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Molecule Transport
Across Cell Membranes:
Electrochemical Quantification
at the Microscale
Associate Head of Graduate Studies
Associate Professor
Department of Chemistry
University of Manitoba
Sabine Kuss, PhD
umanitoba.ca
Molecule Transport across Cell
Membranes:
Electrochemical Quantification at
the Microscale
Dr. Sabine Kuss
Associate Head – Graduate Studies
Associate Professor
Department of Chemistry
umanitoba.ca
Acknowledgements
umanitoba.ca
Drug Resistance Mechanisms
[1] H. Nikaido, Annu. Rev. Biochem., 78, 119–146 (2009).
[2] S. N. Aleksakhina et al Imyanitov, Biochim. Biophys. Acta - Rev. Cancer,
1872, 188310 (2019).
[3] G. Housman et al Cancers (Basel)., 6, 1769–1792 (2014).
à Mechanisms very similar in
mammalian cancer cells
umanitoba.ca
Facts about Drug Resistance
• DR enables cells to withstand antibiotic treatment or chemotherapy [1]
• It is present in every country [2]
• 10 million deaths worldwide each year are attributed to antibiotic
resistance [3]
• DR in cancer is estimated to be responsible for treatment failure in up
to 90% of metastatic cancer patients. [4]
à URGENT NEED FOR NEW TREATMENT AND DIAGNOSTIC
STRATEGIES
[1] H.W. Boucher, G.H. Talbot, J.S. Bradley et al. Clin. Infec. Dis. 48, 1, 2009
[2] World Health Organization, Antimicrobial Resistance – Fact Sheet
Accessed Nov 2019
[3] J. Sun, A.R. Warden, J. Huang et al. Anal. Chem. 91, 7524, 2019.
[4] R. Article. J. Pathol. 205, 275, 2005.
umanitoba.ca
Example for Chemoresistance
• Carboplatin is listed by the WHO as essential
medicine for the treatment of human cancers [1]
• 75% of ovarian cancer patients will relapse
within 18 months [2]
• 85% exhibit chemoresistance against
carboplatin [2]
• Resistance mechanism is unidentified [3]
[1] 1. Cortez, A.J. et al. Can Chemother and Pharmacol 2018. 81(1), 17.
[2] Bowtell, D.D. et al. Nat Rev Can 2015. 15(11), 668.
[3] Howell, S.B. et al. Mol Pharmacol 2010. 77(6), 887
umanitoba.ca
Methods of Transport Quantification
Golubchik A., Lopes L.C., Singh V., Kuss S.: Pharma-molecule transport across bacterial membranes; detection and
quantification approaches by electrochemistry and bioanalytical methods. Angewandte Chemie - International
Edition 2021, 60, 2.
umanitoba.ca
Electrochemical Approaches
umanitoba.ca
R O
R
R
R
e-
E
Electrochemistry at Macroelectrodes
umanitoba.ca
R O
R
R
e-
E
Cyclic Voltammetry
Current Potential
umanitoba.ca
Electrochemical Biomarker
Characterization
• Oxidation/Reduction potentials
• Diffusion behavior in solution/
Interaction with an electrode
• pH dependence
• Detection Limits
• Interferences of ions or competitive
drugs Potential
Current
umanitoba.ca
Cyclic Voltammetry
Ciprofloxacin
Tobramycin
Ciprofloxacin-Tobramycin-Hybrid
Domalaon R. et al. Clin. Microbiol. Rev. 2018, 31 (2), e00077-17
Islam M.R. et al. Electrochem. Comm. 2020. 119C, 106825.
umanitoba.ca
Differential Pulse Voltammetry
Domalaon R. et al. Clin. Microbiol. Rev. 2018, 31 (2), e00077-17
Islam M.R. et al. Electrochem. Comm. 2020. 119C, 106825.
Ciprofloxacin-Tobramycin-Hybrid
umanitoba.ca
Electrochemical Characterization
Domalaon R. et al. Clin. Microbiol. Rev. 2018, 31 (2), e00077-17
Islam M.R. et al. Electrochem. Comm. 2020. 119C, 106825.
umanitoba.ca
Detecting Membrane-Related
Transport Mechanisms
umanitoba.ca
Electrochemical Detection of
Antimicrobial Resistance
Pseudomonas aeruginosa
Lopes L.C., Lima D., Hayat M., Li Y., Kumar A., Kuss S.: Electrochemical
Quantification of Tobramycin Retention in Pseudomonas aeruginosa as Antimicrobial
Susceptibility Indicator. Analytical Chemistry. 2022, 94(37), 12553.
umanitoba.ca
Electrochemical Measurement of
Drug Retention
Lopes L.C., Lima D., Hayat M., Li Y., Kumar A., Kuss S.: Electrochemical
Quantification of Tobramycin Retention in Pseudomonas aeruginosa as
Antimicrobial Susceptibility Indicator. Analytical Chemistry. 2022, 94(37),
12553.
umanitoba.ca
Electrochemical Measurement of
Drug Retention
Lopes L.C., Lima D., Hayat M., Li Y., Kumar A., Kuss S.: Electrochemical
Quantification of Tobramycin Retention in Pseudomonas aeruginosa as
Antimicrobial Susceptibility Indicator. Analytical Chemistry. 2022, 94(37),
12553.
umanitoba.ca
Chemotherapeutic Uptake
A2780-S
H.T.L. Luu, M.W. Nachtigal, S. Kuss. Journal of Electroanalytical
Chemistry, 872C, 114253, 2020.
umanitoba.ca
Carboplatin Uptake/Retention
A2780-S
A2780-CP
H.T.L. Luu, M.W. Nachtigal, S. Kuss. Journal of Electroanalytical
Chemistry, 872C, 114253, 2020.
umanitoba.ca
Detecting Intracellular Mechanisms
through Cell Metabolite Exchange
umanitoba.ca
e-
The Scanning Electrochemical Microscope (SECM)
25 µm Pt electrode
Metal
Glass
umanitoba.ca
Electrochemical Imaging of
Chemoresistance
Kuss S, Trinh D, Mauzeroll J. 2015 Analytical Chemistry 87, 8102−8106.
Ovarian Epithelial Cancer Cells
A2780-cp
Ovarian Epithelial Cancer Cells
A2780-s
umanitoba.ca
Electrochemical
Quantification
Horizontal Line Scans
Across Living Cells
Importance of
temperature Control,
and media composition
Kuss S., Kuss C., Trinh D., Schougaard S.B., Mauzeroll J.
Electrochimica Acta, 2013,110, 42.
umanitoba.ca
SECM Live Cell Imaging
Ovarian Epithelial Cancer Cells
A2780-cp
Ovarian Epithelial Cancer Cells
A2780-s
Slope connected to the kinetics derived from
numerical model
umanitoba.ca
Detecting of Molecule Transport as
Disease Indicator
umanitoba.ca
Impact of Cytochrome C Oxidase Deficiency
• Severe muscle weakness
• Heart, liver and kidney
problems
• Brain damage Brain damage due to COX
Deficiency
Healthy
brain
National Organization for Rare Disorders (NORD). Cytochrome C
Oxidase Deficiency. NORD Report 2021.
Gaillard, F. Normal Brain (MRI). Radiopaedia.
Due to absence or
abnormality of the protein
Cytochrome C Oxidase
umanitoba.ca
Model Systems
Fibroblast cells from connective tissue in muscle
Control
Cells
SCO1 Patient
COX Deficient
Cells
Leary, S. C.
(2007). Cell
Metabolism,
5(1), 9-20. What is the purpose here?
8
Electrochemical monitoring of TMPD interaction with living fibroblasts
The two fibroblast cell lines used in this work included a control cell line (Control
65) of healthy fibroblasts, capable of expressing a fully assembled and functional COX
enzyme. SCO1 cells carry the SCO1 gene mutation that impairs the proper biosynthesis
of COX (3, 11). Both cell types present a similar morphology, characterized by an
elongated shape with a fibrous aspect (Figures 1B and C). To validate the cell lines of
choice, expression levels of fully assembled COX and SCO1 proteins in the Control and
SCO1 cell lines were quantified by Western blot analysis. The SDS-PAGE result displayed
in Figure 2A confirms the reduced level of SCO1 protein (29 kDa) in SCO1 cells. VDAC1
(35 kDa) was used as a loading control and was detected equally in both cell lines.
Accordingly, the BN-PAGE results (Figure 2B) show that cells carrying the SCO1 mutation
present reduced levels of fully assembled COX due to decreased levels of SCO1 protein
(9). Complex I was used as a loading control and was detected equally in both fibroblast
lines. The decreased levels of SCO1 and COX proteins in SCO1 cells, therefore, validate
SCO1 as a COXD cell line. Differential electrochemical signals are expected for COX
activity in Control cells compared to SCO1-deficient fibroblast cells.
Fig. 2. Western blot results of mitoplasts isolated from Control and SCO1 fibroblast cells. (A) Fibroblasts fractionated
by SDS-PAGE and blot were subject to antibodies against SCO1. VDAC1 was used as a loading control. Molecular
weight (MW) protein ladder is indicated on the left. (B) Fibroblasts fractionated by 1D BN-PAGE and blot were subject
to antibodies against complex IV. Complex I was used as a loading control.
Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome
c oxidase deficiency detection in human fibroblasts using scanning
electrochemical microscopy. 2023. Under Revision.
→ SCO1 is a metallo-
chaperone protein required
for proper assembly of
COX
umanitoba.ca
Cytochrome C Oxidase Indicator
TMPD+•
+ e-
- e-
TMPD
L. Michaelis, et al. J. Am. Chem. Soc., vol. 61, no. 8, pp. 1981–1992, Aug. 1939.
D. Menshykau, et a. J. Phys. Chem. C, vol. 112, no. 37, pp. 14428-14438, Aug. 2008.
J. Gordon, et al. J. Pathol. Bacteriol., vol. 31, no. 2, pp. 185–190, 1928.
umanitoba.ca
SECM Imaging of Living Fibroblast Cells
200 µm
Fibroblast cell
Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome
c oxidase deficiency detection in human fibroblasts using scanning
electrochemical microscopy. 2023. Under Revision.
1.10
1.05
1.00
1.05
1.10
1.15
1.20
I
T
/I
T
inf
umanitoba.ca
Electrochemical Detection of COX
Deficiency
Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome
c oxidase deficiency detection in human fibroblasts using scanning
electrochemical microscopy. 2023. Under Revision.
umanitoba.ca
SECM 3D Imaging
Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome
c oxidase deficiency detection in human fibroblasts using scanning
electrochemical microscopy. 2023. Under Revision.
I
T
/I
T
inf
I
T
/I
T
inf
I
T
/I
T
inf
I
T
/I
T
inf
umanitoba.ca
Lines Scans Across
Living Cells 200 µm
Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome
c oxidase deficiency detection in human fibroblasts using scanning
electrochemical microscopy. 2023. Under Revision.
I
T
/I
T
inf
I
T
/I
T
inf
umanitoba.ca
Cell Line Scan Analysis
Kuss S, Trinh D, Mauzeroll J. 2015 Analytical Chemistry 87, 8102−8106.
12 um
Estimated 7-8 um
Kuss S., Kuss C., Trinh D.,
Schougaard S.B., Mauzeroll J.
Electrochimica Acta, 2013,110, 42.
a)
c)
• Topography contribution varies with
increasing scan rate
• Reactivity contribution remains constant
Decoupling of Topography
and Reactivity by
numerical modeling
umanitoba.ca
Kuss S, Trinh D, Danis L, Mauzeroll J. 2015 Analytical Chemistry 87,
8096−8101.
High-speed SECM
SECM at regular
speed
Prof. Dr. Dao Trinh
University La Rochelle, France
log 𝐼!"#$ = 𝑰𝟎 + 𝑨 𝑒(𝒓𝟎 ()*(+"))
𝑃! =
𝑣 𝑎"
𝐷 𝑑
log 𝐼!"#$ = 𝜶 log(𝑃-)
log
(peak
current)
log (velocity)
Normalized velocity:
Decoupling Topography and Reactivity
umanitoba.ca
Numerical Modeling
𝑳𝒐𝒈 (𝑰𝒏𝒐𝒓𝒎) = 𝑰𝟎 + 𝑨𝒆𝒙𝒑 (𝒓𝟎𝑳𝒐𝒈 𝑷𝒔 )
I0, A, and r0 are the fitting parameters which
depend on the apparent heterogenous rate
constant, k0
Slow scan rate
Fast scan
rate
Kuss, S., Trinh, D., Mauzeroll, J. (2015). Analytical Chemistry,
87(16), 8096-8101.
Kuss, S., Kuss, C., Trinh, D., Schougaard, S. B., Mauzeroll, J.
(2013). Electrochimica Acta, 110, 42-48.
umanitoba.ca
Apparent Heterogenous Rate Constants
for Control and SCO1 Cells
• Control 65: 𝑘' = 9 ∗ 10() m/s
• SCO1 Patient: 𝑘' = 7.5 ∗ 10()
m/s
Cytochrome C Reductase
Activity
• Control 65: 𝑘' = 3.2 ∗ 10() m/s
• SCO1 Patient: 𝑘' = 1.5 ∗ 10()
m/s
Cytochrome C Oxidase
Activity
Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome
c oxidase deficiency detection in human fibroblasts using scanning
electrochemical microscopy. 2023. Under Revision.
umanitoba.ca
Towards the Detecting of Reactive
Molecules Transported across
Membranes
umanitoba.ca
Optical Fibers in Electrochemistry
39
N. Thomas, V. Singh, S. Kuss, https://doi.org/10.1016/j.trac.2021.116196
D.A. Van Dyke, H. Yuan Cheng, https://doi.org/10.1021/ac00164a004
Y. Takahashi, H. Shiku, T. Murata, T. Yasukawa, T. Matsue,https://doi.org/10.1021/ac901796r
umanitoba.ca
MORE Fabrication
40
Thomas N., Singh V., Ahmed N., Trinh D., Kuss S.: Single Cell Scanning
Photoelectrochemical Microscopy using Micro-Optical-Ring
Electrodes. Biosensors and Bioelectronics. 2022, 217, 114658.
umanitoba.ca
SPECM Setup
41
Glass
Epoxy Resin
Optical Fiber
Au
Thomas N., Singh V., Ahmed N., Trinh D., Kuss S.: Single Cell Scanning
Photoelectrochemical Microscopy using Micro-Optical-Ring
Electrodes. Biosensors and Bioelectronics. 2022, 217, 114658.
umanitoba.ca
Scanning (Photo)Electrochemical Microscopy (S(P)ECM)
e-
SPECM by
umanitoba.ca
Micro-Optical-Ring-Electrodes (MORE)
umanitoba.ca
TMPD Spectrum through
Optic Fibers and MOREs
Optical Fiber MORE
Thomas N., Singh V., Ahmed N., Trinh D., Kuss S.: Single Cell Scanning
Photoelectrochemical Microscopy using Micro-Optical-Ring
Electrodes. Biosensors and Bioelectronics. 2022, 217, 114658.
umanitoba.ca
Oxygen Detection by
Electrochemistry
Potentiostat
O
2
O
2
O
2
Thomas N., Singh V., Ahmed N., Trinh D., Kuss S.: Single Cell Scanning
Photoelectrochemical Microscopy using Micro-Optical-Ring
Electrodes. Biosensors and Bioelectronics. 2022, 217, 114658.
umanitoba.ca
SPECM Stimulation and Analysis
46
Thomas N., Singh V., Ahmed N., Trinh D., Kuss S.: Single Cell Scanning
Photoelectrochemical Microscopy using Micro-Optical-Ring
Electrodes. Biosensors and Bioelectronics. 2022, 217, 114658.
umanitoba.ca
H2O2
O2 + 2H+ + 2e-
H2O2
H2O2
H2O2
H2O2
Live cell
Petri dish
Potential Future
Biological Applications
of SPECM
• Detection of ROS/RNS
• Identify ROS/RNS involved in cancer
progression
• Monitoring of cancer initiation
umanitoba.ca
Acknowledgements
● Shubhneet Thind
● Dr Dhésmon Lima
● Dr Evan Booy, UM
● Dr Sean McKenna, UM
● Dr Frank Wang
www.bioanalyticschemistry.com
@LBES_UofM
umanitoba.ca
Thank you!
www.bioanalyticschemistry.com

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Molecule Transport across Cell Membranes: Electrochemical Quantification at the Microscale

  • 1. Molecule Transport Across Cell Membranes: Electrochemical Quantification at the Microscale Associate Head of Graduate Studies Associate Professor Department of Chemistry University of Manitoba Sabine Kuss, PhD
  • 2. umanitoba.ca Molecule Transport across Cell Membranes: Electrochemical Quantification at the Microscale Dr. Sabine Kuss Associate Head – Graduate Studies Associate Professor Department of Chemistry
  • 4. umanitoba.ca Drug Resistance Mechanisms [1] H. Nikaido, Annu. Rev. Biochem., 78, 119–146 (2009). [2] S. N. Aleksakhina et al Imyanitov, Biochim. Biophys. Acta - Rev. Cancer, 1872, 188310 (2019). [3] G. Housman et al Cancers (Basel)., 6, 1769–1792 (2014). à Mechanisms very similar in mammalian cancer cells
  • 5. umanitoba.ca Facts about Drug Resistance • DR enables cells to withstand antibiotic treatment or chemotherapy [1] • It is present in every country [2] • 10 million deaths worldwide each year are attributed to antibiotic resistance [3] • DR in cancer is estimated to be responsible for treatment failure in up to 90% of metastatic cancer patients. [4] à URGENT NEED FOR NEW TREATMENT AND DIAGNOSTIC STRATEGIES [1] H.W. Boucher, G.H. Talbot, J.S. Bradley et al. Clin. Infec. Dis. 48, 1, 2009 [2] World Health Organization, Antimicrobial Resistance – Fact Sheet Accessed Nov 2019 [3] J. Sun, A.R. Warden, J. Huang et al. Anal. Chem. 91, 7524, 2019. [4] R. Article. J. Pathol. 205, 275, 2005.
  • 6. umanitoba.ca Example for Chemoresistance • Carboplatin is listed by the WHO as essential medicine for the treatment of human cancers [1] • 75% of ovarian cancer patients will relapse within 18 months [2] • 85% exhibit chemoresistance against carboplatin [2] • Resistance mechanism is unidentified [3] [1] 1. Cortez, A.J. et al. Can Chemother and Pharmacol 2018. 81(1), 17. [2] Bowtell, D.D. et al. Nat Rev Can 2015. 15(11), 668. [3] Howell, S.B. et al. Mol Pharmacol 2010. 77(6), 887
  • 7. umanitoba.ca Methods of Transport Quantification Golubchik A., Lopes L.C., Singh V., Kuss S.: Pharma-molecule transport across bacterial membranes; detection and quantification approaches by electrochemistry and bioanalytical methods. Angewandte Chemie - International Edition 2021, 60, 2.
  • 11. umanitoba.ca Electrochemical Biomarker Characterization • Oxidation/Reduction potentials • Diffusion behavior in solution/ Interaction with an electrode • pH dependence • Detection Limits • Interferences of ions or competitive drugs Potential Current
  • 12. umanitoba.ca Cyclic Voltammetry Ciprofloxacin Tobramycin Ciprofloxacin-Tobramycin-Hybrid Domalaon R. et al. Clin. Microbiol. Rev. 2018, 31 (2), e00077-17 Islam M.R. et al. Electrochem. Comm. 2020. 119C, 106825.
  • 13. umanitoba.ca Differential Pulse Voltammetry Domalaon R. et al. Clin. Microbiol. Rev. 2018, 31 (2), e00077-17 Islam M.R. et al. Electrochem. Comm. 2020. 119C, 106825. Ciprofloxacin-Tobramycin-Hybrid
  • 14. umanitoba.ca Electrochemical Characterization Domalaon R. et al. Clin. Microbiol. Rev. 2018, 31 (2), e00077-17 Islam M.R. et al. Electrochem. Comm. 2020. 119C, 106825.
  • 16. umanitoba.ca Electrochemical Detection of Antimicrobial Resistance Pseudomonas aeruginosa Lopes L.C., Lima D., Hayat M., Li Y., Kumar A., Kuss S.: Electrochemical Quantification of Tobramycin Retention in Pseudomonas aeruginosa as Antimicrobial Susceptibility Indicator. Analytical Chemistry. 2022, 94(37), 12553.
  • 17. umanitoba.ca Electrochemical Measurement of Drug Retention Lopes L.C., Lima D., Hayat M., Li Y., Kumar A., Kuss S.: Electrochemical Quantification of Tobramycin Retention in Pseudomonas aeruginosa as Antimicrobial Susceptibility Indicator. Analytical Chemistry. 2022, 94(37), 12553.
  • 18. umanitoba.ca Electrochemical Measurement of Drug Retention Lopes L.C., Lima D., Hayat M., Li Y., Kumar A., Kuss S.: Electrochemical Quantification of Tobramycin Retention in Pseudomonas aeruginosa as Antimicrobial Susceptibility Indicator. Analytical Chemistry. 2022, 94(37), 12553.
  • 19. umanitoba.ca Chemotherapeutic Uptake A2780-S H.T.L. Luu, M.W. Nachtigal, S. Kuss. Journal of Electroanalytical Chemistry, 872C, 114253, 2020.
  • 20. umanitoba.ca Carboplatin Uptake/Retention A2780-S A2780-CP H.T.L. Luu, M.W. Nachtigal, S. Kuss. Journal of Electroanalytical Chemistry, 872C, 114253, 2020.
  • 22. umanitoba.ca e- The Scanning Electrochemical Microscope (SECM) 25 µm Pt electrode Metal Glass
  • 23. umanitoba.ca Electrochemical Imaging of Chemoresistance Kuss S, Trinh D, Mauzeroll J. 2015 Analytical Chemistry 87, 8102−8106. Ovarian Epithelial Cancer Cells A2780-cp Ovarian Epithelial Cancer Cells A2780-s
  • 24. umanitoba.ca Electrochemical Quantification Horizontal Line Scans Across Living Cells Importance of temperature Control, and media composition Kuss S., Kuss C., Trinh D., Schougaard S.B., Mauzeroll J. Electrochimica Acta, 2013,110, 42.
  • 25. umanitoba.ca SECM Live Cell Imaging Ovarian Epithelial Cancer Cells A2780-cp Ovarian Epithelial Cancer Cells A2780-s Slope connected to the kinetics derived from numerical model
  • 26. umanitoba.ca Detecting of Molecule Transport as Disease Indicator
  • 27. umanitoba.ca Impact of Cytochrome C Oxidase Deficiency • Severe muscle weakness • Heart, liver and kidney problems • Brain damage Brain damage due to COX Deficiency Healthy brain National Organization for Rare Disorders (NORD). Cytochrome C Oxidase Deficiency. NORD Report 2021. Gaillard, F. Normal Brain (MRI). Radiopaedia. Due to absence or abnormality of the protein Cytochrome C Oxidase
  • 28. umanitoba.ca Model Systems Fibroblast cells from connective tissue in muscle Control Cells SCO1 Patient COX Deficient Cells Leary, S. C. (2007). Cell Metabolism, 5(1), 9-20. What is the purpose here? 8 Electrochemical monitoring of TMPD interaction with living fibroblasts The two fibroblast cell lines used in this work included a control cell line (Control 65) of healthy fibroblasts, capable of expressing a fully assembled and functional COX enzyme. SCO1 cells carry the SCO1 gene mutation that impairs the proper biosynthesis of COX (3, 11). Both cell types present a similar morphology, characterized by an elongated shape with a fibrous aspect (Figures 1B and C). To validate the cell lines of choice, expression levels of fully assembled COX and SCO1 proteins in the Control and SCO1 cell lines were quantified by Western blot analysis. The SDS-PAGE result displayed in Figure 2A confirms the reduced level of SCO1 protein (29 kDa) in SCO1 cells. VDAC1 (35 kDa) was used as a loading control and was detected equally in both cell lines. Accordingly, the BN-PAGE results (Figure 2B) show that cells carrying the SCO1 mutation present reduced levels of fully assembled COX due to decreased levels of SCO1 protein (9). Complex I was used as a loading control and was detected equally in both fibroblast lines. The decreased levels of SCO1 and COX proteins in SCO1 cells, therefore, validate SCO1 as a COXD cell line. Differential electrochemical signals are expected for COX activity in Control cells compared to SCO1-deficient fibroblast cells. Fig. 2. Western blot results of mitoplasts isolated from Control and SCO1 fibroblast cells. (A) Fibroblasts fractionated by SDS-PAGE and blot were subject to antibodies against SCO1. VDAC1 was used as a loading control. Molecular weight (MW) protein ladder is indicated on the left. (B) Fibroblasts fractionated by 1D BN-PAGE and blot were subject to antibodies against complex IV. Complex I was used as a loading control. Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome c oxidase deficiency detection in human fibroblasts using scanning electrochemical microscopy. 2023. Under Revision. → SCO1 is a metallo- chaperone protein required for proper assembly of COX
  • 29. umanitoba.ca Cytochrome C Oxidase Indicator TMPD+• + e- - e- TMPD L. Michaelis, et al. J. Am. Chem. Soc., vol. 61, no. 8, pp. 1981–1992, Aug. 1939. D. Menshykau, et a. J. Phys. Chem. C, vol. 112, no. 37, pp. 14428-14438, Aug. 2008. J. Gordon, et al. J. Pathol. Bacteriol., vol. 31, no. 2, pp. 185–190, 1928.
  • 30. umanitoba.ca SECM Imaging of Living Fibroblast Cells 200 µm Fibroblast cell Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome c oxidase deficiency detection in human fibroblasts using scanning electrochemical microscopy. 2023. Under Revision. 1.10 1.05 1.00 1.05 1.10 1.15 1.20 I T /I T inf
  • 31. umanitoba.ca Electrochemical Detection of COX Deficiency Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome c oxidase deficiency detection in human fibroblasts using scanning electrochemical microscopy. 2023. Under Revision.
  • 32. umanitoba.ca SECM 3D Imaging Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome c oxidase deficiency detection in human fibroblasts using scanning electrochemical microscopy. 2023. Under Revision. I T /I T inf I T /I T inf I T /I T inf I T /I T inf
  • 33. umanitoba.ca Lines Scans Across Living Cells 200 µm Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome c oxidase deficiency detection in human fibroblasts using scanning electrochemical microscopy. 2023. Under Revision. I T /I T inf I T /I T inf
  • 34. umanitoba.ca Cell Line Scan Analysis Kuss S, Trinh D, Mauzeroll J. 2015 Analytical Chemistry 87, 8102−8106. 12 um Estimated 7-8 um Kuss S., Kuss C., Trinh D., Schougaard S.B., Mauzeroll J. Electrochimica Acta, 2013,110, 42. a) c) • Topography contribution varies with increasing scan rate • Reactivity contribution remains constant Decoupling of Topography and Reactivity by numerical modeling
  • 35. umanitoba.ca Kuss S, Trinh D, Danis L, Mauzeroll J. 2015 Analytical Chemistry 87, 8096−8101. High-speed SECM SECM at regular speed Prof. Dr. Dao Trinh University La Rochelle, France log 𝐼!"#$ = 𝑰𝟎 + 𝑨 𝑒(𝒓𝟎 ()*(+")) 𝑃! = 𝑣 𝑎" 𝐷 𝑑 log 𝐼!"#$ = 𝜶 log(𝑃-) log (peak current) log (velocity) Normalized velocity: Decoupling Topography and Reactivity
  • 36. umanitoba.ca Numerical Modeling 𝑳𝒐𝒈 (𝑰𝒏𝒐𝒓𝒎) = 𝑰𝟎 + 𝑨𝒆𝒙𝒑 (𝒓𝟎𝑳𝒐𝒈 𝑷𝒔 ) I0, A, and r0 are the fitting parameters which depend on the apparent heterogenous rate constant, k0 Slow scan rate Fast scan rate Kuss, S., Trinh, D., Mauzeroll, J. (2015). Analytical Chemistry, 87(16), 8096-8101. Kuss, S., Kuss, C., Trinh, D., Schougaard, S. B., Mauzeroll, J. (2013). Electrochimica Acta, 110, 42-48.
  • 37. umanitoba.ca Apparent Heterogenous Rate Constants for Control and SCO1 Cells • Control 65: 𝑘' = 9 ∗ 10() m/s • SCO1 Patient: 𝑘' = 7.5 ∗ 10() m/s Cytochrome C Reductase Activity • Control 65: 𝑘' = 3.2 ∗ 10() m/s • SCO1 Patient: 𝑘' = 1.5 ∗ 10() m/s Cytochrome C Oxidase Activity Thind S., Lima D., Booy E., Trinh D., McKenna S., Kuss S.: Cytochrome c oxidase deficiency detection in human fibroblasts using scanning electrochemical microscopy. 2023. Under Revision.
  • 38. umanitoba.ca Towards the Detecting of Reactive Molecules Transported across Membranes
  • 39. umanitoba.ca Optical Fibers in Electrochemistry 39 N. Thomas, V. Singh, S. Kuss, https://doi.org/10.1016/j.trac.2021.116196 D.A. Van Dyke, H. Yuan Cheng, https://doi.org/10.1021/ac00164a004 Y. Takahashi, H. Shiku, T. Murata, T. Yasukawa, T. Matsue,https://doi.org/10.1021/ac901796r
  • 40. umanitoba.ca MORE Fabrication 40 Thomas N., Singh V., Ahmed N., Trinh D., Kuss S.: Single Cell Scanning Photoelectrochemical Microscopy using Micro-Optical-Ring Electrodes. Biosensors and Bioelectronics. 2022, 217, 114658.
  • 41. umanitoba.ca SPECM Setup 41 Glass Epoxy Resin Optical Fiber Au Thomas N., Singh V., Ahmed N., Trinh D., Kuss S.: Single Cell Scanning Photoelectrochemical Microscopy using Micro-Optical-Ring Electrodes. Biosensors and Bioelectronics. 2022, 217, 114658.
  • 44. umanitoba.ca TMPD Spectrum through Optic Fibers and MOREs Optical Fiber MORE Thomas N., Singh V., Ahmed N., Trinh D., Kuss S.: Single Cell Scanning Photoelectrochemical Microscopy using Micro-Optical-Ring Electrodes. Biosensors and Bioelectronics. 2022, 217, 114658.
  • 45. umanitoba.ca Oxygen Detection by Electrochemistry Potentiostat O 2 O 2 O 2 Thomas N., Singh V., Ahmed N., Trinh D., Kuss S.: Single Cell Scanning Photoelectrochemical Microscopy using Micro-Optical-Ring Electrodes. Biosensors and Bioelectronics. 2022, 217, 114658.
  • 46. umanitoba.ca SPECM Stimulation and Analysis 46 Thomas N., Singh V., Ahmed N., Trinh D., Kuss S.: Single Cell Scanning Photoelectrochemical Microscopy using Micro-Optical-Ring Electrodes. Biosensors and Bioelectronics. 2022, 217, 114658.
  • 47. umanitoba.ca H2O2 O2 + 2H+ + 2e- H2O2 H2O2 H2O2 H2O2 Live cell Petri dish Potential Future Biological Applications of SPECM • Detection of ROS/RNS • Identify ROS/RNS involved in cancer progression • Monitoring of cancer initiation
  • 48. umanitoba.ca Acknowledgements ● Shubhneet Thind ● Dr Dhésmon Lima ● Dr Evan Booy, UM ● Dr Sean McKenna, UM ● Dr Frank Wang www.bioanalyticschemistry.com @LBES_UofM