Analytical Chemistry Chemical Cytometry Quantitates Superoxide

on a substrate are preferred. 20-24 The SERS sensor employed in
this work is comprised of a glass substrate coated with gold
nanoparticles. Similar sensors fabricated in the form of glass
nanopipets have been recently demonstrated for minimally invasive
in situ intracellular SERS measurements. 14 In the future, the
results of this study could be potentially extended to NAADP
detection inside cells using the SERS-based approach enabled by
SERS-active nanopipets.
EXPERIMENTAL SECTION
Cell Culture. Breast cancer SkBr3 cells were grown in
McCoy’s 5A modified medium, supplemented with 10% fetal
serum, streptomycin, and penicillin. Cells were purchased from
ATCC.
Acid Extraction of NAADP. For the extraction of NAADP
we used the protocol reported by Lewis et al. 8 All chemicals were
purchased from Sigma-Aldrich. Briefly, SkBr3 cells were treated
with trypsin and suspended in the cell medium. Before treatment
with the agonists, cells were preincubated for 30 min with BAPTA-
AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl
ester). Then in the presence of BAPTA-AM
cells were stimulated for 20 min with the agonists, according to
the technique described in ref 8. This technique has been shown
to trigger the prolonged NAADP synthesis for at least 20 min
during the agonist stimulation. This results in highly amplified
NAADP production. Here we used histamine, adenosine triphosphate
(ATP), and acetylcholine, all at a 5 µM concentration. The
reaction was stopped by adding 0.75 M ice-cold HClO4. Next the
cells were disrupted by sonication and then kept on ice for 10
min. The disrupted cells were centrifuged at 9000g for 10 min.
Supernatant was neutralized with 1 M KHCO3 and vortexed.
The resulting KClO4 precipitate was removed by centrifugation
at 9000g for 10 min. Samples were stored at -80 °C for later
analysis.
Fabrication of the SERS Sensor. Microscope glass slides
were cut into 1 cm × 1 cm pieces and sonicated in a mixture of
NaOH and ethanol. After being washed with plenty of 15 MΩ
deionized water, the slides were dried at room temperature. Next
the slides were dip coated with 0.001% poly-L-lysine, dried at room
temperature for 24 h, and then coated with gold nanoparticles by
dipping them in the gold colloid for 3 h. Poly-L-lysine promotes
the adhesion of the gold nanoparticles to the glass surface. The
mechanism of nanoparticle attachment is based on the electrostatic
interaction between the negatively charged particles and
(15) Vo-Dinh, T.; Yan, F.; Wabuyele, M. B. J. Raman Spectrosc. 2005, 36, 640–
647.
(16) Ivleva, N. P.; Wagner, M.; Horn, H.; Niessner, R.; Haisch, C. Anal. Chem.
2008, 80, 8538–8544.
(17) Kneipp, J.; Kneipp, H.; McLaughlin, M.; Brown, D.; Kneipp, K. Nano Lett.
2006, 6, 2225–2231.
(18) Chourpa, I.; Lei, F. H.; Dubois, P.; Manfait, M.; Sockalingum, G. D. Chem.
Soc. Rev. 2008, 37, 993–1000.
(19) Willets, K. A. Anal. Bioanal. Chem. 2009, 394, 85–94.
(20) McFarland, A. D.; Van Duyne, R. P. Nano Lett. 2003, 3, 1057–1062.
(21) Hartschuh, A.; Qian, H.; Meixner, A. J.; Anderson, N.; Novotny, L. Surf.
Interface Anal. 2006, 38, 1472–1480.
(22) Haynes, C. L.; Van Duyne, R. P. J. Phys. Chem. B 2003, 107, 7426–7433.
(23) Shoute, L. C. T.; Bergren, A. J.; Mahmoud, A. M.; Harris, K. D.; McCreery,
R. L. Appl. Spectrosc. 2009, 63, 133–140.
(24) Deckert, V.; Zeisel, D.; Zenobi, R.; Vo-Dinh, T. Anal. Chem. 1998, 70, 2646–
2650.
Figure 1. (a) Scanning electron micrograph of the SERS sensor,
(b) close-up view of the gold nanoparticles on the SERS sensor, (c)
extinction spectrum of the SERS sensor, and (d) SERS spectrum of
NAADP and a background spectrum from the substrate.
positively charged NH2 functional groups of poly-L-lysine. 14,25,26
After fabrication, the substrates were imaged with a scanning
electron microscope to confirm the nanoparticle distribution on
the surface. SEM images were collected with a field emission Zeiss
Supra 50VP scanning electron microscope at a low accelerating
voltage (0.7-2 kV) without any conductive coating. In addition,
the UV-vis extinction spectra of the substrates were measured
using a home-built setup employing a fiber-optic spectrometer,
HR-4000, Ocean Optics.
SERS Measurements. Raman spectroscopy was performed
using a micro-Raman spectrometer (Renishaw, RM 1000) equipped
with a 632.8 nm HeNe laser (1800 lines/mm grating) and a diode
InGaAs laser operating at 785 nm wavelength (1200 lines/mm
grating). The lasers are manufactured by Renishaw Inc., U.K. The
laser source was focused on the sample through a long working
distance 50× objective to a spot size of approximately 2 µm. The
typical sample volume was 1 µL. The acquisition time for all
spectra was 10 s. Data analysis was performed using the Renishaw
Wire 2.0 software. Experimental data were analyzed using principal
component analysis 27 in the Matlab environment.
RESULTS AND DISCUSSION
Testing the SERS Sensor for Distinguishing between
Different Secondary Ca 2+ Mobilizing Messengers: NAADP,
cADPR, and IP3. Figure 1a shows the SEM image of the SERS
sensor, with the close-up view presented in panel b. The average
diameter of the nanoparticles is on the order of 50 nm. Assembly
of the SERS sensor is based on the wet chemistry two-step
protocol. First, the glass substrates are coated with a positively
charged polymer (poly-L-lysine) layer. The functionalized substrates
are then coated with a monolayer of negatively charged
gold nanoparticles through electrostatic binding from the gold
(25) Freeman, R. G.; Grabar, K. C.; Allison, K. J.; Bright, R. M.; Davis, J. A.;
Guthrie, A. P.; Hommer, M. B.; Jackson, M. A.; Smith, P. C.; Walter, D. G.;
Natan, M. J. Science 1995, 267, 1629–1632.
(26) Nie, S.; Emory, S. R. Science 1997, 275, 1102–1106.
(27) Jackson, J. E. A User’s Guide to Principal Components; John Wiley: New
York, 1991.
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
6771