Graduate Exam Abstract
yusra obeidat
Ph.D. Final
December 7, 2018, 10:00 am - 12:00 pm
Scott 235
A MULTI-SENSOR PLATFORM FOR MEASURING SINGLE_CELL METABOLISM TO IMPROVE SUCCESS RATE OF ASSISTED REPRODUCTIVE TECHNOLOGY (ART)
Abstract: Cell metabolism involves a set of
cellular chemical reactions that are
very important to cell development as
well as its response to environmental
changes around the cell.
Understanding cell metabolism and the
associated metabolic pathways has
been the focus of many research
efforts and it is gaining more attention
recently. In assisted reproductive
technology (ART), understanding
metabolism of oocytes and embryos
provides the possibility of selecting
more viable embryos for transfer and
reducing the number of embryos
transferred in a given in vitro
fertilization (IVF) cycle. Although
stage-specific morphologic markers
and grading systems have been
developed and widely in use, this
approach is unable to reliably assess
the physiological status of the embryo
and it is not only subjective but has a
poor correlation with subsequent
developmental competence.
Therefore, there is an ever-increasing
need for noninvasive quantitative
markers of embryo viability. Analysis of
metabolism has proved to be a
valuable marker of embryo viability
based on animal models. Through
noninvasive analysis of metabolic
markers, it will be feasible to identify
those embryos with the highest
probability of establishing a healthy
pregnancy.
Crucial to cell metabolic process is
a set of analytes that can be used as
indicators of cell metabolism. They
include oxygen, glucose, and lactate.
Some techniques for measuring
dissolved oxygen (DO), glucose and
lactate rely on fluorescent labels.
These techniques are incredibly labor
intensive, and the pipet construction
used is complex comparing with solid
state and electrochemical methods.
Injecting a cell with fluorescent label
can also lead to experimental error,
since biochemical mechanisms inside
of the cell may interact with the label.
Other techniques include the use of
scanning electrochemical microscopy
(SECM) for studying the metabolism of
single cells, but it has its drawbacks
including probe fouling, complex
instrumentation, as well as calibration
can also be challenging. Furthermore,
electrochemical microphysiometers
were used for monitoring changes in
glucose and lactate concentrations in
cell cultures, but these techniques
need larger sample volumes and might
need difficult calibration.
Due to the lack of quantitative and
real-time monitoring of cell
metabolism, the success rate of in-
vitro fertilization (IVF) is still low, with
very low percentage of embryos
transferred resulting in a term
pregnancy [1-2]. Therefore, more work
needs to be done for testing embryos
metabolism in-vitro to improve the
culture conditions and reduce the
effect of environmental stresses and
chose the media that balance all
nutrients the cell needs during
development.
In this work, a multi-sensor
platform has been used for measuring
single cell metabolism to improve
success rate of assisted reproductive
technology. The work presents the
development of a multi- sensors
system to measure concentrations of
dissolved oxygen (DO), glucose, and
lactate and the pH level, at single cell
level in real-time. DO was measured
amperometrically using a three-
electrode system of working (WE),
counter (CE) and reference (RE)
electrodes. Glucose and lactate were
measured enzymatically by measuring
the current generated from the
oxidation of hydrogen peroxide
generated from the catalysis of
glucose or lactate at the WEs with their
catalysis enzymes. pH was measured
potentiometrically using two electrodes
system of Indium Tin Oxide (ITO) WE
and Au pseudo RE. A micro-chamber
containing all four sensors was
designed and manufactured to
investigate single cell immersed in a
respiration medium. The micro-
chamber design is an important part of
the platform that provides sufficient
changes of the target analytes in the
micro-environment that enables the
sensors to measure tiny changes of
the target analytes due to cell
respiration. This setup helps to
measure the analytes with a change in
concentration ranges from (0.001 to
20) fmol/s with high specificity which is
comparable with what was published
in literature. The specificity of our
sensors was clearly determined by
monitoring the switch in metabolism to
glycolysis induced by adding
oligomycin as an inhibitor for ATP-
synthase. The ability to measure the
extracellular acidification rate (ECAR)
in addition to lactate production can
help to differentiate the respiratory acid
production from glycolytic acidification.
The ability of the sensor to detect a
metabolic shift from oxidative
phosphorylation (OXPHOS) to
glycolysis was demonstrated in
embryos by an ablation of oxygen
consumption and an increase in
lactate production as well as ECAR
following addition of oligomycin. The
increase in pH change rate after
adding oligomycin and its slowdown
after FCCP further indicates the
dependence of cell on glycolysis and
the increase of lactate production. The
results of bovine or equine embryos
show that the embryos metabolism
change with development as expected
and the amounts of glucose and
oxygen uptakes and lactate production
increase at later stages of
developments, which match the
existing biological knowledge about
metabolism of increasing the need for
ATP production at later stages of
development.
The importance of our work came
from the need for real-time
measurement of multiple metabolites
in addition to the ECAR during cell
metabolism. The platform of our multi-
sensors system allows tracking the
embryo development in the same
chamber and the change of metabolic
activity during development. The
analysis of all analytes as well as the
change in pH level can give better
insight to the "switch" in metabolism
induced by inhibitors such as
oligomycin, and at the different time
points of embryo development. Our
system is capable to provide single-
cell metabolism measurement with
more complete panel than what
commercially available devices such
as Seahorse provides. Our results
provide a clearer insight into the
mechanism of OXPHOS and glycolysis
for single cells and a more complete
analysis to include inter-sensor
interference for improved accuracy.
This multi-sensor system has some
potential applications include
evaluating effects of metabolic
therapies on oocyte bioenergetics,
study the effect of aging on embryos
development and monitoring
mitochondrial function throughout
oocyte maturation and blastocyst
development to predict embryo viability
to compliment assisted reproductive
technologies and can also be used in
comparing normal cells and cancerous
cells metabolisms.
Adviser: Tom Chen
Co-Adviser: NA
Non-ECE Member: Stuart Tobet, Dept of Biomedical sciences
Member 3: Sudeep Pasricha
Addional Members: George Collins
Publications:
Accepted journal papers:
1. Obeidat, Y., Evans, A., Tedjo, W., Chicco, A., Carnevale, E., Chen, T., 2018. Monitoring Oocyte/Embryo Respiration Using Electrochemical-Based Oxygen Sensors, Sensors and amp; Actuators: B. Chemical. https://doi.org/10.1016/j.snb.2018.07.157.
2. Yusra Obeidat, Giovana Catandi, Elaine Carnevale, Adam J. Chicco, August DeMann, Stuart Field and Tom Chen. 2018b. A Multi-Sensor System for Measuring Bovine Embryo Metabolism, Biosensors and Bioelectronic, https://doi.org/10.1016/j.bios.2018.09.071
conference papers:
1.Y. Obeidat and T. Chen, “Characterization of an O2 Sensor Using Microelectrodes”. IEEE sensors. 2016, Orlando, FL, Oct. 30 – Nov. 2.
to be submitted journal papers:
1. Yusra Obeidat, Ming-hoa Cheng, Giovana Catandi, Elaine Carnevale, Adam J. Chicco, August DeMann, Stuart Field and Tom Chen. 2018b. Understanding mitochondrial bioenergetics and the role of glycolysis in equine embryos, Biosensors and Bioelectronic,
Program of Study:
ECE-799-001
ECE-569-001
ECE-538-L01
ECE-538-001
ECE-526-001
ECE-656-001
ECE-614-001
ECE-571-001 and ECE-575-L01
