Sensitive Mesofluidic Immunosensor for Detection of

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21 Sensitive Mesofluidic Immunosensor for Detection of Circulating Breast Cancer Cells onto AntibodyCoated Long Alkylsilane Self-Assembled Monolayers François Breton and Phuong-Lan Tran Contents 21.1 Introduction................................................................................................................................... 377 21.2 Materials....................................................................................................................................... 379 21.3 Methods......................................................................................................................................... 379 21.3.1 Glass Surface Silanization................................................................................................ 379 21.3.2 Physical Characterization of the Silane Films and Quality Control................................ 379 21.3.3 Physical Characterization of Antibody-Coated Surfaces................................................. 380 21.3.4 Characteristics of Laminar Flow and Simulation in a Parallel Plate Flow Chamber...... 380 21.3.5 Isolation of MCF7 Breast Cancer Cells Spiked in Normal Blood Leukocytes................381 21.3.6 Immunofluorescence Staining and Identification of MCF7 Cells by Fluorescence Microscopy....................................................................................................................... 382 21.4 Results........................................................................................................................................... 382 21.4.1 Physical Characteristics of the Antibody-Coated AHTS Surface................................... 382 21.4.2 Simulation of the Fluid Flow in a Single Parallel Plate Laminar Flow Chamber........... 383 21.4.3 Isolation of MCF7 Breast Cancer Cells Spiked in Background Normal Blood Leukocytes....................................................................................................................... 383 21.4.4 Identification of MCF7 Breast Cancer Cells Spiked in Normal Blood Leukocytes........ 385 21.5 Discussion..................................................................................................................................... 385 21.6 Future Trends................................................................................................................................ 386 Acknowledgments................................................................................................................................... 386 References............................................................................................................................................... 387

21.1  Introduction Breast cancer is the first leading cause of cancer death in women. Release of tumor cells into blood circulation may occur at early stage of the disease and may be responsible for its progression [1]. Metastasis in breast cancer patients leads to cancer-related death because early dissemination of tumor cells usually remains undetected at initial diagnosis on clinical, imaging, and biochemical examination. Thus, detection of circulating tumor cells (CTCs) in a blood sample becomes of potential value despite their rarity (median, ≤1 CTC/mL). CTCs have been successfully detected and isolated from blood in patients with metastatic carcinomas [2,3]. A simple blood test would allow the detection and analysis of CTCs to be frequently repeated. It would also help to noninvasively stage the disease at diagnosis as well as to monitor therapy and long-term patient management [4,5]. Several reports have shown that efficacy of treatment could be measured by the number of CTCs in blood [6–8]. CTCs provide easy access to patients’ cancer cells for performing several molecular analyses. Studies on alterations in CTC oncogenes suggested

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heterogeneity among cancer cells from a single patient, for example, with respect to heterogeneity of HER-2 and uPA gene copy number and expression in breast cancer [9,10]. Thus, identifying the CTC subpopulations contained in a blood sample could help to identify targets for treatment and exploring mechanisms that underlie metastases and drug sensitivity. Increasing the useful information on CTCs might help to tailor systemic therapies to the individual needs of a cancer patient. During the past decades, most research on CTCs has been focused on the development of reliable methods for CTC enrichment and identification, which could overcome severe technical limitations [11–14]. The most currently used methods for CTC detection include quantitative immunomagnetic separation followed by immunocytochemistry detection [2,3,15,16] or RT-PCR to indicate the qualitative presence of CTCs in peripheral blood [17–19]. EpCAM is an epithelial cell adhesion molecule antigen that is overexpressed in breast cancer metastases [20]. Immunomagnetic beads coated with EpCAM antibody facilitate CTCs isolation. All cells that are magnetically labeled are identified by cytokeratin positivity, DAPI nuclear staining, and CD45 (leukocyte-specific antigen) negativity (Cell Search System, Veridex, Johnson & Johnson, Raritan, NJ). Other techniques used to isolate and enumerate CTCs in blood samples are based on cell size selection such as cell filtration [21] and flow cytometry [22,23]. Alternative methodologies addressing microfluidic procedures (i.e., at a micrometer scale) have been reported. CTC detection is performed on CTC-Chips made within a 967 mm2 surface of 78,000 functionalized microposts (diameter × height: 100 μm × 100 μm) coated with anti-EpCAM antibody [24], in a biochip consisting of microchannels also coated with anti-EpCAM antibody [25], or in a label-free microdevice separating CTCs from blood constituents on biorheological property differences [26]. Most existing techniques are associated with protocols that allow capturing only cancer EpCAMpositive epithelial cell population in peripheral blood. This could be a limiting factor and leads to missing information on cancer EpCAM-negative cell subpopulations if more than one cancer cell subpopulations are present [27]. Therefore, an advanced tool for detection of various breast cancer cell subpopulations for “tailored” therapy should be investigated [28]. Faced up to challenge a highly sensitive diagnostic method for cancer disease, we developed a new mesofluidic (i.e., at a millimeter scale) immunosensor to immobilize breast cancer cells based on antigen-mediated adhesion of cells to specific antibody-binding surfaces, in a laminar flow field, using a parallel plate, millimeter scale, laminar flow chamber for fluid circulation [29,30]. The floor of the flow chamber can be achieved by surface chemical patterning for cell immobilization. Bioengineered surfaces have proved their performances in chip technology for immobilization of proteins including antibodies, DNA, or cells [31–34]. Self-assembled monolayers (SAMs) have drawn attention to devise such surfaces due to their promising surface properties [35]. Thus, surface chemical patterning can be functionalized by grafting long-chain organosilicon compounds on a Si/SiO2 solid support to form dense organized SAMs (for a review, [36]). The quality of such functionalized surfaces is dependent on a wide variety of parameters including chain length, temperature, solvent, and reaction time. It has been shown that C22-derivatized SiO2 surfaces are readily applied to immobilize covalently oligonucleotides [37,38] or red blood cells [39]. Therefore, surface functionalization of the flow chamber floor is carried out by grafting long alkyl organosilicon chain, 21-aminohenicosyl trichlorosilane (AHTS), onto a standard microscopy glass slide to form dense organized SAMs. Control quality of the homogeneous AHTS SAMs displayed an excellent resistance to both physical constraints (temperature and shear stress) and chemical constraints (UV and hydrolysis), as well as preservation of the antibody functional activity [29,37,40]. Then tethering EpCAM antibody to AHTS SAMs provides selectivity and specificity of cell capture [29,41]. Accordingly, (1) design of the flow chamber at a millimeter scale, hence the so-called mesofluidic designation, enables to reduce the rheological phenomena not yet well-mastered in microfluidics; (2) optimization of the flow kinetics of the immunosensor for minimal shear forces, and maximal contact between cells and the active surface, provides high yield of captured MCF7 breast cancer cells spiked in background leukocytes [29] or metastatic breast cancer CTCs [30]; and (3) simultaneous combination of, in parallel, four independent parallel plate laminar flow chambers [30], onto which each independent surface is grafted with a specific monoclonal antibody, would allow patterning various antibodies for capturing breast cancer CTC subpopulations in a single blood sample.

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21.2  Materials 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and (2-[morpholino]ethanesulfonic acid) (MES)-buffered saline, 0.09% NaCl, pH 4.7, were purchased from Perbio Science (Brebières, France), and human serum albumin (HSA) from Sigma-Aldrich (Saint Quentin Fallavier, France). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI), fetal calf serum (FCS), penicillin, streptomycin, and Hank’s balanced salt solution (HBSS) were supplied by Gibco BRL (Invitrogen, France). The antihuman EpCAM antibody was purchased from R&D Systems (Lille, France), and Alexa-conjugated goat anti-rabbit IgG and Alexa-conjugated goat anti-mouse IgG from Molecular Probes (Invitrogen, France). ACCUSPIN tubes were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France), and EDTA Vacutainer tubes from Becton Dickinson (Pont de Claix, France).

21.3  Methods 21.3.1  Glass Surface Silanization Glass substrate and silane film preparation were performed as previously described [29,37,40]. Standard microscopy glass slides were first silanized under argon atmosphere with N-(21-trichlorosilanylhenicosyl) phthalimide (N-protected AHTS), then deprotection of the amino group of AHTS was performed as described [29,37,40]. Figure 21.1A displays both N-protected AHTS and AHTS after deprotection.

21.3.2  Physical Characterization of the Silane Films and Quality Control Quality controls of the silanized surfaces were performed as described [29,40]. Fourier transform infrared (FTIR) spectroscopy was used as a quantitative quality control tool for routine monitoring of the buildup process of the films (Nicolet FT/IR Nexus 870 apparatus equipped with a mercury–cadmium telluride [MCT] detector and purged with dry air). Figure 21.1B shows the FTIR spectrum of long alkyl chains AHTS and displays both methylene antisymmetric (νasCH2) and symmetric (νsCH2) stretching modes near 2851 and 2923 cm−1, respectively, which indicate that N-protected AHTS forms quasi-compact and

O Cl3Si

N 21 O

N-Protected AHTS

(A)

Cl3Si

NH2 21

Transmittance (%)

100.1 100 99.9 99.8

99.6 3000

AHTS

(B)

Before deprotection After deprotection

99.7 2950

2900

2850

Wavenumbers (cm–1)

2800

FIGURE 21.1  (A) Representation of N-(21-trichlorosilanylhenicosyl)-phthalimide (N-protected AHTS) and 21-aminohenicosyl trichlorosilane (AHTS). (From Navarre, S. et al., Langmuir, 17, 4844, 2001; From Bennetau, B., Bousbaa, J., and Choplin, F. CNRS FR Patent 0000695.) (B) Fourier transform infrared (FTIR) spectra for grafted long AHTS film on glass slide, respectively, before deprotection (straight line) and after deprotection (dotted line) of the amino group. (From Biosens. Bioelectron., 24, Ehrhart, J.C., B. Bennetau, L. Renaud, J.P. Madrange, L. Thomas, J. Morisot, A. Brosseau, S. Allano, P. Tauc, and P.L. Tran., A new immunosensor for breast cancer cell detection using antibody-coated long alkylsilane self-assembled monolayers in a parallel plate flow chamber, 467–474, Copyright 2008 Elsevier.)

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ordered monolayers. After deprotection (Figure 21.1B, dotted line), the FTIR spectrum shows a shift of the CH2 vibration bands (νasCH2 and νsCH2) to 2921 and 2850 cm−1, respectively. This shift clearly indicates a better order in the alkyl chains when the protecting group is removed. Modified surfaces by AHTS were studied for their wetting properties by contact angle measurements using a Krüss goniometer. Droplets (1.5–2 μL) of Nanopure water were placed randomly on the surface and contact angle values were determined within 1 min after droplet deposition. The value of contact angles was observed at 61.4° ± 1.0°, proving optimal spreading of water on AHTS surface. The physical characterization and quality control of silane films were also performed by atomic force microscopy (AFM) using a modified Digital Instruments contact NII head and a NanoscopeEcontroller [40]. For the amplitude modulation mode, a lock-in amplifier (Perkin-Elmer 7280 DSP) produces the excitation of the cantilever piezoceramic and records the variations of amplitude and phase [40]. The frequency modulation data are recorded with a Nanosurf electronics including a phase lock loop. All the data were recorded using Si cantilevers (average stiffness of about 50 N/m), a resonance frequency around 150 kHz, and a quality factor of Q ≈ 400 at 200 nm from the surface. The images were recorded with an “NCLW” tip and approach-retract curves with “Supersharp” tips [40].

21.3.3  Physical Characterization of Antibody-Coated Surfaces Antibody immobilization on the AHTS-grafted glass surface was performed with the monoclonal antihuman EpCAM antibody in MES-buffered saline, 0.09% NaCl, pH 4.7 at a concentration of 200 μg/mL in the presence of EDC, overnight at 4°C. All slides were then washed in phosphate-buffered saline (PBS) and stored at 4°C under Ar atmosphere up to 1 month until use. Quality controls were performed as described [29]. Antibody-coated AHTS slides were incubated with a secondary goat anti-mouse Alexa-conjugated IgG overnight at 4°C. After thorough washing in PBS, the slides were analyzed by AFM using the Explorer microscope (VEECO, Santa Barbara, CA) in tapping mode. A soft Si cantilever (AURORA NanoDevices Inc., Nanaimo, Canada) was used with a nominal force constant of ∼40 N/m, a resonance frequency of 300 kHz, and a tip radius

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