Analytical Chemistry Chemical Cytometry Quantitates Superoxide

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the amount of oxygen available in the solution is not sufficient for full conversion of glucose conversion to H2O2 with enzyme (see also eq 1). In effect, oxygen is depleted from the solution by the enzyme reaction. To prevent the effect of oxygen depletion in measurements employing immobilized enzymes on electrodes, electrodes have been prepared with a semipermeable coating to reduce the amount of glucose diffusing to the electrode. Other approaches have involved the use of mediators, molecules which take on the role of oxygen in the reaction. 3,35 In early experiments, we also observed that the signal was dramatically decreased and calibration curves were not linear at higher concentrations of glucose when the mixing ratio between glucose and GOx was 1:1; instead, the signal tended to level off. It was decided to increase the flow rate of GOx with respect to glucose sample to a ratio of 3:1, diluting the effective glucose concentration in the channel by a factor of 4 to prevent oxygen depletion. All experiments were performed three times and a repeatable output signal was observed (as shown in the inset of Figure 6). As the concentrations of GOx solution increased, oxidation current gradually increased from approximately 20 nA (10 U/mL) to reach a maximum of 100 and 120 nA (150 U/mL) in channels with no grooves and slanted grooves, respectively. At higher GOx concentrations, the current leveled off and then decreased in both channels with no grooves and slanted grooves. These results indicated that increasing concentrations of enzyme provided better conversion to H2O2 and product within a reaction time of approximately 4 s. However, at concentrations of GOx greater than 150 U/mL, the system exhibited less stable signal, as reported elsewhere; 8 this could be due to the interaction of GOx and H2O2 produced. Therefore, a 150 U/mL concentration of GOx solution was chosen as an optimum condition for further experiments. Compared with slanted grooves in the channel, the absence of grooves in the channel resulted in lower current values. It can be concluded that the chaotic flow pattern results in more efficient mixing caused by transverse flow in the channel. 3.4. Comparison of System Response for Premixed Glucose-GOx Solutions and Reaction of Glucose and GOx in the Channel. In order to investigate dependence on flow rate, the response measured for premixed glucose-GOx solutions was compared with that measured for the reaction of glucose and GOx in the mixing/reaction channel as a function of flow velocity. For the latter experiments, a 5.6 mM glucose sample and a 150 U/mL GOx solution were introduced at a 1:3 mixing ratio in a slanted grooves channel and the flow velocity was varied from 0.95 mm/s (0.4 µL/min) to 14.3 mm/s (6 µL/min). The amperometric responses for premixed solutions and reaction in the mixing channel are presented in Figure 7. There are two dominant factors in this continuous flow-through reaction system which affect detector response, namely flow velocity and reaction time. The effect of flow velocity was measured by introducing premixed solution (5.6 mM glucose sample and 150 U/mL GOx solution had already reacted to produce H 2O2) in both inlets and varying the flow rate. The results showed that as the flow velocity increased, the recorded current values also increased gradually. It was also observed that current values increased as a function (35) Dixon, B. M.; Lowry, J. P.; O’Neill, R. D. J. Neurosci. Methods 2002, 119, 135–142. 6762 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 Figure 7. Amperometric response in a channel with slanted grooves, arising from a 1:3 reaction ratio of 5.6 mM glucose and 150 U/mL GOx as a function of flow velocity (from 0.4 to 6 µL/min). The response due to premixed solution (5.6 mM glucose and 150 U/mL GOx) was compared with the glucose-GOx reaction in the mixing channel (a single device was used for both curves, n ) 3 for each point). of flow velocity when experimenting with 0.5 mM H2O2 solutions (data not shown). This is very different from the decreasing signal observed for GOx immobilized on electrodes at higher flow rates, which Lowry et al. ascribed to the removal of H2O2 from the electrode surface. 36 On the other hand, Wu et al. 37 observed currents which increased as a function of flow rate at two closely spaced oxygen sensors in a microchannel. This latter report supports our hypothesis that the observed increase in current values for premixed solutions is due to the thinner diffusion layers at the electrode surface which result at higher flow rates. These layers are stagnant, and molecules must diffuse through them to reach the electrode surface. As diffusion layers decrease in thickness, the time required for diffusion to the electrode surface also decreases, resulting in the delivery of analyte to that surface to be less limited by diffusion. Higher rates of electron exchange and thus higher recorded currents are the result. In the case of glucose reaction with GOx in the mixing channel, a decreasing signal as a function of flow velocity was observed. This decrease in current is primarily due to lower H2O2 concentrations, the result of shorter reaction times. The maximum reaction of glucose and GOx was observed at very low flow rates (lower than 0.4 µL/min). Thus, based on reaction times of 22 and 4 s (0.4 and 2 µL/min, respectively) from the Y-junction to the electrodes, the most efficient conversion of glucose with GOx can be achieved either at very low flow rates and/or in longer reaction channels. 3.5. Calibration Curve. A calibration curve was obtained when a 150 U/mL GOx solution was reacted with glucose sample in a channel at a total flow rate of 2 µL/min, with a 1:3 flow rate (and thus 1:4 dilution) ratio of glucose and GOx. Figure 8 shows the amperometric response as a function of glucose concentration (36) Lowry, J. P.; McAteer, K.; El Atrash, S. S.; Duff, A.; O’Neill, R. D. Anal. Chem. 1994, 66, 1754–1761. (37) Wu, J.; Ye, J. Lab Chip 2005, 5, 1344–1347.
in channels with either no grooves or slanted grooves. The data exhibit a linear relationship between current and glucose concentrations of 0-20 mM, with a sensitivity of 5.3 and 6.9 nA/mM in channels with no grooves and slanted grooves, respectively. The current density was calculated by dividing sensitivity by the active area of the working electrode, yielding 26.5 and 34.5 mA/ M · cm2 for channels with no grooves and slanted grooves, respectively. The linearity of the curves was tested by plotting observed current values versus predicted current values and examining the distribution of points around the resulting diagonal line (data not shown). In both cases, points were very symmetrically distributed around the line. The slope of the plot for the no-groove case was 0.9992, with an R2 of 0.9983. In the groove case, the slope was 1.0032, with an R2 Figure 8. Calibration curves obtained using 150 U/mL GOx solution at a total flow rate of 2 µL/min (0.5 µL/min glucose: 1.5 µL/min GOx) in two channel types, without grooves and with slanted grooves (one device, n ) 3). Glucose concentrations were in the clinical range of interest. of 0.9989. In both cases, excellent linearity was thus observed. In our approach, the flow velocity and time for reaction of glucose sample and GOx enzyme strongly affect the sensitivity (38) Ghosal, S. Anal. Chem. 2002, 74, 771–775. (39) Mei, Q.; Xia, Z.; Xu, F.; Soper, S. A.; Fan, Z. H. Anal. Chem. 2008, 80, 6045–6050. of the sensor signal, allowing flow conditions to be tuned to obtain optimal results. One of the big advantages of this microfluidic reactor approach is that by varying flow rates of glucose and GOx, it is possible not only to avoid the oxygen depletion effect without any electrode treatment but to tune the sensitivity for the application. However, continuous perfusion of glucose and GOx to the device does lead to a slightly decreased signal over time. This might be caused by GOx adsorbing either onto the microchannel or electrode surface as described elsewhere. 38 This effect will be further investigated as part of the development of systems for long-term measurement of glucose in in vivo applications. 4. CONCLUSION We have successfully demonstrated an enzymatic glucose reactor based on chaotic mixing in a microfluidic channel network for continuous glucose monitoring. Together with another recent report describing improved lucerifase detection in a chaotic mixer, 39 our example of glucose detection is one of the first examples of this type of mixer being applied to the enhancement of a biochemical reaction at the nL scale. A linear calibration curve was obtained in both microchannels with slanted grooves and no grooves, using a 150 U/mL GOx enzyme solution. Higher sensitivity was obtained with slanted groove arrays compared to micromixers with no grooves, due to enhanced mixing. The factors which determine sensitivity are flow velocity and extent of reaction. Though there is a loss of GOx due to the continuous flow to the outlet, this disadvantage is alleviated by the use of micofluidics for nL liquid handling and the application of low flow rates (2 µL/min for the optimized system). The low flow rates used are also compatible with microdialysis sampling and are required to achieve a high recovery of glucose from the subcutaneous tissue and reduce solution consumption. The possible influence of interfering substances such as ascorbic acid, uric acid, and acetaminophen on glucose determination are now under investigation, prior to testing the system in vivo in rats. Received for review January 8, 2010. Accepted June 24, 2010. AC1000509 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6763
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: Figure 5. Comparison of cyclic volt
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
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Page 65 and 66: Polymerase (1 U per sample). Reacti
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analytically useful signals at less
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carbon black and RP-C18 for the ext
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solution in an equal volume, and 1
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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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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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