Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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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.
Figure 4. SERS analysis of NAADP concentration modulation in cell extracts. Each graph contains 12 spectra collected at different locations on the SERS sensor for each sample to demonstrate the data reproducibility. (a) SERS spectrum of the untreated cells, marked as the control. (b-d) SERS spectra of cell extracts with induced NAADP release by treating cells with (b) histamine, (c) ATP, and (d) acetylcholine. All three agonists had a concentration of 5 µM. The sample volume used in this experiment was on the order of 2 µL. The data acquisition time was 10 s. A 785 nm excitation laser was used. The spectra are normalized and offset for clarity. Figure 5. (a) Concentration-dependent SERS spectra of an aqueous solution of NAADP. The bottom spectrum represents the background signal from the SERS sensor. (b) Principal component analysis of SERS data collected on cells treated with acetylcholine, an aqueous solution of 100 µM NAADP, and untreated control cells. Each point in the principal component space represents an SERS spectrum with the distance between data points proportional to the degree of similarity between the spectra. (c) Pareto chart showing the amount of information about data variability explained by the first two principal components. The results demonstrate that there is a strong correlation between the SERS spectra of cells with the modulated NAADP concentration and that of the pure 100 µM NAADP solution. In order to quantify the NAADP concentration in treated cells, we compare their SERS spectra with the reference NAADP spectra collected from the pure NAADP aqueous solution. Concentrationdependent SERS spectra of NAADP are presented in Figure 5a. (30) Zou, S. L.; Schatz, G. C. Chem. Phys. Lett. 2005, 403, 62–67. As is often the case in SERS, the spectral signature changes with concentration. 31 The 733 cm -1 peak of adenine, for example, dominates the spectrum at higher concentrations. The reduc- (31) Kim, S. K.; Joo, T. H.; Suh, S. W.; Kim, M. S. J. Raman Spectrosc. 1986, 17, 381–386. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6773
Page 1 and 2: Anal. Chem. 2010, 82, 6745-6750 Let
Page 3 and 4: purchased from Invitrogen-Molecular
Page 5 and 6: Figure 3. Electropherograms of TPP-
Page 7 and 8: Anal. Chem. 2010, 82, 6751-6755 Res
Page 9 and 10: (pH 8.0) with cysteine and cystamin
Page 11 and 12: Figure 4. Ion mobility mass spectra
Page 13 and 14: GOx glucose + O298 gluconic acid +
Page 15 and 16: coater at 2000 rpm for 20 s, and th
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Page 19 and 20: in channels with either no grooves
Page 21 and 22: indicators of atmospheric processin
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Page 27: on a substrate are preferred. 20-24
Page 31 and 32: Anal. Chem. 2010, 82, 6775-6781 Hig
Page 33 and 34: tion, 2 µL of proprionaldehyde wer
Page 35 and 36: Figure 4. Analysis of 2a by HPLC-MS
Page 37 and 38: eaction of 1a with PBH can be condu
Page 39 and 40: Numerous references had demonstrate
Page 41 and 42: Figure 1. TEM images of the prepare
Page 43 and 44: Figure 4. Schematic representation
Page 45 and 46: esult in a big SPR signal change wi
Page 47 and 48: also reduces chemical noise, which
Page 49 and 50: Table 1. Extraction Yields, Liquid
Page 51 and 52: scanning of AMPP amides of the anal
Page 53 and 54: Anal. Chem. 2010, 82, 6797-6806 δ
Page 55 and 56: Figure 1. Schematic view of the pre
Page 57 and 58: from the specimen and enclosed in a
Page 59 and 60: Figure 4. (A) IRMS mass-44 chromato
Page 61 and 62: Table 3. Ambient Measurement Result
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Page 65 and 66: Polymerase (1 U per sample). Reacti
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Page 75 and 76: Table 2. Concentrations and Ratios
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mg/mL protein, followed by separati
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Figure 2. Productivity of SEQUST an
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Figure 4. High-resolution MS/MS spe
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Figure 6. Characterization of PSMs
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containing T-T mismatches. 23 Based
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Figure 1. Extinction spectra of sol
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Figure 3. (A) The value of Ex650 nm
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Figure 5. Extinction spectra and co
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wished to explore the dehydration o
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constant medium for separation; we
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Figure 2. Standards of (Pi)n, n ) 1
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Figure 5. Quantitative calibration
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Anal. Chem. 2010, 82, 6847-6853 Met
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Figure 1. 226 Ra spectrum by liquid
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Table 1. Counting Properties and De
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Table 3. Analysis of 226 Ra in Sedi
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mercial microarray scanner and fabr
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Figure 2. Optical transmission meas
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Figure 5. Volcano plots detailing t
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data as well. The 41 genes in Table
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corresponding compound if its chemi
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Figure 1. Schematic illustration of
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Table 1. Absolute Quantification Re
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ment. Therefore, the long-time drea
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Figure 1. Chemically actuated micro
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Figure 2. Influence of a surfactant
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Figure 5. Device to eject and mix s
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Anal. Chem. 2010, 82, 6877-6886 Imm
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NaCl, phosphate buffer saline (PBS)
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Figure 2. Product ion mass spectra
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-70 °C resolved the problem, givin
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Table 1. Intraday Precision and Acc
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Anal. Chem. 2010, 82, 6887-6894 Fer
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Figure 1. Infrared spectra of (A) u
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Figure 3. Cyclic voltammograms obta
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Figure 6. Calculated charge from ch
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Anal. Chem. 2010, 82, 6895-6903 Ele
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Table 1. Chemical Structure, pKa Va
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pH with a tilted baseline (Figure 1
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Table 2. Linearity and Detection Li
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ascorbic acid (AA), uric acid (UA),
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the multielement capabilities, the
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RESULTS AND DISCUSSION Sulfur Detec
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Table 2. Molecular Properties and C
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Anal. Chem. 2010, 82, 6911-6918 Dir
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dilution and hybridization buffer.
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solution under appropriate incubati
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Figure 4. Standardization curve for
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Anal. Chem. 2010, 82, 6919-6925 Ele
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the ×10 objective, to have a large
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Figure 2. With a suitable removal o
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the NB signal in a much better foot
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Scheme 1. Reactions of Selenium Rea
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Figure 2. (a) ESI-MS spectrum showi
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Figure 4. (a) ESI-MS spectrum showi
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Anal. Chem. 2010, 82, 6933-6939 Dif
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trode 28 by a finite element using
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Figure 4. Comparison between simula
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Figure 6. Comparison between simula
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educed in the vicinity of double bo
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Figure 2. Normalized product ion ab
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Figure 4. EID (a) and IRMPD (b) of
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Anal. Chem. 2010, 82, 6947-6957 Ide
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Figure 1. Schematic flowchart showi
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difference, ppm compound Table 1. I
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isoforms, its successful use, in th
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Figure 5. Extracted ion current ESI
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m/z 1172.935 was observed for Ser14
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linked products via affinity tags.
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Scheme 2. Fragmentation Mechanism o
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Figure 1. (A) ESI-LTQ-CID-MS 2 prod
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Figure 3. (A) ESI-LTQ-CID-MS 2 prod
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Figure 5. (A) MALDI-TOF/TOF product
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Anal. Chem. 2010, 82, 6969-6975 Ana
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Figure 3. Equilibrium response as a
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Figure 6. The average measured resp
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for the fill time, and we find that
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nucleotide tails. 3-5 Thus, the amo
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allow the use of higher aptamer con
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quent ligation of the aptamers afte
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Anal. Chem. 2010, 82, 6983-6990 Imp
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Figure 2. Configuration editor wind
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Figure 4. Dependencies between volu
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Since the time for liquid expulsion
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Anal. Chem. 2010, 82, 6991-6999 Int
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Figure 1. Representation of the Car
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Table 2. Capillary Electrophoresis
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Figure 4. (A) Typical raw electroph
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field. DNA profiles were delivered
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Another approach to handle this lim
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(principal components, PCs) in whic
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Figure 5. Relevant score plots and
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CONCLUSIONS In this paper we have i
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electroactive-species loaded liposo
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Figure 2. Qdot-based FLFTS response
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Figure 6. Fluorescence imaging of Q
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Anal. Chem. 2010, 82, 7015-7020 Sel
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Table 1. Effect of 1 D-LC Condition
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Figure 3. Peak-production rate vers
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Anal. Chem. 2010, 82, 7021-7026 Cel
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luciferase (T7 control vector) was
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Figure 3. Inhibitory effects of luc
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Anal. Chem. 2010, 82, 7027-7034 Pat
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Electrochemistry. Electrochemical m
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Figure 2. SEM images of Au substrat
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Table 3. Film Thickness Measurement
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Anal. Chem. 2010, 82, 7035-7043 Qua
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Figure 1. (A) FL spectra of BSPOTPE
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Figure 4. (A) Variation in the FL i
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Figure 6. (A) FL spectra of BSPOTPE
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Scheme 1. Proposed Mechanism for Fl
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one or more drawbacks including poo
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Figure 2. Free fluorophores do not
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Anal. Chem. 2010, 82, 7049-7052 Dev
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Figure 2. Comparative analysis of d
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Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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