Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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applied potential is the most widely used strategy for the reduction step, but simple immersion of the substrate in a solution of an aryldiazonium salt can also lead to formation of surface layers. This spontaneous, or open-circuit potential (OCP), reaction has been reported for a range of metals, semiconductors, and carbon materials 4 and appears to involve electron transfer from the substrate to the aryldiazonium cation in solution. Currently, there are few examples of patterned organic layers prepared by reduction of aryldiazonium salts. In the earliest report, we used mechanical scribing with an atomic force microscope (AFM) tip to remove regions of electrografted film from a carbon substrate. 23 A second aryldiazonium salt was then electrografted to the bare regions, creating a surface with dual chemical functionality. In a soft lithographic approach, we patterned a carbon substrate by adhering a poly(dimethylsiloxane) (PDMS) mold to the surface (either bare or film-coated) to form microchannels. 24 The channels were subsequently filled, either with aryldiazonium salt solution for site-specific electrografting or with reagents used for electrochemical or chemical conversion of the pre-existing surface film. Most recently, we demonstrated that conventional photolithography can be coupled with electrografting to give large areas of micrometer-sized patterns of modifiers on highly doped silicon. 22 Charlier, Palacin, and co-workers, 25 and Cougnon, Bélanger, and co-workers 26 have established that the scanning electrochemical microscope is a useful tool for localized surface grafting from aryldiazonium salts, while the former research group has also reported elegant patterning methods specific to silicon substrates. In one example, they used ionic implantation to create locally doped areas of silicon and, thus, achieved site-specific electrografting of an aryldiazonium salt. 27 In another example, they illuminated p-type silicon through a mask to locally increase the substrate conductivity and allow electrografting to proceed. 28 Palacin and co-workers have also explored the use of a patterned agarose hydrogel containing an aryldiazonium salt solution sandwiched between two electrodes as an electrochemical “printing” method. 29 Finally, Corgier and Bélanger have adapted the methods of colloidal nanolithography to electrograft organic groups to the nanoscale spaces between polystyrene beads assembled on carbon and gold surfaces. 30 All of the patterning methods described above involve electrochemical generation of aryl radicals at an externally applied potential. In an earlier communication, we established that spontaneous, OCP reduction of aryldiazonium salts by carbon substrates can also be used. 31 We showed that microcontact (22) Flavel, B. S.; Garrett, D. J.; Lehr, J.; Shapter, J. G.; Downard, A. J. Electrochim. Acta 2010, 55, 3995–4001. (23) Brooksby, P. A.; Downard, A. J. Langmuir 2005, 21, 1672–1675. (24) Downard, A. J.; Garrett, D. J.; Tan, E. S. Q. Langmuir 2006, 22, 10739– 10746. (25) Ghorbal, A.; Grisotto, F.; Charlier, J.; Palacin, S.; Goyer, C.; Demaille, C. ChemPhysChem 2009, 10, 1053–1057. (26) Cougnon, C.; Gohier, F.; Belanger, D.; Mauzeroll, J. Angew. Chem., Int. Ed. 2009, 48, 4006–4008. (27) Charlier, J.; Palacin, S.; Leroy, J.; Del Frari, D.; Zagonel, L.; Barrett, N.; Renault, O.; Bailly, A.; Mariolle, D. J. Mater. Chem. 2008, 18, 3136–3142. (28) Charlier, J.; Clolus, E.; Bureau, C.; Palacin, S. J. Electroanal. Chem. 2008, 622, 238–241. (29) Mouanda, B.; Eyeffa, V.; Palacin, S. J. Appl. Electrochem. 2009, 39, 313– 320. (30) Corgier, B. P.; Belanger, D. Langmuir 2010, 26, 5991–5997. (31) Garrett, D. J.; Lehr, J.; Miskelly, G. M.; Downard, A. J. J. Am. Chem. Soc. 2007, 129, 15456–15457. 7028 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 printing (MCP) using PDMS stamps and aryldiazonium salt inks gave micrometer-scale patterns of modifiers on glassy-carbon-like thin films (pyrolyzed photoresist film; PPF). MCP is a very simple and relatively fast patterning method and, in these respects, has obvious advantages over the electrochemical methods outlined above. 32 In this paper, we investigate the characteristics (surface concentration, thickness, homogeneity, and chemical reactivity) of layers prepared using MCP and OCP reduction of aryldiazonium salts on PPF. We demonstrate that the method can be extended to metal (Au and Cu) and semiconductor (Si) substrates and provide guidelines concerning its general applicability in terms of substrate/diazonium cation combinations and the characteristics of the resultant films. We also demonstrate a unique feature of MCP of aryldiazonium salts: covalently coupled two-component surfaces can be prepared simply by printing a second layer onto a previously modified surface. EXPERIMENTAL SECTION Materials. Aqueous solutions were prepared using Millipore Milli-Q water (>18 MΩ cm). Tetrafluoroborate salts of 4-nitrobenzenediazonium (NBD) and 4-carboxybenzenediazonium (CBD) were synthesized using standard procedures. 33 4-Aminobenzenediazonium salt (ABD) was synthesized as a 20 mM solution in 0.5 M HCl. 34 Procedures for preparing citrate-capped Au nanoparticles (∼13 nm diameter), 35 PPF, 36 and planar Au films (Au/ NiCr/Si), 37 and drying acetonitrile (ACN) have been described previously. Uncut single walled carbon nanotubes (SWCNTs) (Carbon Nanotechnologies Incorporated) were acid-treated by adding 25 mg to 27 mL of 3:1 concentrated H2SO4 and HNO3 and sonicating for 10 h while adding ice to the ultrasonicator bath to maintain a temperature close to 20 °C. Following sonication, the solution was poured into 500 mL of distilled water. After standing overnight, the solution was filtered under suction through Millipore 0.22 µm hydrophilic polyvinylidene fluoride filter membranes and then washed with copious amounts of water. The dried SWCNT cakes were peeled from the filters and resuspended in DMSO to give a1mgmL -1 stock solution. Si(100) wafers (1-20 Ω cm, Silicon Quest and Micro Materials) were cut into ∼15 × 15 mm 2 tiles, immersed in 40% HF (Sigma- Aldrich) for 3 min (Caution: HF is hazardous; handle with care and appropriate personal protective clothing), washed with methanol, dried in a stream of N2 gas, and used within 10 min of HF treatment. Small pieces of Cu plate were immersed in 16 M HNO3 for 10 s, washed with water, immersed in 17 M acetic acid for 30 s, and dried in a stream of N2 gas. 38 Fabrication and solvent extraction of PDMS stamps followed previously described procedures. 24 The stamps were either nonpatterned or had a test pattern with micrometer-sized features. (32) Xia, Y. N.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 551–575. (33) Saunders, K. H.; Allen, R. L. M. Aromatic Diazo Compounds, 3rd ed.; Edward Arnold: London, 1985. (34) Lyskawa, J.; Belanger, D. Chem. Mater. 2006, 18, 4755–4763. (35) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735–743. (36) Brooksby, P. A.; Downard, A. J. Langmuir 2004, 20, 5038–5045. (37) Lehr, J.; Williamson, B. E.; Flavel, B. S.; Downard, A. J. Langmuir 2009, 25, 13503–13509. (38) Chamoulaud, G.; Belanger, D. J. Phys. Chem. C 2007, 111, 7501–7507.
Electrochemistry. Electrochemical measurements were made at room temperature in an N2 atmosphere. The electrochemical cell exposed a circular area (0.18 cm 2 for PPF or 0.79 cm 2 for Au) of substrate to the cell solution, as described previously. 36 The auxiliary and reference electrodes were Pt and SCE, respectively. Cyclic voltammograms were recorded with a scan rate of 100 mV s -1 . Surface concentrations of grafted nitrophenyl (NP) groups were estimated from cyclic voltammograms of modified surfaces in 0.10 M H2SO4. The area under the irreversible reduction peak at Ep,c ≈ -0.6 V and the area under the oxidation peak at Ep,a ≈ 0.3 V was determined by fitting the data with polynomial baselines and mixed Lorentzian-Gaussian curves, using the Levenberg-Marquardt algorithm implemented via Linkfit software. 36 The surface concentration was then calculated using Faraday’s law, as previously described, 36 with an estimated ±20% uncertainty in the absolute surface concentration. Microcontact Printing. Within2hofuse, PDMS stamps were treated with O2 plasma (100 W at 0.1 Torr for 5 min). Stamps were immersed in 20 mM aryldiazonium cation ink for 2 min, dried to tackiness in a stream of N2 gas, and placed on the substrate for 30 min. Unless stated otherwise, all printed samples were ultrasonicated for 5 min in Milli-Q water and dried in a stream of N2 prior to analysis or further treatment. Each stamp was used with only one type of ink. The compositions of printing inks are denoted “diazonium cation/solvent”. Control samples were prepared in the same way but using a solution from which the aryldiazonium salt had been omitted (“blank” ink). Characterization of Films and Patterns. AFM (Digital Instruments Dimension 3100) depth-profiling measurements were performed on modified PPF samples by scratching with the AFM tip to remove a section of film, as described previously. 36 Three average line profiles were obtained from each of two 10 × 1.25 µm 2 scratches per sample. Each line profile gave two thickness values: one from the step down into the scratch and the other from the step out of the scratch. Thus, the reported thickness for each sample is a mean of 12 values, and the uncertainties are two standard deviations of the mean. Condensation figures were obtained by allowing water vapor to condense on surfaces and imaging using an Olympus BX60 inverted light microscope equipped with a polarizer and an Olympus DP10 camera. Scanning electron microscopy (SEM) images were obtained using a JEOL 7000 high-resolution instrument with an accelerating voltage of 15 kV. The procedure for measuring water contact angles has been described previously. 31 Measurements were made using two 1 µL drops of Milli Q water placed on each duplicate sample or control. The stated values are the means of the four measurements, and the uncertainties are two standard deviations of the mean. RESULTS AND DISCUSSION This section is divided into four parts. The first describes printing with aryldiazonium salt inks on PPF, Au, and Si substrates using nonpatterned PDMS stamps. The second part demonstrates that printing with patterned PDMS stamps gives micrometer-scale patterned layers on Au, PPF, Si, and Cu. The reactivity of the printed layers is described in the third part, and finally, we show Figure 1. First (s) and second scan (---) cyclic voltammograms in 0.1MH2SO4 of a PPF surface printed with NBD/1 M H2SO4 ink for 30 min. that a second modifier can be printed onto an already modified surface to create patterned, covalently coupled, two-component surfaces. Nonpatterned Printing on PPF, Gold, and Silicon. Preliminary results establishing successful MCP of PPF using DMFand 1 M H 2SO4-based aryldiazonium salt inks have been reported earlier. 31 In both that work and the present study, PPF was used as the carbon substrate because aryldiazonium salts spontaneously graft to its surface at OCP from acidic aqueous solution 31 and its low surface roughness facilitates measurement of film thickness. 39 Here, two aryldiazonium cations were chosen for detailed examination of the printing process. The NBD derivative was selected as a relatively easily reduced species (in cyclic voltammograms, Ep,c ≈ 0VvsSCE in 0.1 M H2SO4 at a scan rate of 100 mV s -1 ) that is additionally convenient because the resultant grafted NP moieties can be readily detected and quantified by electroreduction. The CBD cation was selected as a more difficult to reduce species (Ep,c ≈ -0.3 V vs SCE in 0.1 M H2SO4 at a scan rate of 100 mV s -1 ), but which gives grafted carboxyphenyl (CP) films whose chemical reactivity makes them useful as tether layers for subsequent coupling reagents. CP groups are not electroactive in the potential range accessible in 0.1 M H2SO4, but their presence can be detected by water contact angle measurements. Figure 1 shows consecutive cyclic voltammograms of a PPF surface printed with NBD/1 M H2SO4 ink using a nonpatterned stamp. The first scan, from 0.8 to -0.9 V, shows the characteristic, irreversible reduction (Ep,c ≈ -0.60 V) of surfaceattached NP groups to aminophenyl (eq 1) and hydroxyaminophenyl groups (eq 2) but no evidence for physisorbed aryldiazonium cations, which, if present, would be reduced at Ep,c ≈ 0 V. The return scan shows the reversible oxidation (Ep,a ) 0.35 V) of hydroxyaminophenyl to nitrosophenyl groups (eq 3), 40 with the nitrosophenyl/hydroxyaminophenyl redox couple appearing as the only significant feature in subsequent cycles. These results are consistent with immobilized NP groups, spontaneously grafted to the surface during printing. Table 1 lists electroactive-NP surface concentrations, ΓNP, determined for three nonpatterned NBD/1 M H2SO4 samples from the (39) Ranganathan, S.; McCreery, R. L. Anal. Chem. 2001, 73, 893–900. (40) Ortiz, B.; Saby, C.; Champagne, G. Y.; Belanger, D. J. Electroanal. Chem. 1998, 455, 75–81. Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 7029
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Anal. Chem. 2010, 82, 6745-6750 Let
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Anal. Chem. 2010, 82, 6751-6755 Res
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Figure 1. GC/MS total ion chromatog
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Table 2. Concentrations and Stable
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on a substrate are preferred. 20-24
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Anal. Chem. 2010, 82, 6775-6781 Hig
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Numerous references had demonstrate
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Figure 1. TEM images of the prepare
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also reduces chemical noise, which
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Table 1. Extraction Yields, Liquid
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scanning of AMPP amides of the anal
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Anal. Chem. 2010, 82, 6797-6806 δ
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Figure 4. (A) IRMS mass-44 chromato
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Table 3. Ambient Measurement Result
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Anal. Chem. 2010, 82, 6807-6813 Dir
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Polymerase (1 U per sample). Reacti
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Table 2. Concentrations and Ratios
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Anal. Chem. 2010, 82, 6821-6829 Mac
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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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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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Figure 2. Optical transmission meas
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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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Table 2. Linearity and Detection Li
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ascorbic acid (AA), uric acid (UA),
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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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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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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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difference, ppm compound Table 1. I
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isoforms, its successful use, in th
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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 3. (A) ESI-LTQ-CID-MS 2 prod
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Anal. Chem. 2010, 82, 6969-6975 Ana
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Page 263 and 264: CONCLUSIONS In this paper we have i
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Page 293 and 294: Figure 1. (A) FL spectra of BSPOTPE
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Page 299 and 300: Scheme 1. Proposed Mechanism for Fl
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Page 307 and 308: Figure 2. Comparative analysis of d
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