Analytical Chemistry Chemical Cytometry Quantitates Superoxide

More documents

Recommendations

Info

Figure 6. Logarithmic plot of fold change comparisons between glass microarray, PC microarray, and sequencing results for five genes randomly selected from the list of genes found to be differentially expressed on the PCs but not on the glass slides. Fold change values for glass and PC microarrays were calculated by determining the ratio between average microarray expression level for trifoliate samples to average microarray expression level for cotyledon samples. A similar ratio was calculated using number of reads for sequencing data. The PC microarray results show similar directions and magnitudes of change compared to sequencing data for all 5 genes, while glass microarray data for CHP089 and CHP004 does not agree with the sequencing data. to the experimental protocol. While the initial photolithography process needed to fabricate the silicon mold of the grating has high costs, a single round of photolithography on silicon can be translated into thousands of devices that are fabricated uniformly over large areas. By fabricating the mold on an 8 in. wafer, there is a large degree of flexibility in fitting PCs to preferred labware formats such as microscope slides and microtiter plates. Because PCs were cut to fit standard microscope slides, they could be processed with existing protocols and scanned with commercially available equipment, allowing for convenient adoption of PC substrates in a standardized experiment. Not only does the nanoreplica molding process provide a convenient form factor for the substrates, it also enables the excellent level of optical uniformity required to ensure that every spot on the microarray experiences the same level of enhancement. This is key to ensure that the data obtained from PCs does not have a higher level of variation than the data obtained from glass slides. The signal enhancement factor primarily used in previous work in this field is defined as spot intensity subtracted by the local background observed on the enhancement substrate divided by the same value observed on the glass slide or control substrate. The signal enhancement factor observed from Cy-5 spots with high expression genes in this microarray experiment was approximately 60×, which is identical to the enhancement demonstrated in previous work with this substrate. 14 Thus, the PC signal enhancement compares favorably to experiments with metal island films that have yielded signal enhancement factors of 10-40×. 5,6 However, the signal enhancement factor is not an ideal measurement to assess the practical utility of the substrate. This work has focused on SNR enhancement rather than signal enhancement because microarray data analysis programs use SNR values to classify spots as detected or not detected. It is possible to achieve 6860 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 good signal enhancement without achieving similar SNR enhancement if a substrate enhances fluorescence but has a large noise value thus voiding any advantages of fluorescence enhancement. Without knowledge of a substrate’s impact on the SNR observed from spots, it is difficult to ascertain whether a substrate will benefit a target assay. The PC not only attains a large signal enhancement but it also achieves an SNR enhancement of approximately 10× (measured over all spots in the experiment), suggesting that the array can detect hybridization at concentrations 10× lower than can be detected on glass substrates. The noise in a DNA microarray experiment arises primarily from the following sources: sample variation, nonspecific binding, instrumentation, and substrate fluorescence. Variation in the amount of nucleic acid sample captured is accounted for by hybridizing multiple arrays and figures prominently into tests of significance for differential expression experiments. Nonspecific binding is controlled largely by blocking and hybridization conditions and is assessed by evaluating negative control spots. The noise observed from instrumentation can be characterized by measurements of dark noise, but in this experiment, this represents only
data as well. The 41 genes in Tables S-4 and S-2 in the Supporting Information corresponding to genes detected as differentially expressed on the PC slides had an average RPKM value of 56 in the leaf sample and 67 in the seed sample, whereas the 26 genes detected as differentially expressed on both PC and glass slides had much higher average RPKMs in both the leaf (341 PRKM) and seed (3965 RPKM) samples, respectively. Thus, both microarray and sequencing data suggest the PC can reliably quantify genes with expression levels at least 1 order of magnitude lower than measured with conventional glass microarrays. By expanding the dynamic range of the microarray experiment, the number of genes for which statistically significant changes in expression could be observed improved from 27 to 68 genes, or from 13 to 34% of the genes probed in the experiment. Because the gene expression follows a power law distribution, modest enhancements in the performance of the assay can dramatically increase the number of genes researchers are able to probe in this microarray format. This data thus suggests that the detection capabilities of microarray protocols currently used today can be greatly expanded by substitution of conventional substrates with enhanced fluorescence substrates such as PCs. The increased SNRs provided by PCs may allow researchers to perform experiments that are currently problematic on glass slides. Because lower amounts of bound sample can be detected with the PC, sample sizes may be reduced to volumes that would be difficult to probe using normal glass substrates. This may be particularly helpful for profiling gene expression in smaller tissue samples or small populations of rare cells such as stem cells. Alternately, the reduction in experimental variation afforded by this substrate may allow researchers to confidently identify differentially expressed genes with fewer replicates, which may also prove useful with small sample sizes or rare cells. This approach is not limited to conventional DNA microarray experiments. Any surface-bound biomolecular assay can be performed on these PCs for improved performance, as is illustrated in previous work with immunoassays. 25 This substrate can also potentially be adapted to improve reliability of novel technologies such as next-generation genomic sequencing platforms, since these instruments make extensive use of fluorescent molecules. CONCLUSION We have demonstrated that enhanced fluorescence is capable of significantly improving a DNA microarray that probes changes (25) Mathias, P. C.; Ganesh, N.; Cunningham, B. T. Anal. Chem. 2008, 80, 9013–9020. in gene expression between samples. By using a PC substrate with uniform optical characteristics over microscope slide-sized areas, the SNR from microarray spots was increased by an order of magnitude compared to commercial glass substrates. This SNR enhancement translated into a greater number of genes detected above the noise level and allowed for the detection of statistically signficant changes in low expression genes. After evaluating differential expression in soybean trifoliate tissue versus cotyledon tissue, more than twice as many genes were characterized as differentially expressed on the PCs compared to the glass slides, and many of these were validated by high-throughput mRNA sequencing data. Using a PC substrate for microarray experiments thus opens the possibility to interrogate the roles of genes that previously could not be reliably quantified in a high-throughput fashion. ACKNOWLEDGMENT This work was supported by the National Institutes of Health (grant no. GM086382A), the National Science Foundation (grant no. CBET 07-54122), SRU Biosystems, and the Illinois Soybean Association. Any opinions, findings, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Institutes of Health or the National Science Foundation. The authors thank Stephen Schulz, Brenda Hugh, Frank Jackson, and Kurt Albertson at SRU Biosystems for attaching photonic crystal substrates to microscope slides. The authors thank the staff at the Micro and Nanotechnology Laboratory at the University of Illinois at Urbana-Champaign. The authors also thank Sean Bloomfield for bioinformatics assistance and the staff of the Keck Center at the Biotechnology Center at University of Illinois for Illumina sequencing. SUPPORTING INFORMATION AVAILABLE Tables S-1 and S-2 list genes found to have statistically significant changes in expression on glass and photonic crystal slides and Tables S-3 and S-4 show corresponding RNA transcript levels. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 31, 2010. Accepted July 2, 2010. AC100841D Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6861
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
Page 17 and 18:
Figure 5. Comparison of cyclic volt
Page 19 and 20:
in channels with either no grooves
Page 21 and 22:
indicators of atmospheric processin
Page 23 and 24:
Figure 1. GC/MS total ion chromatog
Page 25 and 26:
Table 2. Concentrations and Stable
Page 27 and 28:
on a substrate are preferred. 20-24
Page 29 and 30:
Figure 4. SERS analysis of NAADP co
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
Page 63 and 64:
Anal. Chem. 2010, 82, 6807-6813 Dir
Page 65 and 66: Polymerase (1 U per sample). Reacti
Page 67 and 68: of which was constant for all ampli
Page 69 and 70: analytically useful signals at less
Page 71 and 72: carbon black and RP-C18 for the ext
Page 73 and 74: solution in an equal volume, and 1
Page 75 and 76: Table 2. Concentrations and Ratios
Page 77 and 78: Anal. Chem. 2010, 82, 6821-6829 Mac
Page 79 and 80: mg/mL protein, followed by separati
Page 81 and 82: Figure 2. Productivity of SEQUST an
Page 83 and 84: Figure 4. High-resolution MS/MS spe
Page 85 and 86: Figure 6. Characterization of PSMs
Page 87 and 88: containing T-T mismatches. 23 Based
Page 89 and 90: Figure 1. Extinction spectra of sol
Page 91 and 92: Figure 3. (A) The value of Ex650 nm
Page 93 and 94: Figure 5. Extinction spectra and co
Page 95 and 96: wished to explore the dehydration o
Page 97 and 98: constant medium for separation; we
Page 99 and 100: Figure 2. Standards of (Pi)n, n ) 1
Page 101 and 102: Figure 5. Quantitative calibration
Page 103 and 104: Anal. Chem. 2010, 82, 6847-6853 Met
Page 105 and 106: Figure 1. 226 Ra spectrum by liquid
Page 107 and 108: Table 1. Counting Properties and De
Page 109 and 110: Table 3. Analysis of 226 Ra in Sedi
Page 111 and 112: mercial microarray scanner and fabr
Page 113 and 114: Figure 2. Optical transmission meas
Page 115: Figure 5. Volcano plots detailing t
Page 119 and 120: corresponding compound if its chemi
Page 121 and 122: Figure 1. Schematic illustration of
Page 123 and 124: Table 1. Absolute Quantification Re
Page 125 and 126: ment. Therefore, the long-time drea
Page 127 and 128: Figure 1. Chemically actuated micro
Page 129 and 130: Figure 2. Influence of a surfactant
Page 131 and 132: Figure 5. Device to eject and mix s
Page 133 and 134: Anal. Chem. 2010, 82, 6877-6886 Imm
Page 135 and 136: NaCl, phosphate buffer saline (PBS)
Page 137 and 138: Figure 2. Product ion mass spectra
Page 139 and 140: -70 °C resolved the problem, givin
Page 141 and 142: Table 1. Intraday Precision and Acc
Page 143 and 144: Anal. Chem. 2010, 82, 6887-6894 Fer
Page 145 and 146: Figure 1. Infrared spectra of (A) u
Page 147 and 148: Figure 3. Cyclic voltammograms obta
Page 149 and 150: Figure 6. Calculated charge from ch
Page 151 and 152: Anal. Chem. 2010, 82, 6895-6903 Ele
Page 153 and 154: Table 1. Chemical Structure, pKa Va
Page 155 and 156: pH with a tilted baseline (Figure 1
Page 157 and 158: Table 2. Linearity and Detection Li
Page 159 and 160: ascorbic acid (AA), uric acid (UA),
Page 161 and 162: the multielement capabilities, the
Page 163 and 164: RESULTS AND DISCUSSION Sulfur Detec
Page 165 and 166: Table 2. Molecular Properties and C
Page 167 and 168:
Anal. Chem. 2010, 82, 6911-6918 Dir
Page 169 and 170:
dilution and hybridization buffer.
Page 171 and 172:
solution under appropriate incubati
Page 173 and 174:
Figure 4. Standardization curve for
Page 175 and 176:
Anal. Chem. 2010, 82, 6919-6925 Ele
Page 177 and 178:
the ×10 objective, to have a large
Page 179 and 180:
Figure 2. With a suitable removal o
Page 181 and 182:
the NB signal in a much better foot
Page 183 and 184:
Scheme 1. Reactions of Selenium Rea
Page 185 and 186:
Figure 2. (a) ESI-MS spectrum showi
Page 187 and 188:
Figure 4. (a) ESI-MS spectrum showi
Page 189 and 190:
Anal. Chem. 2010, 82, 6933-6939 Dif
Page 191 and 192:
trode 28 by a finite element using
Page 193 and 194:
Figure 4. Comparison between simula
Page 195 and 196:
Figure 6. Comparison between simula
Page 197 and 198:
educed in the vicinity of double bo
Page 199 and 200:
Figure 2. Normalized product ion ab
Page 201 and 202:
Figure 4. EID (a) and IRMPD (b) of
Page 203 and 204:
Anal. Chem. 2010, 82, 6947-6957 Ide
Page 205 and 206:
Figure 1. Schematic flowchart showi
Page 207 and 208:
difference, ppm compound Table 1. I
Page 209 and 210:
isoforms, its successful use, in th
Page 211 and 212:
Figure 5. Extracted ion current ESI
Page 213 and 214:
m/z 1172.935 was observed for Ser14
Page 215 and 216:
linked products via affinity tags.
Page 217 and 218:
Scheme 2. Fragmentation Mechanism o
Page 219 and 220:
Figure 1. (A) ESI-LTQ-CID-MS 2 prod
Page 221 and 222:
Figure 3. (A) ESI-LTQ-CID-MS 2 prod
Page 223 and 224:
Figure 5. (A) MALDI-TOF/TOF product
Page 225 and 226:
Anal. Chem. 2010, 82, 6969-6975 Ana
Page 227 and 228:
Figure 3. Equilibrium response as a
Page 229 and 230:
Figure 6. The average measured resp
Page 231 and 232:
for the fill time, and we find that
Page 233 and 234:
nucleotide tails. 3-5 Thus, the amo
Page 235 and 236:
allow the use of higher aptamer con
Page 237 and 238:
quent ligation of the aptamers afte
Page 239 and 240:
Anal. Chem. 2010, 82, 6983-6990 Imp
Page 241 and 242:
Figure 2. Configuration editor wind
Page 243 and 244:
Figure 4. Dependencies between volu
Page 245 and 246:
Since the time for liquid expulsion
Page 247 and 248:
Anal. Chem. 2010, 82, 6991-6999 Int
Page 249 and 250:
Figure 1. Representation of the Car
Page 251 and 252:
Table 2. Capillary Electrophoresis
Page 253 and 254:
Figure 4. (A) Typical raw electroph
Page 255 and 256:
field. DNA profiles were delivered
Page 257 and 258:
Another approach to handle this lim
Page 259 and 260:
(principal components, PCs) in whic
Page 261 and 262:
Figure 5. Relevant score plots and
Page 263 and 264:
CONCLUSIONS In this paper we have i
Page 265 and 266:
electroactive-species loaded liposo
Page 267 and 268:
Figure 2. Qdot-based FLFTS response
Page 269 and 270:
Figure 6. Fluorescence imaging of Q
Page 271 and 272:
Anal. Chem. 2010, 82, 7015-7020 Sel
Page 273 and 274:
Table 1. Effect of 1 D-LC Condition
Page 275 and 276:
Figure 3. Peak-production rate vers
Page 277 and 278:
Anal. Chem. 2010, 82, 7021-7026 Cel
Page 279 and 280:
luciferase (T7 control vector) was
Page 281 and 282:
Figure 3. Inhibitory effects of luc
Page 283 and 284:
Anal. Chem. 2010, 82, 7027-7034 Pat
Page 285 and 286:
Electrochemistry. Electrochemical m
Page 287 and 288:
Figure 2. SEM images of Au substrat
Page 289 and 290:
Table 3. Film Thickness Measurement
Page 291 and 292:
Anal. Chem. 2010, 82, 7035-7043 Qua
Page 293 and 294:
Figure 1. (A) FL spectra of BSPOTPE
Page 295 and 296:
Figure 4. (A) Variation in the FL i
Page 297 and 298:
Figure 6. (A) FL spectra of BSPOTPE
Page 299 and 300:
Scheme 1. Proposed Mechanism for Fl
Page 301 and 302:
one or more drawbacks including poo
Page 303 and 304:
Figure 2. Free fluorophores do not
Page 305 and 306:
Anal. Chem. 2010, 82, 7049-7052 Dev
Page 307 and 308:
Figure 2. Comparative analysis of d
show all

Analytical Chemistry Chemical Cytometry Quantitates Superoxide

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?