Limits...
Measuring localized redox enzyme electron transfer in a live cell with conducting atomic force microscopy.

Alfonta L, Meckes B, Amir L, Schlesinger O, Ramachandran S, Lal R - Anal. Chem. (2014)

Bottom Line: A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII.An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed.Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, ‡Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , P.O. Box 653, Beer-Sheva, 84105, Israel.

ABSTRACT
Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of E. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells.

Show MeSH
Cyclic voltammograms collected at everymode of bacterium selection.Background: before engaging the surface. Surface mode: engaging witha single bacterium. Withdrawn mode: withdrawing upon attachment ofa bacterium. Scan rates were 0.05 V s–1; referenceelectrode: Ag/AgCl wire.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215851&req=5

fig2: Cyclic voltammograms collected at everymode of bacterium selection.Background: before engaging the surface. Surface mode: engaging witha single bacterium. Withdrawn mode: withdrawing upon attachment ofa bacterium. Scan rates were 0.05 V s–1; referenceelectrode: Ag/AgCl wire.

Mentions: A bacterium was pickedup by the conducting AFM probe through the following steps as summarizedin Scheme 2. Afterlocalization of a bacterium, the gold-coated conducting AFM probewas allowed to remain in controlled contact with the bacterium surfacewithout imaging to facilitate gold–thiol bond formation betweenthe linker 1 site specifically attached to ADHII andthe probe for ∼5 min (Scheme 2B). Thechemical structure of the thiol containing linker 1 isshown in the inset of Scheme 2. The tip wasthen disengaged from the surface (Scheme 2C).Successful attachment of the bacterium to the tip inhibited reimagingof the surface. This was further confirmed through observed changesin cyclic voltammograms. The activity of the probe before imagingthe bacteria was recorded (Figure 2, background).During the selection of a single bacterium, electrochemical measurementswere conducted to check whether electrochemical activity could bedetected (Figure 2, surface mode). This servedas our first indication of a successful transfer of a bacterium fromthe surface to the tip. Upon selecting and attaching a bacterium tothe AFM tip, we commenced with our electrochemical analysis of electrontransfer (ET) processes (Figure 2, withdrawnmode). Coupling both the AFM imaging abilities with the electrochemicalsignals demonstrated that we have collected a redox active bacteriumoff the surface. However, this did not exclude the possibility thatonly part of the bacteria was removed.


Measuring localized redox enzyme electron transfer in a live cell with conducting atomic force microscopy.

Alfonta L, Meckes B, Amir L, Schlesinger O, Ramachandran S, Lal R - Anal. Chem. (2014)

Cyclic voltammograms collected at everymode of bacterium selection.Background: before engaging the surface. Surface mode: engaging witha single bacterium. Withdrawn mode: withdrawing upon attachment ofa bacterium. Scan rates were 0.05 V s–1; referenceelectrode: Ag/AgCl wire.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215851&req=5

fig2: Cyclic voltammograms collected at everymode of bacterium selection.Background: before engaging the surface. Surface mode: engaging witha single bacterium. Withdrawn mode: withdrawing upon attachment ofa bacterium. Scan rates were 0.05 V s–1; referenceelectrode: Ag/AgCl wire.
Mentions: A bacterium was pickedup by the conducting AFM probe through the following steps as summarizedin Scheme 2. Afterlocalization of a bacterium, the gold-coated conducting AFM probewas allowed to remain in controlled contact with the bacterium surfacewithout imaging to facilitate gold–thiol bond formation betweenthe linker 1 site specifically attached to ADHII andthe probe for ∼5 min (Scheme 2B). Thechemical structure of the thiol containing linker 1 isshown in the inset of Scheme 2. The tip wasthen disengaged from the surface (Scheme 2C).Successful attachment of the bacterium to the tip inhibited reimagingof the surface. This was further confirmed through observed changesin cyclic voltammograms. The activity of the probe before imagingthe bacteria was recorded (Figure 2, background).During the selection of a single bacterium, electrochemical measurementswere conducted to check whether electrochemical activity could bedetected (Figure 2, surface mode). This servedas our first indication of a successful transfer of a bacterium fromthe surface to the tip. Upon selecting and attaching a bacterium tothe AFM tip, we commenced with our electrochemical analysis of electrontransfer (ET) processes (Figure 2, withdrawnmode). Coupling both the AFM imaging abilities with the electrochemicalsignals demonstrated that we have collected a redox active bacteriumoff the surface. However, this did not exclude the possibility thatonly part of the bacteria was removed.

Bottom Line: A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII.An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed.Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, ‡Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , P.O. Box 653, Beer-Sheva, 84105, Israel.

ABSTRACT
Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of E. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells.

Show MeSH