Analytical Chemistry Chemical Cytometry Quantitates Superoxide

More documents

Recommendations

Info

Figure 1. Schematic of the developed sandwich ELISA procedure for the HFA/AHSG assay. Plate functionalization was performed by treatment with 1% (w/v) KOH and then silanization with 2% (v/v) APTES. KOH oxidizes the organic moiety of the polymer generating a carbonyl (-CO) or a hydroxyl (-OH) group according to the nature of the polymer. The alkoxy groups of APTES subsequently react with the carbonyl or hydroxyl groups of the surface in a hydrolysis-dependent reaction. 11,12 Anti-HFA/AHSG antibody was then captured using EDC and sulfo-NHS chemistry on the aminated 96-well plate, where the EDC-SNHS was used in a ratio of 1:100 with the antibody. The EDC-SNHS-activated antibody molecules were then immobilized on the aminated ELISA plate, where a bond was formed between amine groups on the plate and the activated carboxyl groups of the antibody. 100 µL of 1.0% (w/v) KOH at 37 °C for 10 min followed by five washings with 300 µL of DIW. The KOH-treated wells were then functionalized with amino groups by incubation with 100 µL of 2% (v/v) aminopropyltriethoxysilane (APTES) per well at 80 °C for 1 h inside a vacuum-desiccator, in order to achieve maximum silanization. The reaction mechanism following the KOH activation and APTES-based surface functionalization involves mild oxidation followed by a hydrolytic APTES polymerization on the oxidized surface through its alkoxy groups. 11,12 The desiccator was equilibrated to room temperature for 20 min. The amine-functionalized plate was subsequently washed five times with 300 µL of DIW in order to remove excess unbound 3-APTES from the surface. Afterward, the anti-HFA/AHSG (R2-HS-glycoprotein; 990 µL of4µg/mL) was incubated with 10 µL of a premixed solution of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (4 mg/mL) and N-hydroxysulfosuccinimide (SNHS) (11 mg/mL) for 15 min at 37 °C. The resulting EDC cross-linked anti- HFA/AHSG antibody solution was added to each of the functionalized wells (100 µL) and incubated for 1hat37°C. The anti- HFA/AHSG-coated wells were then washed five times with 300 µL of PBS. The methodology pertaining to the conventional assay is described in detail in the Supporting Information methods section. ELISA on Anti-HFA/AHSG Antibody-Immobilized Microtiter Plates. Plates with covalent and passively immobilized anti- HFA antibody were blocked with 1% (v/v) BSA diluted in 0.1 M 7050 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 phosphate buffered saline (PBS), pH 7.4, for 30 min at 37 °C and subsequently washed five times with 300 µL of PBS. Varying concentrations of HFA/AHSG (from 4.8 pg/mL to 20 ng/mL) were prepared in 0.1 M PBS, pH 7.4, and 100 µL of each of these concentrations were incubated in the antibody-coated plates for 1hat37°C and, subsequently washed five times with PBS. One hundred microliters of biotinylated anti-HFA/AHSG detection antibody (200 ng/mL) was added and incubated for 1hat37°C followed by five PBS washes. HRP-conjugated streptavidin (100 µL per well), at a dilution of 1:200, was added and then incubated for 20 min at 37 °C followed by five washes with PBS. The 3,3′,5,5′tetramethylbenzidine (TMB) substrate was subsequently added (as per the manufacturer’s recommendations), and the reaction was stopped after 20 min by addition of 50 µL of1NH 2SO4. The absorbance was recorded at a primary wavelength of 450 nm with a reference wavelength of 540 nm. The dual wavelength system of absorbance measurement in 96-well plates is designed to eliminate or greatly reduce the optical imperfections at the well-to-well level. All the experiments were carried out in triplicate. The control for this study was 0 ng/mL HFA in 0.1 M PBS, pH 7.4, and the absorbance of the control was subtracted from all the assay values. The respective analytical sensitivity (detection limit) of the assay was determined where analytical sensitivity was calculated using the formula [mean absorbance of blank +3σ (standard deviation of the blank)]. Additionally, data sets obtained from the modified
Figure 2. Comparative analysis of developed sandwich ELISA (black circles) with the conventional format. Anti-human fetuin A (anti-HFA) antibody was covalently bound to the amine-modified surface in the developed ELISA, whereas antibody adsorbed on the unmodified surface was used in the conventional assay format. The conventional assay was performed with the manufacturer-recommended protocol (white circles) and the protocol developed in this study (inverted triangle). The error bars represents standard deviations. Table 1. Comparison of Assay Performance Parameters (Precision and Sensitivity) for Conventional and Developed ELISAs a intraday precision range (CV %), (n ) 5) interday precision range (CV %), (n ) 3) sensitivity (pg/mL) developed ELISA 2.4-10.4 1.7-17.6 39 conventional ELISA 4.7-17.4 3.6-20.0 624 a Intraday precision was calculated from the five repeats (n ) 5) of the same assay on a single day but at different times, while interday precision was calculated from assay repeats on three different days (n ) 3). All the assays were carried out in triplicate. Analytical sensitivity was calculated using the formula [average absorbance of the blank + 3(SDblank)]. and conventional ELISA were analyzed using standard curve analysis of Sigma Plot software, version 11.0. RESULTS AND DISCUSSION The range of detection of HFA/AHSG for the developed ELISA (Figure 2) was 9-20 pg/mL (r 2 ) 0.99). An analytical sensitivity of 39 pg/mL was recorded for developed ELISA, where analytical sensitivity was calculated using the formula [average absorbance of the blank + 3(SDblank)]. Assay variability parameters for this high-sensitivity assay for HFA are described in Table 1, where intraday assay variability was calculated from 5 repeats performed on a single day, and interday assay variability was calculated from three repeats performed on three different days in triplicate. The percentage recovery for lower concentrations (4.88-625 pg/mL) was 50-80% while for higher concentrations (1.2-20 ng/mL) it was 75-100%. The percentage recovery was calculated as the obtained value/expected value × 100. The half-effective concentration (EC50) obtained from the dose-response curve, 13 which is a measure of analyte-ligand (13) Armbruster, D. A.; Schwarzhoff, R. H.; Hubster, E. C.; Liserio, M. K. Clin. Chem. 1993, 39, 2137–2146. interaction, was found to be on an average of 3.3 ng/mL. The EC50 for all the assay repeats was found to be in the range of 3-3.5 ng/mL. A lower EC50 (3.3 ng/mL), which was obtained for the developed ELISA, suggests a strong interaction between HFA and anti-HFA antibody. This interaction behavior is attributed to the significant increase in the total amount of immobilized antibody achieved using the covalent immobilization strategy, which increased the availability of anti-HFA antibody per HFA molecule, thus increasing the resultant HFA capture on the well surface. Conversely, EC50 for the conventional ELISA format was 23 ng/mL, (r 2 ) 0.99), which was significantly higher than the developed ELISA. This suggests a less sensitive conventional assay (Supplementary Table 2, Supporting Information). The most important factor that may be attributed to the enhanced sensitivity of the modified assay is the covalent immobilization of the anti-HFA/AHSG capture antibody since the covalently crosslinked antibodies should not leach out during the assay procedure in comparison to the use of passively adsorbed antibodies, where leaching may occur more easily. Performance of the developed sandwich ELISA procedure was compared with the conventional protocols (Figure 2), both involving passively adsorbed antibody on normal unmodified microtiter plates (one performed using the modified protocol and the other as per the manufacturer’s recommendations). All the assays were performed simultaneously on the same day under same set of conditions in order to minimize variability, where variability was reported as percentage coefficient of variation (% CV) (Table 1). The technique employed here for chemically modifying the polymeric surface was developed and standardized by our group, and a standard EDC-based cross-linking strategy was deployed to immobilize anti-HFA/AHSG antibody. The developed sandwich ELISA procedure decreased the overall assay duration from 20 to 6 h, which is more than a 3-fold decrease. Thus, the reported procedure is a rapid ELISA format having the additional benefit of being generic in nature, i.e., it can be used with sandwich ELISAs on different polymeric matrixes for a range of different analytes. Hence, using the surface modification technique reported here, it is possible to develop rapid and high sensitivity assays on various categories of solid supports including those that are chemically inert. To the best of our knowledge, this developed ELISA procedure is the most sensitive assay reported for the detection of HFA/ AHSG (Supplementary Table 1, Supporting Information), which is based on the comparison between different commercially available ELISA kits for HFA. The sensitivity of the assay developed by our group may be attributed to the use of monoclonal antibody at the capture stage, which was covalently immobilized on the surface, and a polyclonal antibody to detect the antigen. The covalently cross-linked monoclonal antibodies that are used as capture antibodies in this study were found to capture significantly higher amounts of HFA, which has improved the detection limit and the overall assay sensitivity of the developed ELISA procedure in comparison to the conventional format. However, the commercially available ELISA kits, as described in Supplementary Table 1 (Supporting Information), have different capture and detection antibody combinations, which Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 7051
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 and 116:
Figure 5. Volcano plots detailing t
Page 117 and 118:
data as well. The 41 genes in Table
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: Anal. Chem. 2010, 82, 7049-7052 Dev
show all

Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Create successful ePaper yourself

Delete template?

Save as template?