Materials & Methods
Reagents
All chemicals were purchased from Sigma-Aldrich (MO, USA) unless specified otherwise. QSY21 was purchased from Life Technologies (NY, USA). Anti-EpCAM and anti-HER2 monoclonal antibodies were purchased from AbCAM (MA, USA). Carboxy-poly(ethylene)-thiol (HOOC-PEG-SH, molar weight: 5000) and methoxy-PEG-thiol (mPEG-SH, molar weight: 5000) were purchased from Laysan Bio, Inc. (AL, USA). SK-BR-3 cells were purchased from ATCC (VA, USA). Human whole blood was purchased from Research Blood Components, LLC (MA, USA). IO NPs (SHP 25) were obtained from Ocean Nanotech (AR, USA).
Synthesis & Characterization of IO–Au Nanoovals
IO–Au nanoovals (NOVs) were synthesized using a seed-mediated growth method[20] with modifications. First, 10 µl of 10 mM diamminesilver ions (Ag[NH3]2 +), which were prepared by mixing ammonia with silver nitrate (AgNO3), were added to 100 µl of 1 mg/ml negatively charged polymer-coated IO NPs and stirred for 30 min. The Ag+-adsorbed IO NPs were purified by centrifugation (10,000 rpm, 8 min) and reconstituted with 0.25 ml water, followed by addition of 100 µl of 10 mM sodium borohydride (NaBH4) to form Ag-decorated IO NPs. After 40 min, the Ag-decorated IO NPs were purified by three centrifugations and washings and redispersed in 500 µl water. Second, 1.5 ml of growth solution containing 0.4 mM chloroauric acid (HAuCl4), 0.1 M cetyltrimethylammonium bromide and 0.08 mM AgNO3 were prepared, followed by addition of 23 µl of 40 mM ascorbic acid. Then, 22 µl of the Ag-decorated IO NP solution was injected and the solution changed to a purple color within a few minutes, indicating the growth of IO–Au NOVs. The absorption spectra of the NPs were measured using a VIS-NIR absorption spectrometer (Ocean Optics, FL, USA). The magnetic properties were measured using a vibration sample magnetometer (Dexing Magnets, China). The size and morphology of the NPs were examined with a JEM1200EX® II transmission electron microscopy (TEM; JEOL Ltd, Japan).
Preparation & Characterization of Antibody-Conjugated IO–Au SERS NOVs
First, 50 µl of 0.1 mM QSY21 was added to 1 ml of 0.1 nM IO–Au NOVs (QSY21:IO–Au = 50,000). The mixture was vortexed in the dark for 15 min to allow the adsorption of the dye onto the NPs. This was followed by the addition of 20 µl of 0.05 mM HOOC-PEG-SH (HOOC-PEG-SH:IO–Au = 10,000). The bifunctional PEG was attached to the NPs via Au–S bonds. After vortexing for 20 min, 10 µl of 0.05 mM mPEG-SH (mPEG-SH:IO–Au = 5000) was added to saturate the surface of the NPs. The mixture was vortexed for 1 h in the dark at room temperature. The functionalized IO–Au SERS NOVs were centrifuged and washed three times (10,000 rpm, 10 min) to separate unbound molecules. The NPs were redispersed in 100 µl of pH 5.5 2-(N-morpholino)ethanesulfonic acid buffer for ligand conjugation. To conjugate anti-EpCAM or anti-HER monoclonal antibodies to the IO–Au SERS NOVs, 3 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 3 mg of sulfo-N-hydroxysuccinimide (sulfo-NHS) were added to 100 µl of 1 nM functionalized IO–Au SERS NOVs in pH 5.5 2-(N-morpholino)ethanesulfonic acid buffer. The mixture was vortexed for 15 min, followed by the addition of 900 µl of PBS and centrifugation (10,000 rpm, 10 min). The NP pellet was redispersed in 200 µl of PBS, followed by the addition of 50 µl of 0.2 mg/ml of antibodies. EDC and sulfo-NHS were used as cross-linking agents to couple carboxyl groups on the NPs to primary amines on the antibodies. EDC reacts with -COOH groups to form O-acylisourea intermediates. The intermediate further reacted with sulfo-NHS to form a semistable amine-reactive NHS-ester. The solution was vortexed for 2 h at room temperature to complete the coupling reaction, and then stored at 4°C. Prior to use, the solution was centrifuged and washed. The antibody-conjugated IO–Au SERS NOVs were resuspended in 100 µl of PBS. Surface modification at each step was monitored by dynamic light scattering measurement with a Particle Size Analyzer (Brookhaven Instruments Corp., NY, USA).
Construction of an Integrated System for On-line Cell Isolation & Detection
The major components of the system were a syringe pump (New Era Pump Systems Inc., NY, USA), a quartz capillary (inner diameter = 100 µm, outer diameter = 110 µm), two cylindrical neodymium–iron–boron magnets (K&J Magnetics Inc., PA, USA) and a portable Raman spectrometer (Enwave Optronics, CA, USA). The capillary was connected to the syringe with a plastic cap. Magnet-1 was 20 mm in diameter and 25 mm in thickness, with a surface field of 4800 Gauss. Its function is to separate and capture tumor cells under a high flow velocity (>3 cm/s). Magnet-2 was 0.2 mm in diameter and 3 mm in thickness, with a surface field of 500 Gauss. Its function is to capture and confine the purified tumor cells at low flow velocity (<0.5 cm/s) to a fine region for SERS detection. It was fixed in a styrofoam holder. Both magnets were set up on the motorized XYZ stage of the Raman spectrometer. The capillary was tightly attached to the top side of the magnets. During cell capture and detection, the two magnets were attached to the system one at a time. The Raman spectrometer has an excitation laser with a wavelength at 785 nm and adjustable power up to 250 mW. The laser beam spot is 200 µm at its focus.
Cell Culture & Labeling
Human breast cancer cells SK-BR-3 were cultured in RPMI 1640 medium with 10% fetal bovine serum at 37°C under 5% CO2. To label cells with the antibody-conjugated IO–Au NPs, 10,000 SK-BR-3 cells in 1 ml of PBS were incubated with 5 pM anti-EpCAM/QSY21/IO–Au NPs and 5 pM anti-HER2/QSY21/IO–Au NPs for 30 min with gentle vortexing at room temperature. The cells were purified by repeated centrifugation and washing (1500 rpm, 3 min). Unconjguated IO–Au SERS NPs were used as the control. The NP-treated cells were fixed with 4% paraformaldehyde. Cellular binding was examined by dark field imaging with an Olympus® IX71 inverted microscope (Olympus America, PA, USA).
Determination of Capture Efficiency
To determine the capture efficiency of tumor cells by magnet-1, SK-BR-3 cells were labeled with a cocktail of anti-EpCAM/IO–Au SERS NOVs and anti-HER2/IO–Au SERS NOVs, as described above. A total of 1 ml of PBS containing 10,000 prelabeled cancer cells were introduced into the flow system and pumped through the capillary at a variety of flow velocities in the presence of magnet-1. Cells in a stock PBS solution were counted with a hemocytometer and then diluted with PBS to achieve the cell solution with the desired cell number. Uncaptured cells were quantified by cell counting with a hemocytometer. To determine the capture efficiency of the NPs by magnet-1, 1 ml of 10 pM mPEG-stabilized IO–Au NOVs was introduced into the flow system and pumped through the capillary at a variety of flow velocities in the presence of magnet-1. The uncaptured NPs were quantified by absorption spectroscopy. The capture efficiency of tumor cells or NPs was presented as the percentage of trapped cells or NPs with respect to the loaded tumor cells or NPs.
Detection of Prelabeled SK-BR-3 Cells Spiked Into Whole Blood
SK-BR-3 cells were dispersed in PBS and labeled with a cocktail of 5 pM anti-EpCAM/QSY21/IO–Au NOVs and 5 pM anti-HER2/QSY21/IO–Au NOVs, as described above. After purification and fixation, the cells were redispersed in PBS and counted with a hemocytometer. The cells were subject to a series of dilutions with PBS or human whole blood to make 1-ml solutions containing 10, 20, 50, 100, 250 or 500 cells. The sample was transferred to a 1.0-ml syringe and placed on a syringe pump. With magnet-1 in position, the solution was pumped at 6 cm/s. After 10 min, when all the solution was pumped, a vial containing fresh PBS was placed at the end of the capillary. A total of 100 µl of PBS was pulled from the vial to resuspend the purified tumor cells while magnet-1 was removed. Then, magnet-2 was placed under the capillary and the tumor cells were pumped through the capillary at 0.2 cm/s. After 30 min, when all the solution was pumped, the Raman spectrometer was turned on and the SERS spectrum was collected in real time (10 ms per spectrum). The position of magnet-2 was adjusted so that the tumor cells were all exposed to the laser beam, giving maximal signals. The focus of the laser was further adjusted to optimize the SERS signals. PBS or whole blood only were used as the negative controls. The SERS spectrum with the maximal intensity for each sample was used for quantitative studies. The spectrum was baseline corrected to subtract the SERS background (broad continuum emission) using a multisegment polynomial fitting. The plot of SERS intensity at 1496 cm−1 versus the cell number was fitted linearly using OriginPro® 8 (OriginLab Corp., MA, USA). The LOD was determined to be the cell concentration required to give a signal equal to the negative control plus three times that of the standard deviation of the negative control.
Detection Of SK-BR-3 Cells Spiked Into PBS & Into Whole Blood
A total of 1 ml of PBS or human whole blood containing 10, 20, 50, 100, 250 or 500 fixed SK-BR-3 cells was incubated with 5 pM anti-EpCAM/QSY21/IO–Au NOVs and 5 pM anti-HER2/QSY21/IO–Au NOVs for 30 min with gentle vortexing at room temperature. The mixture was then transferred into the flow system and subjected to isolation and detection using the procedures described above. PBS or whole blood containing the same concentration of conjugated NOVs but not tumor cells was used as the negative control. The plot of SERS intensity at 1496 cm−1 versus the number of SK-BR-3 cells was fitted linearly using Origin 8 and the LOD was calculated as the number of cells that gave a signal equal to the negative control plus three times that of the standard deviation of the negative control.
Nanomedicine. 2014;9(5):593-606. © 2014 Future Medicine Ltd.
