Analytical Chemistry Chemical Cytometry Quantitates Superoxide

More documents

Recommendations

Info

Fluorescence Measurements for Model Titrations. Fluorescence measurements were conducted using a Nanodrop 3300 fluorospectrometer (Thermo Scientific). The intensity of FAM emission was measured at 520 nm with LED excitation at 485 nm. The buffer used for titrations was 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2. Titrations sets (14-16 points) were carried out in duplicate, and each fluorescence measurement was repeated six times. Fixed amounts of Loop or FreeA and FreeB strands were incubated with variable amounts of Cb overnight at room temperature prior to fluorescence measurements. Proximity Ligation Assays. Temperature control was achieved using a Mastercycler-EP gradient thermal cycler (Eppendorf). Thrombin and oligonucleotide solutions were made as described. 1 An amount of 4 µL of a reaction mixture containing human thrombin, aptA, and aptB was incubated at 37 °C for 15 min before adding 1 µL of connector (C16,PLA, C18,PLA, orC20,PLA). An additional 30 min of incubation at 22 °C was performed to stabilize complexes. These 5 µL samples were then brought to a total volume of 55 µL for the ligation and amplification mixture, which contained 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.9 mM MgCl2, 0.4 Weiss unit T4 DNA ligase, 73 µM ATP, 0.18 mM dNTPs, 0.45 µM forward and reverse PCR primers, 45 nM Taqman probe for the 5′ nuclease assay, and 1.5 units of AmpliTaq Gold polymerase (ABI). The reactions were transferred to a real-time PCR instrument (CFX96, Bio-Rad) for temperature cycling: 5 min at 22 °C for ligation, 10 min at 95 °C, and then cycling for 15 s at 95 °C and60sat60°C, repeated 45 times. Signal-to-background (S/BG) values were calculated from the relative number of ligation products detected in a sample with thrombin to that in a sample without thrombin. The relative quantity of the ligated products was calculated by using the comparative threshold cycle (Ct) method. 11 For measurements of PLA background, 15 pM aptA and 20 pM aptB were used, with different connector concentrations as follows: C16,PLA ) 178 nM; C18,PLA ) 112 nM; C20,PLA ) 45 nM. For asymmetric PLA, 900 pM aptA and 1200 pM aptB were used with 480 nM C16,PLA; and for symmetric PLA, 15 pM aptA and 20 pM aptB were used with 400 nM C16,PLA (same conditions as previously reported 1 ). Connector Kd Calculations. Base-pairing dissociation constants for the invariable segment (Kd,A) and for each variable segment (Kd,B) of the connectors were calculated using the wellestablished, thermodynamic nearest-neighbor model. 12 Dissociation constants were derived from thermodynamic parameters calculated via the BioPHP melting temperature calculator (http://insilico.ehu.es/tm.php), a free online service that uses SantaLucia’s method. 12 Source code is freely downloadable at http://www.biophp.org/. Kd,B values were also experimentally confirmed; in Table 2, the column labeled “Kd,B” shows the BG calculated values, and the column labeled “Kd,eff” shows the experimentally confirmed values. These values are in good agreement, as further shown in the Supporting Information (Figures S-3 and S-4). (11) Livak, K. J. User Bulletin No. 2: ABI PRISM 7700 Sequence Detection System; PE Applied Biosystems: Foster City, CA, 1997; pp 11-15. (12) SantaLucia, J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1460–1465. 6978 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Table 2. Analysis of Connector Sequences Used in the Experimental Model of Proximity Hybridization a connector, C b length (bases) Kd,B BG S (nM) Kd,eff (nM) Kd,eff (nM) S/BG [Cn] opt (nM) C20 20 16 18.6 ± 2.7 3.25 ± 0.48 5.72 45 C19 19 135 149 ± 11 5.54 ± 0.93 26.9 80 C 18 18 539 446 ± 59 9.52 ± 1.33 46.8 110 C 17 17 3370 2550 ± 220 7.12 ± 1.67 358 170 C16 16 28 600 7100 ± 740 8.47 ± 0.38 838 480 C 15 15 297 000 17.5 ± 6.3 20 000 a Notes: Kd,B, calculated dissociation constant for variable segment of connector in 100 mM salt and 1 mM Mg2+ BG S ; Kd,eff and Kd,eff, measured effective dissociation constants of background and signal, respectively; [Cn]opt, optimal connector concentration for PLA; unvaried Kd,A ) 14.8 nM. RESULTS AND DISCUSSION Signal and Background in Proximity Ligation. A schematic of the original, aptamer-based PLA 1,6 is shown in Figure 1A, in the context of protein detection with aptamer probes. The quantitative capability of PLA is most heavily dependent upon two steps: the cooperative hybridization of the four-part complex, aptamer A-target-aptamer B-connector (aptA-TaptB-C20,PLA), followed by the proximity-dependent ligation of the two aptamers using a DNA ligase. This process results in an amount of ligated product that is proportional to the original amount of protein analyte (“signal” ligations), albeit with some analyte-independent ligation products present (“background” ligations). Although qPCR does not differentiate signal from background ligations, under optimized conditions 1,6 the signal will be dominant over background to allow highly sensitive, indirect detection of the protein analyte. Signal enhancement over background in PLA is based on the proximity effect. The effective concentrations of two aptamers are significantly increased due to their proximity, afforded by simultaneous binding to the same protein. 3-5 The chief interference in PLA is target-independent, background ligation (Figure 1A, bottom). This background ligation has been reported to result from two sources. 1,6,13 First, based simply on binding equilibria, a fraction of aptamers will always hybridize with the 20-base connector sequences (C20,PLA), even in the absence of protein analyte, resulting in target-independent ligations that increase the background levels in the assay. 1,6 Second, the T4 DNA ligase is capable of ligating a small fraction of ssDNA sequences, even in the absence of a connector sequence. 13,14 This will also increase assay background (not shown in the figure). Importantly, the presence of this background limits the amount of usable aptamer (or antibody) probes, which affects the sensitivity and partially defines the upper limit of the dynamic range. 6 Methods to widen dynamic range would address a major weakness of PLA and significantly improve the applicability of the assay. Design of the Experimental Model of Proximity Hybridization. We hypothesized that by decreasing connector binding affinities for aptamers using an asymmetric connector (Figure 1B), the amount of background ligation products would be greatly reduced compared to signal ligation products. In turn, this should (13) Leslie, D. C.; Sohrabi, A.; Ikonomi, P.; McKee, M. L.; Landers, J. P. Electrophoresis 2010, 31, 1–8. (14) Kuhn, H.; Frank-Kamenetskii, M. D. FEBS J. 2005, 272, 5991–6000.
allow the use of higher aptamer concentrations, which can increase the upper limit of dynamic range and improve sensitivity. This hypothesis was based on the inherent signal enhancement given by cooperative binding in the proximity effect, 3-5 which does not apply to background. To understand the impact of connector binding affinity on S/BG ratio in PLA, we developed a simple experimental model using DNA hybridization and fluorescence quenching (Figure 1C). First, we define an “asymmetric connector” in the model system as a connector with one side shortened, compared to the original PLA connector, C20,PLA. Herein, model connector sequences are represented by Cb, where b is equal to the total number of bases in the connector that are complementary to the Loop sequence. For example, the symmetric connector used in the model is represented by C20, similar to the 20-base connector used in the original PLA work, 1,6 whereas an asymmetric connector with only six bases in the variable region is represented by C16. Sequences are noted in Table 1, with variable regions in bold. (Note that connectors used later for PLA or asymmetric PLA are differentiated from model connectors using the subscript “PLA,” such as C20,PLA or C16,PLA.) In our model system, signal and background were treated separately. The equilibria shown in Figure 1C represent signal complex formation with a sequential binding mode and background complex formation with an independent binding mode. These are not blind assumptions but are based upon Hill coefficients which were determined by nonlinear least-squares fitting of our experimental data (discussed in detail below). To model target-dependent signal in PLA, a single DNA loop was hybridized with connector sequences (Figure 1C, top). The aptA-T-aptB complex in PLA was modeled using a 100nucleotide DNA loop (Loop) that is capable of hybridizing with a connector sequence at both ends, representing the ideal case in which aptamers possess infinite affinities for their protein analyte (Kd’s ≈ 0). To measure the extent of hybridization, the Loop was covalently labeled with IABkFQ quencher at the 5′end (black circle) and with FAM at the 3′-end (gray circle). Quenching of FAM fluorescence ensued when both the 5′- and 3′-ends of the Loop were hybridized to a connector sequence, mimicking the proximity hybridization complex in PLA (i.e., Loop-Cb represents aptA-T-aptB-C20,PLA). Thus, a decrease in Loop fluorescence was referred to as “signal” in this case. The model of background ligations in PLA (Figure 1C, bottom) follows from the model of signal. The FAM- and IABkFQ-labeled Loop sequence was essentially split into two free strands (FreeA and FreeB), representing aptamers unbound by protein analytes. Upon combination of both free strands and a connector sequence, the extent of fluorescence quenching represented target-independent hybridization in PLA and was proportional to the amount of background ligation products that would be expected (i.e., FreeA-Cb-FreeB represents aptA-C20,PLA-aptB). Thus, a decrease in fluorescence was referred to as “background” in this case. Complex Formation with Asymmetric Connectors. Moving toward our goal of reduced background in PLA, the experimental model (Figure 1C) was used to determine effective dissociation constants (Kd,eff) of signal and background complexes at different connector lengths (Cb, where 15 e b e 20). The assumption was that the Kd,eff values could represent the relative amounts of signal and background. Separate titrations of the Loop and Free strands with the Cb strand gave the corresponding Kd,eff values of the complexes. The S/BG was then calculated as S/BG ) K S a,eff BG Ka,eff ) K BG d,eff S Kd,eff This S/BG represents the ideal case in which aptamers have infinite affinities for the target (Kd’s ≈ 0). Connectors’ affinities for the FreeB strand (or for one arm of the Loop) were reduced by decreasing the number of complementary bases to FreeB or the Loop arm (gray segments in Figure 1B; bolded text in Table 1). Base-pairing dissociation constants for the invariable segment (Kd,A) and for each variable segment (Kd,B) of the connectors were calculated (Table 2) using the well-established, thermodynamic nearest-neighbor model. 12 Starting with the 20-base connector (C20) similar to that used in standard PLA, the variable segment was decreased in length, resulting in five different asymmetric connectors (C15 through C19). Titrations with these asymmetric connectors and the symmetric control connector (C20) were carried out on both the signal and background models (Figure 1C), and the decrease in fluorescence was used to assay stability. Background Reduction in the Experimental Model. Modeling of cooperative complex formation has proven difficult, particularly in the realm of predicting the affinity of bivalent ligands based on known affinities of each individual ligand. 3-5 Rather than attempting to develop an extensive model of the system shown in Figure 1C, titration data was analyzed using the Hill equation, 15 a simplified model that has found wide use in biochemistry, physiology, and pharmacology to analyze binding equilibria and ligand-receptor interactions. 16 According to Weiss, 16 the Hill coefficient is best described as an “interaction coefficient” that reflects the extent of cooperativity among multiple ligand binding sites. Since it is expected that cooperativity is inherent to the formation of the aptA-T-aptB-C20 complex in PLA, or the Loop-Cb complexes in our model, we employed nonlinear least-squares (NLLS) fitting of our data to the Hill equation (eq 2) to assay this cooperativity and to determine Kd,eff values. S ) S i + (S f - S i ) [C b ] n (K d,eff ) n + [C b ] n where S is the fluorescence signal measured as a function of [Cb], the connector concentration, Si and Sf are the initial and final signals, respectively, and Kd,eff represents the effective dissocia- S tion constant of the Loop-Cb complex (Kd,eff) or the FreeA- BG Cb-FreeB complex (Kd,eff). In this way, fluorescence measurements during titration with increasingly higher [Cb] could be fit to the Hill equation (eq 2) and the Kd,eff values could be extracted. Furthermore, each extracted Hill coefficient, n, represented the extent of cooperativity in that particular system. Our expectation was that the Loop-Cb complexes (signal) (15) Hill, A. V. J. Physiol. 1910, 40, iv–vii. (16) Weiss, J. N. FASEB J. 1997, 11, 835–841. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 (1) (2) 6979
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: nucleotide tails. 3-5 Thus, the amo
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?