Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 2. SERS spectra of aqueous solutions of NAADP, cADPR,
and IP3. The bottom spectrum was collected from the mixture of all
three Ca 2+ mobilizing second messengers (3 µM concentration of
each). The excitation laser wavelength was 633 nm. The data
acquisition time was 10 s. The data were collected immediately after
1 µL of the sample was placed on the SERS sensor. The spectra
are normalized and offset for clarity.
colloid. The average interparticle distance is controlled by the time
that the substrates are exposed to the gold colloid. Here, the
average distance is approximately 75 nm. The UV-vis spectrum
of the SERS-enabled substrate, with the maximum extinction at
around 540 nm, is shown in Figure 1c.
The selectivity of the SERS sensor for calcium messengers
was studied with three different samples with 10 µM concentration:
NAADP, IP3, and cADPR. The mixture of all three
messengers was also analyzed. Figure 1 shows that each
messenger has its characteristic SERS spectrum. Moreover, the
analysis of the mixture of all three messengers (bottom spectrum
in Figure 2) shows that it is possible to distinguish the features
of each component.
For example, the adenine moiety of NAADP represents itself
in the spectrum of the mixture with the 733 cm -1 peak, similar
to that observed in the spectrum of the control NAADP
solution. The contribution from cADPR and IP3 to the mixture
spectrum is confirmed by the presence of 898 cm -1 , 1257 cm -1
(amide II), and 1416 cm -1 (C-H stretch) peaks, which are
present in the spectra collected from pure solutions. The 898
cm -1 peak in the spectrum of cADPR can be attributed to
ribose. 17,28 Interestingly, although cADPR contains adenine, it
shows only as a weak signal at 733 cm -1 . This is likely due to
the circular structure of the cADPR molecule, where adenine
is located between two ribose groups. Further analysis of the
Raman spectra is beyond the scope of this work. The key result
demonstrated above is that detection of a specific analyte
(NAADP in our case) is clearly possible using spectral
signatures as a whole obtained from SERS-enabled substrates.
It is important to note that, for the enzymatic cycling assay,
samples must be purified from NADP, which interferes with
(28) Kneipp, J.; Kneipp, H.; Rice, W. L.; Kneipp, K. Anal. Chem. 2005, 77, 2381–
2385.
6772 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
Figure 3. SERS spectra of aqueous solutions of NAD, NADP, and
NAADP at a 100 µM concentration. The data were collected using
the 785 nm excitation laser; the signal acquisition time was 10 s.
NAADP detection. SERS, however, makes it possible to distinguish
between NAADP and its metabolites, such as nicotinamide
adenine dinucleotide (NAD), NADP, and cADPR. Such specificity
to the molecular structure is unattainable, as far as we know, by
any other technique previously applied for NAADP detection.
Figure 3 illustrates the difference between the SERS spectra of
NAD, NADP, and NAADP at a 100 µM concentration. Furthermore,
SERS substrates employed in this work can be utilized in
a wide range of excitation wavelengths. Figures 2 and 3 clearly
illustrate that SERS signals obtained with 633 and 785 nm
excitations have good signal-to-noise ratios. Therefore, the substrates
can be used in conjunction with different lasers, depending
on availability and on the demands of a particular application.
Multiwavelength spectroscopy can also be employed to further
improve differentiation of NAADP spectral signatures using
appropriate pattern recognition.
SERS Detection of an Agonist-Induced Change of the
NAADP Concentration in Cells. Next we studied an agonistinduced
change of the NAADP concentration in breast cancer
SkBr3 cells using the SERS sensor. An increase of the NAADP
concentration was triggered by treating cells with three different
agonists with a 5 µM concentration: ATP, acetylcholine, and
histamine. The protocol for inducing NAADP concentration
modulation results in a final NAADP concentration that is
significantly increased as compared to its basal level. 8,10 The latter
has been estimated to be on the order of tens of nanomolar. The
acid extraction protocol established in ref 8 was used to obtain
NAADP samples. Figure 4a shows the SERS spectrum collected
from the untreated cell extracts, denoted as the control, and those
of the treated cells (Figure 4b-d). Multiple spectra were acquired
from each sample for further analysis. Importantly, the data
collected with the SERS sensor show a good repeatability, as can
be seen in Figure 4. This is expected given the fixed configuration
of the gold nanoparticles on the sensor’s surface. 29,30
(29) Hering, K.; Cialla, D.; Ackermann, K.; Dorfer, T.; Moller, R.; Schneidewind,
H.; Mattheis, R.; Fritzsche, W.; Rosch, P.; Popp, J. Anal. Bioanal. Chem.
2008, 390, 113–124.