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