP patterning is the construction of a localized position enclosed with strong electric fields to allow cell capturing through a repulsive force balanced from every direction. We have previously fabricated periodic and alternate cell lines incorporating two types of cells using n-DEP.46 Using both p- and n-DEP, individual cell types with different dielectrophoretic properties were separately patterned at different positions.47,48 The direction and intensity of the driving force acting on the cells depend on the dielectric properties of the cells and solutions, electrode configurations, particle size, and applied voltage and frequency. We can easily and reversibly fabricate two different patterns with cells by controlling the direction of the dielectrophoretic force through the applied frequency. However, only one feature has been used in most cases of cell patterning. We recently developed a rapid and simple sandwich-type immunosensing system using n-DEP-based accumulation and redispersion of microparticles.49 52 The particles were irreversibly captured on the substrate to form sandwich-type immunocomplexes in the presence of specific analytes because the particles modified with the antibody rapidly accumulated and pressed against the substrate modified with the antibody by n-DEP. Unreacted particles were automatically removed from the substrate by deregulation of DEP. The use of n-DEP manipulation of microparticles allowed separation-free sensing of unreacted target molecules within 3 min. In this study, we employed DEP to demonstrate rapid and simple detection of surface antigens on cells. The use of the p-DEP can reduce the transport time and remove the cell diffusion for the capture of target cells by antibodies on electrodes for the cell manipulation. Recently, the advantage of p-DEP was applied to assist the immuno-capture by the reduction of the transport time of bacteria cells.53 56 We positively applied n-DEP to remove nonspecific cells uniformly from the electrodes. Figure 1 schematically depicts a cross-sectional view of the DEP device and the

Fabrication of the Dielectrophoretic Patterning Device. A dielectrophoretic patterning device was constructed with an ITO electrode and an ITO band electrode. The band electrode was fabricated by conventional photolithography and chemical etching using an etchant solution (ITO-02, Kanto Chemical, Tokyo, Japan) for 10 15 min under ultrasonication. Each element was 2 mm long, was 12 μm wide, and placed at a distance of 50 μm from adjacent bands. A 5 μm thick insulating layer of negative photoresist (SU-8 3005, MicroChem Corp. Newton, MO) was fabricated on the ITO pattern to define the electrode areas exposed to the solution. A 30 μm thick polyester film (Nitto Denko, Osaka, Japan) was used as a spacer and sandwiched between the ITO electrode and the band electrode to construct the patterning device. Modification of the Band Electrode with the Anti-CD33 Antibody. We modified the band electrode with mouse antiCD33 monoclonal antibody (anti-CD33, AbD Serotec, Kidlington, U.K.) to identify the cells with the CD33 cell surface antigen. A goat antimouse IgG polyclonal antibody (antimouse IgG, AbD Serotec) was used as a negative control. The band electrode was immersed in anhydrous ethanol containing 2 v/v% 3(glycidoxypropyl)trimethoxysilane (Sigma-Aldrich Chemical, St. Louis, MO) for 1 h at 25 °C to introduce a terminal epoxy group that readily reacts with the amino group of biomolecules. After washing with ethanol, the band electrode was treated with carbonate buffer (pH 9.6) containing 100 μg mL 1 antibody for 16 h at 4 °C. After washing with phosphate-buffered saline (PBS, pH 7.5), the electrode was immersed in carbonate buffer containing 10 mg mL 1 bovine serum albumin (BSA, Wako, Osaka, Japan) for 24 h at 4 °C to eliminate nonspecific binding of cells. Finally, the electrode modified with the antibody was washed and stored in PBS at 4 °C before use. Modeling and Calculations. The distribution of the electric field strength for the patterning device was calculated by the finite element method solver (COMSOL Multiphysics, Stockholm, Sweden). The ITO electrode was mounted on the band electrode. The width of the bands and the distance from adjacent 7208

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Analytical Chemistry bands were set at 12 and 50 μm, respectively. The potential difference and the distance between the ITO electrode and the band electrode were set at 10 V and 35 μm, respectively. Manipulation of Cells by DEP. HL60 cells (CD33+ cells) were grown in RPMI 1640 medium (Invitrogen Japan KK, Tokyo, Japan), containing 10% fetal bovine serum (Invitrogen), 50 units mL 1 penicillin (Invitrogen), and 50 μg mL 1 streptomycin (Invitrogen), at 37 °C under 5% CO2. HL60 cells (4  107 cells mL 1) were suspended in DEP medium consisting of 250 mM sucrose and adjusted the conductivity to 80 mS m 1 with 250 mM HEPES buffer (pH 7.4) for manipulation of cells by DEP. The ac voltage in the p-DEP frequency region (typically 15 Vpp and 10 MHz) was applied between the ITO electrode and the band electrode to accumulate cells on the bands. Then, n-DEP (5 15 Vpp and 100 kHz) was used to remove the cells from the bands to the gap region. Cell viability was investigated by dynamic staining of cells with propidium iodide (PI).57,58 Cells were suspended in the DEP medium containing 1 μM PI and regularly manipulated by p- and n-DEP to monitor cell viability. Manipulation of cells by DEP was performed under an optical and fluorescent microscope (IX70, Olympus, Tokyo, Japan) equipped with a CCD camera (DP72, Olympus). A cell suspension (1.0 1.5 μL) was introduced into the patterning device. The ac voltage was applied between the ITO electrode and the band electrode to form the alternating electric field in the device (function generator 7075, Hioki E.E. Co., Ueda, Japan). Cell Binding Based on Manipulation of the Mixture of Cells by DEP. HL60 cells were manipulated by p- and n-DEP in the patterning device consisting of the band electrode modified with anti-CD33 or antimouse IgG. We prepared nonspecific cells treated with anti-CD33 and used them as CD33-negative cells. Furthermore, the cells were labeled with fluorescent molecules to distinguish antibody-treated nonspecific cells. Cells were successively incubated in 10 μg mL 1 of anti-CD33 and 1 μM of CFDA SE (Invitrogen) in PBS for 15 min at 37 °C under 5% CO2. Suspensions of specific and nonspecific cells were mixed at different ratios to study the relationship between the number of CD33positive cells in the suspension and the number of cells captured on the band electrode. The cell mixture accumulated on the band electrode by p-DEP for 60 s. The binding efficiency of the cells was calculated after application of the ac voltage for n-DEP for 180 s. The efficiency was defined as the ratio of the average cell density on the band electrode immediately before and 180 s after the ac voltage for n-DEP was applied to remove unbound cells. We calculated each point of cell numbers as an average of three data points. Cell Binding Assay under Static Condition. A buffer containing 20 μg mL 1 of anti-CD33 was introduced into a 96-well microtiter plate (Thermo Fisher Scientific Inc., Waltham, MA) for 16 h at 4 °C to immobilize the antibody on the surface by physical adsorption. After the plate was washed with PBS, the plate was treated with 10 mg mL 1 BSA for 2 h at 4 °C to prevent nonspecific adsorption. After a second washing, the cell suspension (3  106 cells mL 1) was applied to the wells and incubated at 37 °C under 5% CO2. The plate was gently washed to remove unbound cells. The number of captured cells was calculated using microscope images.

’ RESULTS AND DISCUSSION Cell Manipulation by p- and n-DEP. Dielectrophoretic patterning with HL60 cells was studied using a patterning device

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Figure 2. Series of optical images showing cell manipulations by p- and n-DEP (A) before and (B) 5 s after the application of the ac voltage (intensity, 15 Vpp; frequency, 10 MHz) for p-DEP manipulation and (C) 2 s and (D) 10 s after the frequency was switched to 100 kHz for n-DEP manipulation. (E) Cross-sectional view of the numerically calculated electric field formed in the patterning device calculated from the digital simulation. Upper and lower gray bars show the ITO electrode and the band electrode, respectively.

consisting of an ITO electrode and a band electrode without antibody immobilization. We introduced a suspension (conductivity, 80 mS m 1) of HL60 cells in the patterning device and subsequently applied an ac voltage of 15 Vpp at a frequency of 10 MHz to induce accumulation of cells on the band electrode using p-DEP. Figure 2A,B shows the optical microscope images before and 5 s after application of the voltage, respectively. The cells dispersed randomly in the channel (Figure 2A) rapidly accumulated on the band electrode within 5 s (Figure 2B) of the application. After the frequency was switched to 100 kHz, most of the cells moved toward the gap region because of the repulsive force caused by n-DEP, resulting in the formation of another line pattern within 10 s (Figure 2B D). Moreover, the formation of line patterns reproduced as the frequency was switched between p- and n-DEP. The results clearly indicated that the different cell patterns could be easily created by applying voltages of different frequencies. Figure 2E shows the cross-sectional area of the electric field formed in the patterning device calculated from digital simulation in which a voltage is applied to the ITO electrode and the band electrode. Regions with high electric field were found in areas at the edge of the bands (areas labeled as P in Figure 2E). Thus, the suspended cells moved to area P when the ac voltage in the p-DEP frequency region was applied. However, regions with low electric field were found in the gap region between the band electrodes (areas labeled as N in Figure 2E). These results indicated that the application of the ac voltage in the p- or n-DEP frequency region to the electrode forced the cells to move to area P or N, which was in good agreement with the experimental results (Figure 2A D). Characterization of Dielectrophoretic Force Induced on HL60. The operating direction of the dielectrophoretic force on HL60 cells was studied at various frequencies and shown in Figure S1 in the Supporting Information. The ac voltage (15 Vpp) 7209

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Figure 3. Optical images of cells patterned by p- and n-DEP: (A) cells accumulated on the band electrode modified with anti-CD33 by p-DEP; (B) cell pattern captured on the band electrode modified with antiCD33 after separating unbound cells by n-DEP; (C) cells accumulated on the band electrode modified with antimouse IgG by p-DEP; and (D) cell pattern captured on the band electrode modified with antimouse IgG after separating unbound cells by n-DEP. Conductivity of the medium, 80 mS/m; applied voltage and frequency for p-DEP, 15 Vpp and 10 MHz; applied voltage and frequency for n-DEP, 5 Vpp and 100 kHz; duration of voltage application p-DEP and n-DEP, 60 and 180 s.

was applied in solutions with different conductivities, which were prepared by adding 10 mM PBS to the DEP medium. In solutions with low conductivities (0.2 mS m 1), the attractive force of p-DEP was derived at all frequencies. In solutions with middle conductivities (10 100 mS m 1), the attractive force was derived at higher frequencies. As the conductivity of the solutions increased, the crossover frequencies shifted to the higher frequency region. However, in solutions with high conductivities (>500 mS m 1) no attractive force was observed, indicating that the crossover frequencies shifted to frequencies beyond the experimental region. Therefore, we selected conductivity (10 100 mS m 1) for manipulation of cells by both p- and n-DEP. The results obtained corresponded well to the calculation of the real portion of the Clausius Mossotti factor by which the direction of the dielectrophoretic force was decided in a previous report.36 Unfortunately, nonspecific cells treated with anti-CD33 exhibit the similar DEP behavior in all frequency regions used in this study; hence, the modification of the cell surface with antibody did not allow for a sufficient difference for DEP properties. Capture of HL60 Cells with CD33 Antigen Based on Manipulation by DEP. We investigated the number of cells accumulated on the band electrode using cell binding efficiency. Figure 3 shows the optical images of cells accumulated on the band electrode (Figure 3A,C) and the captured cells after the separation of unbound cells by n-DEP (Figure 3B,D). Again, the uniformly dispersed cells initially started to move toward the band electrode to form clear line patterns because of a strong attractive p-DEP force (Figure 3A,C). The number of cells attracted to the bands increased with the duration of voltage application and saturated within 5 s. Surface modification by the antibody had almost no significant effect on accumulation behavior of the cells. However, removal of cells was markedly inhibited by the use of band electrode modified with anti-CD33, which corresponded to

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Figure 4. Investigation of the optimal voltage duration for cell accumulation by p-DEP. Cell binding efficiency was plotted 180 s after the ac voltage was switched for manipulation by n-DEP. Modified antibodies: (a) anti-CD33 and (b) antimouse IgG.

the CD33 surface antigen expressed on HL60 cells. When the cells were accumulated on the band electrode modified with antimouse IgG, almost all the cells moved to the gap region after the frequency was switched to the n-DEP region frequency after 60 s (Figure 3D). In contrast, some cells accumulated on the band electrode modified with anti-CD33 remained even after the frequency was switched for n-DEP (Figure 3B). These results suggested that the cells with CD33 surface antigens react with the antibodies to irreversibly capture at that position. The number of cells on the band electrode rapidly decreased within 30 s after the frequency was switched and then reached a steady-state value. The time course of accumulation and removal of the cells is shown in Figure S2 in the Supporting Information. The cell binding efficiency was estimated from the steady-state value and found to be 46.9 ( 3.4%. Slight undesired binding originating from the nonspecific adsorption was observed on the bands modified with antimouse IgG (8.7 ( 1.5%). Time as short as 30 s was required for removing unbound cells. Therefore, the cells with CD33 cell surface antigens can be rapidly identified from the cell suspension by spatial separation based on immunoreaction and manipulation by DEP. The effects of intensity of the applied voltage were studied to optimize the cell binding assay with manipulation by DEP and summarized in Figure S3 in the Supporting Information. In the investigation of the applied voltage for p- and n-DEP, the highest cell binding efficiency captured by specific immunoreactions were obtained from the application of 15 Vpp and 5 Vpp, respectively. We also investigated the effect of the duration of ac voltage application for p-DEP accumulation. We applied an ac voltage (15 Vpp) for different durations in a range from 30 s to 10 min to accumulate cells for p-DEP. As described above, the cells formed patterns within approximately 5 s after voltage application and remained constant for subsequent voltage application. During this immunoreaction time, the capture of cells proceeded at the band electrode. The applied frequency was then switched for removal of unbound cells, and the cell binding efficiency was measured. Figure 4 shows the cell binding efficiency 180 s after the ac voltage was switched. The application of the ac voltage for p-DEP for 30 s was sufficient to ensure the capture of cells at the band electrode modified with anti-CD33 (Figure 4a). Signal intensity linearly increased with increasing immunoreaction time. Furthermore, the cell binding efficiency on the band 7210

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Figure 5. Cell binding to the band electrode modified with anti-CD33 from mixed suspensions of specific and nonspecific HL60 cells. Treated cells were stained with a fluorescent molecule (CFDA SE). Photographs were obtained by combining the optical and fluorescent images. Initial ratios in the original suspensions were set at (A) 0% and (B) 50% of the specific cells. (C) Ratio of the cells captured on the band electrode.

electrode modified with antimouse IgG linearly increased at a lower level with the duration of ac voltage application for p-DEP (Figure 4b). The slope obtained in Figure 4b is very close to that in Figure 4a. These results clearly indicated that the cells directed and in contact with the band electrode specifically reacted within 30 60 s with the modified antibody and were then nonspecifically adsorbed. Therefore, the duration of 30 60 s is sufficient to capture the cells via immunoreactions. Subsequently, we investigated the effect of the electric fields on cell viability by dynamic cell staining with PI. HL60 cells were successively manipulated by p-DEP (15 Vpp at 10 MHz) for 60 s and n-DEP (5 Vpp at 100 kHz) for 180 s. The ratio of PI-positive (dead) cells was estimated immediately before and 180 s after the application of the ac voltage for n-DEP and found to be 2.5 ( 0.7% and 2.5 ( 0.8%, respectively. The ratios evaluated were nearly the same as those of the stained cells in the original cell suspensions. The results suggested that cell viability is not adversely affected by manipulation by DEP under the optimal conditions of ac voltage and duration. Cell Binding Using a Microtiter Plate. To confirm the rapidity of the present method and the conductivity required for the assay, the cell binding assay was performed using a microtiter plate modified with anti-CD33. HL60 cells suspended in 300 mM PBS (conductivity, 1.3 S m 1) were introduced and incubated in microwells modified with anti-CD33 or antimouse IgG. No cell was captured on the plate modified with anti-CD33 for 10 min (see Figure S4A,b in the Supporting Information). The captured cell density gradually increased with increasing incubation time and was saturated at 30 min (see Figure S3A,a in the Supporting Information). We also investigated the effect of conductivity of the cell suspension. The cells were incubated in PBS with different conductivities for 30 min. In solutions with high conductivities (over 200 mS m 1), the cells were sufficiently captured on the microwells (see Figure S3B in the Supporting Information). However, the captured cell density decreased with decreasing conductivity and almost no cells were observed

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at 70 mS m 1. Therefore, at least 80 mS m 1 was required to capture cells on the surface via immunoreactions. Unfortunately, it is difficult to use a medium with such high conductivity that is optimal for cell binding with the immobilized antibody because manipulations by both p- and n-DEP are indispensable for the present strategy. Nevertheless, the time required for the detection of surface antigens is dramatically decreased to only 60 s. Effect of Nonspecific Cells to Specific Cell Binding. Mixtures of HL60 cells specific and nonspecific to anti-CD33 were used to determine the number of cells captured to the band electrode modified with anti-CD33. Figure 5A,B shows photographs obtained by combining optical and fluorescent images, which were obtained 180 s after the switching of the direction for n-DEP. Almost no nonspecific cells with a fluorescent signal were captured on the band electrode, whereas the specific cells were captured on the band electrode, even in the presence of treated cells (Figure 5B). Figure 5C shows the ratio of cells captured on the band electrode. The ratio of the captured cells linearly increased with the increasing ratio of specific cells in the prepared mixture suspension. The results indicated that the presence of the cells without the target antigen does not obstruct specific cell binding from detecting cells with surface antigens. Furthermore, the linear relationship could be useful to determine the present ratio of cells with surface antigen in the suspension. Unfortunately, the relatively high binding efficiency of nonspecific cells (i.e., at 0% specific) was observed. This might be due to the increase of the contact area of the cell surface to the capture element by the flat deformation of cells for the strong DEP suppression.

’ CONCLUSIONS We propose a novel method of a rapid and simple cell binding assay based on the manipulation of cells by DEP. On application of the ac voltage at a frequency in the p-DEP region, cells were forced to accumulate on the band electrode yielding line formations. Cells were repelled from the band electrode and formed another line pattern in the gap region between the band electrodes after the force direction was switched in n-DEP. Therefore, the cells accumulated by p-DEP were easily directed to another site by controlling the DEP forces by frequency. The formation of the second line pattern by the cells was markedly inhibited by immunoreactions between surface antigens and the specific antibody immobilized on the microband array electrode. HL60 cells with surface antigen (CD33) were irreversibly captured on the band electrode modified with a specific antiCD33; as a result, the captured cells were not removed after the application of a negative dielectrophoretic force. Importantly, the capture of cells through immunoreactions was assisted by rapid accumulation with p-DEP regulation on the band electrode. This immunoreaction at the surface was accelerated by p-DEP and completed within 30 s, whereas a long incubation time was required to capture cells through antibodies immobilized on the microtiter plate. The time required for the separation of unbound cells by n-DEP was estimated by rapid discrimination systems and found to be around 30 s. The total time required for detecting the antigens expressed on the cell surface was as short as 60 s. Moreover, no pretreatments, such as cell staining with fluorescent molecules, are required for the proposed method. The preparation of the antibody array with the microband electrode may allow the appearance of multiple assay systems combined with rapid DEP manipulation methods. In addition, 7211

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’ ASSOCIATED CONTENT

bS Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author

*Tomoyuki Yasukawa: phone, +81-791-58-0171; fax, +81-79158-0493; e-mail, [email protected]. Fumio Mizutani: e-mail, [email protected].

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dx.doi.org/10.1021/ac201789m |Anal. Chem. 2011, 83, 7207–7212