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Fluorescence-enabled electrochemical microscopy with dihydroresorufin as a fluorogenic indicator.

Oja SM, Guerrette JP, David MR, Zhang B - Anal. Chem. (2014)

Bottom Line: The use of dihydroresorufin has enabled us to study a series of reducible analyte species including Fe(CN)6(3-) and Ru(NH3)6(3+).FEEM is used to quantitatively detect the reduction of ferricyanide down to a concentration of approximately 100 μM on a 25 μm ultramicroelectrode.We also demonstrate that dihydroresorufin, as a fluorogenic indicator, gives an improved temporal response and significantly decreases diffusional broadening of the signal in FEEM as compared to resazurin.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Washington , Seattle, Washington 98195-1700, United States.

ABSTRACT
Recently, we introduced a new electrochemical imaging technique called fluorescence-enabled electrochemical microscopy (FFEM). The central idea of FEEM is that a closed bipolar electrode is utilized to electrically couple a redox reaction of interest to a complementary fluorogenic reaction converting an electrochemical signal into a fluorescent signal. This simple strategy enables one to use fluorescence microscopy to observe conventional electrochemical processes on very large electrochemical arrays. The initial demonstration of FEEM focused on the use of a specific fluorogenic indicator, resazurin, which is reduced to generate highly fluorescent resorufin. The use of resazurin has enabled the study of analyte oxidation reactions, such as the oxidation of dopamine and H2O2. In this report, we extend the capability of FEEM to the study of cathodic reactions using a new fluorogenic indicator, dihydroresorufin. Dihydroresorufin is a nonfluorescent molecule, which can be electrochemically oxidized to generate resorufin. The use of dihydroresorufin has enabled us to study a series of reducible analyte species including Fe(CN)6(3-) and Ru(NH3)6(3+). Here we demonstrate the correlation between the simultaneously recorded fluorescence intensity of resorufin and electrochemical oxidation current during potential sweep experiments. FEEM is used to quantitatively detect the reduction of ferricyanide down to a concentration of approximately 100 μM on a 25 μm ultramicroelectrode. We also demonstrate that dihydroresorufin, as a fluorogenic indicator, gives an improved temporal response and significantly decreases diffusional broadening of the signal in FEEM as compared to resazurin.

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Voltage pulsing experimentcomparing the FEEM detection of ferricyanideusing dihydroresorufin to the detection of ferrocyanide using resazurinat a 25 μm Au electrode. (a) Comparison of the normalized fluorescenceintensity over time of the resazurin system and dihydroresorufin system.The voltage pulse was in the “on” state (a potentialsufficient to drive the coupled reactions of interest) for 8 s andthen switched to the “off” state for 8 s. (b) Linescansacross the electrode over the course of the voltage pulse. (c) Fluorescenceimages of the electrode over the course of the voltage pulse. Themaximum intensity on the color scale was set to the maximum pixelintensity recorded at the electrode at 8 s.
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fig6: Voltage pulsing experimentcomparing the FEEM detection of ferricyanideusing dihydroresorufin to the detection of ferrocyanide using resazurinat a 25 μm Au electrode. (a) Comparison of the normalized fluorescenceintensity over time of the resazurin system and dihydroresorufin system.The voltage pulse was in the “on” state (a potentialsufficient to drive the coupled reactions of interest) for 8 s andthen switched to the “off” state for 8 s. (b) Linescansacross the electrode over the course of the voltage pulse. (c) Fluorescenceimages of the electrode over the course of the voltage pulse. Themaximum intensity on the color scale was set to the maximum pixelintensity recorded at the electrode at 8 s.

Mentions: In spite of the relatively high limit of detection of FEEM usingdihydroresorufin, we found that this system gives rise to severalsignificant benefits. As already demonstrated, the electrochemicalcurrent signal can be reported by directly monitoring the total fluorescencesignal rather than the time derivative of this signal as with ourprevious report. More importantly, FEEM using dihydroresorufin asthe fluorogenic reporter appears to give enhanced spatial and temporalresolution than FEEM using resazurin as the reporter. A simple potentialstep experiment, shown in Figure 6, demonstratesthis point. In this experiment, two 25 μm diameter Au electrodeswere connected to form a closed BPE. One pole was placed in a solutionthat was 250 μM in both ferricyanide and ferrocyanide with 1M KCl as supporting electrolyte. The opposite pole was placed in either100 μM dihydroresorufin, 67 mM glucose, and 0.5 M NaOH or 100μM resazurin in 50 mM phosphate buffer. An 8 s pulse at a potentialsufficient to drive the coupled redox reactions was applied (“on”state), followed by an 8 s period at which the potential was adjustedbelow the onset potential (“off” state). For the coupleddihydroresorufin oxidation/ferricyanide reduction, the “on”state was +0.3 V and the “off” state was +1.2 V. Forthe coupled resazurin reduction/ferrocyanide oxidation, the “on”state was +1.2 V and the “off” state was +0.3 V. Figure 6a shows the normalized fluorescence intensity recordedover the length of the pulse cycle for both coupled reactions. Asseen, the fluorescence response of the two fluorogenic indicatorsis markedly different. The dihydroresorufin system reaches a steady-statefluorescence intensity within 2 s of the start of the “on”pulse, while the fluorescence intensity of the resazurin system increasesthroughout the “on” pulse, failing to reach a steady-statein 8 s. When the potential is switched to the “off”state, the fluorescence intensity of the dihydroresorufin system decaysto its initial intensity within 1 s, while the intensity of the resazurinsystem shows an immediate spike, followed by a slow decay, failingto return to its initial intensity within 8 s.


Fluorescence-enabled electrochemical microscopy with dihydroresorufin as a fluorogenic indicator.

Oja SM, Guerrette JP, David MR, Zhang B - Anal. Chem. (2014)

Voltage pulsing experimentcomparing the FEEM detection of ferricyanideusing dihydroresorufin to the detection of ferrocyanide using resazurinat a 25 μm Au electrode. (a) Comparison of the normalized fluorescenceintensity over time of the resazurin system and dihydroresorufin system.The voltage pulse was in the “on” state (a potentialsufficient to drive the coupled reactions of interest) for 8 s andthen switched to the “off” state for 8 s. (b) Linescansacross the electrode over the course of the voltage pulse. (c) Fluorescenceimages of the electrode over the course of the voltage pulse. Themaximum intensity on the color scale was set to the maximum pixelintensity recorded at the electrode at 8 s.
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Related In: Results  -  Collection

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fig6: Voltage pulsing experimentcomparing the FEEM detection of ferricyanideusing dihydroresorufin to the detection of ferrocyanide using resazurinat a 25 μm Au electrode. (a) Comparison of the normalized fluorescenceintensity over time of the resazurin system and dihydroresorufin system.The voltage pulse was in the “on” state (a potentialsufficient to drive the coupled reactions of interest) for 8 s andthen switched to the “off” state for 8 s. (b) Linescansacross the electrode over the course of the voltage pulse. (c) Fluorescenceimages of the electrode over the course of the voltage pulse. Themaximum intensity on the color scale was set to the maximum pixelintensity recorded at the electrode at 8 s.
Mentions: In spite of the relatively high limit of detection of FEEM usingdihydroresorufin, we found that this system gives rise to severalsignificant benefits. As already demonstrated, the electrochemicalcurrent signal can be reported by directly monitoring the total fluorescencesignal rather than the time derivative of this signal as with ourprevious report. More importantly, FEEM using dihydroresorufin asthe fluorogenic reporter appears to give enhanced spatial and temporalresolution than FEEM using resazurin as the reporter. A simple potentialstep experiment, shown in Figure 6, demonstratesthis point. In this experiment, two 25 μm diameter Au electrodeswere connected to form a closed BPE. One pole was placed in a solutionthat was 250 μM in both ferricyanide and ferrocyanide with 1M KCl as supporting electrolyte. The opposite pole was placed in either100 μM dihydroresorufin, 67 mM glucose, and 0.5 M NaOH or 100μM resazurin in 50 mM phosphate buffer. An 8 s pulse at a potentialsufficient to drive the coupled redox reactions was applied (“on”state), followed by an 8 s period at which the potential was adjustedbelow the onset potential (“off” state). For the coupleddihydroresorufin oxidation/ferricyanide reduction, the “on”state was +0.3 V and the “off” state was +1.2 V. Forthe coupled resazurin reduction/ferrocyanide oxidation, the “on”state was +1.2 V and the “off” state was +0.3 V. Figure 6a shows the normalized fluorescence intensity recordedover the length of the pulse cycle for both coupled reactions. Asseen, the fluorescence response of the two fluorogenic indicatorsis markedly different. The dihydroresorufin system reaches a steady-statefluorescence intensity within 2 s of the start of the “on”pulse, while the fluorescence intensity of the resazurin system increasesthroughout the “on” pulse, failing to reach a steady-statein 8 s. When the potential is switched to the “off”state, the fluorescence intensity of the dihydroresorufin system decaysto its initial intensity within 1 s, while the intensity of the resazurinsystem shows an immediate spike, followed by a slow decay, failingto return to its initial intensity within 8 s.

Bottom Line: The use of dihydroresorufin has enabled us to study a series of reducible analyte species including Fe(CN)6(3-) and Ru(NH3)6(3+).FEEM is used to quantitatively detect the reduction of ferricyanide down to a concentration of approximately 100 μM on a 25 μm ultramicroelectrode.We also demonstrate that dihydroresorufin, as a fluorogenic indicator, gives an improved temporal response and significantly decreases diffusional broadening of the signal in FEEM as compared to resazurin.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Washington , Seattle, Washington 98195-1700, United States.

ABSTRACT
Recently, we introduced a new electrochemical imaging technique called fluorescence-enabled electrochemical microscopy (FFEM). The central idea of FEEM is that a closed bipolar electrode is utilized to electrically couple a redox reaction of interest to a complementary fluorogenic reaction converting an electrochemical signal into a fluorescent signal. This simple strategy enables one to use fluorescence microscopy to observe conventional electrochemical processes on very large electrochemical arrays. The initial demonstration of FEEM focused on the use of a specific fluorogenic indicator, resazurin, which is reduced to generate highly fluorescent resorufin. The use of resazurin has enabled the study of analyte oxidation reactions, such as the oxidation of dopamine and H2O2. In this report, we extend the capability of FEEM to the study of cathodic reactions using a new fluorogenic indicator, dihydroresorufin. Dihydroresorufin is a nonfluorescent molecule, which can be electrochemically oxidized to generate resorufin. The use of dihydroresorufin has enabled us to study a series of reducible analyte species including Fe(CN)6(3-) and Ru(NH3)6(3+). Here we demonstrate the correlation between the simultaneously recorded fluorescence intensity of resorufin and electrochemical oxidation current during potential sweep experiments. FEEM is used to quantitatively detect the reduction of ferricyanide down to a concentration of approximately 100 μM on a 25 μm ultramicroelectrode. We also demonstrate that dihydroresorufin, as a fluorogenic indicator, gives an improved temporal response and significantly decreases diffusional broadening of the signal in FEEM as compared to resazurin.

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