In this webinar, Dr. Sabine Kuss will discuss the importance of transmembrane molecule exchange and how to detect and quantify membrane transport of molecules in cells.
Complex biological processes, such as the transport of molecules across cell membranes, are difficult to understand using purely biological methodologies. Investigating cellular transport processes is challenging, because of the highly complex chemical composition of cells and the diffusion of molecules in and around cells at low concentrations. The development and advancement of electroanalytical methods over the last two decades has enabled the monitoring of living cells and their interaction with the environment, including external stimuli, such as pharma-molecules.
This presentation emphasizes electrochemical and electrophysiological methods of detection and quantification but also makes a comparison to other bioanalytical approaches. Join us to discover a substantial diversity in methods used to monitor the transport of cell metabolites, crucial for cell survival, and pharmaceutical compounds, involved in cell characteristics such as drug resistance.
Key Topics Include:
- Understanding transmembrane molecule transport through bioanalytical methods
- Electrochemical approaches to monitor molecule transport across cell membranes
- What bioanalytical and especially electrochemical approaches can reveal
- Challenges associated with instrument limitations
Pests of jatropha_Bionomics_identification_Dr.UPR.pdf
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.
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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
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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.
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.
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
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.
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.
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