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Optical pH Surveillance in a Microfluidic System for Cell Culture Evaluation of VisiSens pH sensor foils integrated in microfluidic systems

Authors:
  • PreSens Precision Sensing

Abstract and Figures

pH surveillance within microfluidic systems is a challenging problem. Liquid quantities available for measurement are limited and also relatively inaccessible within the microchannel. In addition, the absence of turbulent mixing implicates that pH gradients build up easier than in macroscopic settings which in turn makes single point measurements rather unreliable for describing the state in the whole system. By using the VisiSens fluorescent sensor foil mounted within the microfluidics, pH can be monitored via ratiometric image analysis. Gradients can easily be detected, and by carefully calibrating the system prior to experiments pH can be quantitatively determined.
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APPLICATIONS
Optical pH Surveillance in a Microfluidic
System for Cell Culture
Evaluation of VisiSens pH sensor foils integrated in microfluidic systems
A. Schopf
1,2
, C. Boehler
1,2
, M. Asplund
1,2
1
Laboratory for Biomedical Microtechnology, Department of Microsystems Engineering (IMTEK),
University of Freiburg, Germany
2
Brain Tools Cluster of Excellence, University of Freiburg, Germany
pH surveillance within microfluidic systems is a challenging problem. Liquid quantities available for
measurement are limited and also relatively inaccessible within the microchannel. In addition, the
absence of turbulent mixing implicates that pH gradients build up easier than in macroscopic
settings which in turn makes single point measurements rather unreliable for describing the state
in the whole system. By using the VisiSens fluorescent sensor foil mounted within the microfluidics,
pH can be monitored via ratiometric image analysis. Gradients can easily be detected, and by
carefully calibrating the system prior to experiments pH can be quantitatively determined.
It is a well-established fact that liquids in a microfluidic
system follow the rules of laminar flows, preventing
mixing, in contrast to the macroscopic world where two
liquids in contact rapidly mix. This can lead to the for-
mation of substantial concentration gradients. While
laminar flows in many cases are a technological advantage
of microsystems it can also be a cumbersome problem for
experiments where uniform conditions are essential, e. g.
ensuring an even pH distribution in microfluidic cell culture
environment. The integration of microsensors into such
systems is possible but single spot measurements do not
give information about the distribution over the surface.
There is the risk that hot spots in the fluidics, meaning
regions were gradients build up, would be overlooked by
such an approach. With foil based optical sensors, such as
the VisiSens pH sensitive foils (SF-HP5R, PreSens) surface
measurement of pH is possible via fluorescence ratiome-
tric imaging. By integrating the foil in a transparent micro-
fluidic channel, surface measurements can be performed
and regions of distinctly different pH will stand out from
the rest in the final processed image. In order to quantify
the difference, the ratios in the image must first be
translated to pH via a separate calibration measurement.
Set-up for pH Measurements
A test set-up was assembled using a rigid stainless steel
stand with the camera clamped to it. It was facing the
sample mounted on a 3D micromanipulator screwed to the
bottom of the steel stand (Fig. 1). The sample could thus
easily be moved in the x-y and z axis for alignment to the
camera optics. The full system was enclosed in a box to
exclude ambient light during measurements. The fluidic
channel consisted of a silicon rubber gasket clamped
between a lid of transparent poly-methyl- methacrylate
and a glass cover slip. The sensor foil was glued onto the
glass slide prior to mounting the gasket, and imaging was
Fig. 1: Schematic illustration of the measurement set-up: The VisiSens camera is
mounted on a rigid stand facing upwards. The sample is firmly fixed into a frame
(base plate) connected to a micromanipulated stage above the camera. The full
set-up is enlosed in a dark box.
performed through the glass from below. A typical image
captured with the VisiSens USB microscope can be seen in
Fig. 2A. The contour of the gasket can easily be distin-
guished on the picture. The sharp edges typically seen at
the beginning of the measurements become more blurred
over time as liquid soaks the sensor foils and diffuses
within the sensor layer (Fig. 2B). Calibration buffers were
prepared from sodium dihydrogen phosphate dihydrate
and di-sodium-hydrogen phosphate dihydrate and
adjusted to five different pH values. The example images in
Fig 2 C-D are produced by first filling the channel with the
most acidic buffer and then adding the basic buffer to the
right side of the channel.
Calibration
Ratiometric image analysis is an efficient method to get a
quick impression of gradients or hot spots appearing
within the channel. An example can be seen in Fig. 2 C-D,
where the mixing of an acidic (pH = 4.5) and a basic (pH =
Application Note
www.PreSens.de
PreSens Precision Sensing GmbH
Josef-Engert-Str. 11
93053 Regensburg, Germany
Phone +49 941 94272100
Fax +49 941 94272111
info@PreSens.de
Bring to light whats inside. Ask our experts:
Fig. 2: Fluorescence imaging in a PDMS based channel shaped gasket. Intially the
PDMS gasket is clearly visible (A). The boundaries between channel edges (red) and
the liquid filled region in the middle (green) are well defined. After several hours of
duty the foil has soaked also under the PDMS gasket (B). The response time of the
foil is fast enough to follow the dynamics of mixing. Samples were taken at 2 s
intervals after filling the channel with a base and subsequently loading acid into one
of the side ports (C, D).
8.5) liquid in the central region of the channel was visual-
ized. Nevertheless, it is often necessary to translate the
signal to specific pH values. Calibration is performed by
filling the channel with buffers of known pH and image
recording after the foil has equilibrated with the new
solution. A non-linear calibration curve is established by
which the calculated ratios can be interpreted to pH
values. Four possible interferences need special consi-
deration in order to obtain an accurate calibration mea-
surement: foil equilibration time, buffer contamination,
colorimetric interference and progressive weakening of the
signal over time due to photobleaching. If the equilibration
is incomplete, there could be interference with the signal
of the previous buffer filling. Our tests showed that one
should wait for at least 8 min between filling the liquid into
the channel and capturing the image for calibration, but
each user should invest a few initial measurements to
figure out optimal parameters in their case. Also rinsing
between buffer fillings preferably with the next buffer to be
measured avoids substantial contamination from one
buffer to the next. Fig. 3 shows our results when we
conducted calibration with and without rinsing. There is a
clear lag in the signal from one measurement to the next
when the channel is not rinsed, while in the rinsed version
the signal does not depend on the sequence in which the
buffers are measured. Therefore, we recommend rinsing
in-between calibration points to improve the accuracy of
the calibration. Furthermore, colorimetric pH indicators,
such as phenol red, can interfere with the pH sensor foil´s
signal. Such substances are routinely added to full cell
culture media as a quick surveillance method for the
health status of the culture. We observed a major shift in
the sensor signal when we performed calibration in buffers
with or without phenol red (Fig. 4). This clearly shows the
importance of working in phenol red free medium when
Fig. 3: Foil response to a sequence of buffers when stepped from low to high (black
squares) and then from high to low (red triangles). Without intermediate rinsing
between buffer changes there is a clear assymetry (A) explained by buffer
contamination with preceeding buffer remains. When intermediate rinsing is used
the signal is largely symmetric (B).
Fig. 4: Calibration performed with (black line) and without (blue dotted line) phenol
red added to the medium. It is clear that the additional pH sensitive pigments have a
substantial influence on the measured signal.
performing measurements with the pH sensor foils. Also
photobleaching of the fluorescent sensor dye is a factor
that should not be neglected when using the sensor over
longer time periods. Prolonged illumination can cause a
signal drift over time and therefore re-use of the foils is not
recommended for high precision measurements. A second
calibration after long-term measurements should be
performed. This way it is possible to estimate how singal
drift might have influenced the measurements.
Conclusion
For rapid qualitative analysis of pH gradients the VisiSens
pH sensor foil is an efficient method as can be seen in Fig.
2 C-D. However, for quantitative pH measurement within
microscale fluidic systems the above mentioned points
must be taken into consideration. It is clear that accurate
pH determination via the foil requires a carefully planned
calibration sequence both prior and after experiments. On
the other hand, an excessively long calibration procedure
will contribute to signal weakening over time due to
photobleaching, so the right balance has to be found.
pH Sensor Foils in Microfluidic Systems
Application Note
... Following each measurement series an additional calibration round was performed to account for any bleaching effects of the foil fluorescence. This procedure is described in detail elsewhere [30]. ...
Article
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Direct current (DC) stimulation can be used to influence the orientation and migratory behavior of cells and results in cellular electrotaxis. Experimental work on such phenomena commonly relies on electrochemical dissolution of silver:silver-chloride (Ag:AgCl) electrodes to provide the stimulation via salt bridges. The strong ionic flow can be expected to influence the cell culture environment. In order to shed more light on which effects that must be considered, and possibly counter balanced, we here characterize a typical DC stimulation system. Silver migration speed was determined by stripping voltammetry. pH variability with stimulation was measured by ratiometric image analysis and conductivity alterations were quantified via two electrode impedance spectroscopy. It could be concluded that pH shifts towards more acidic values, in a linear manner with applied charge, after the buffering capability of the culture medium is exceeded. In contrast, the influence on conductivity was of negligible magnitude. Silver ions could enter the culture chamber at low concentrations long before a clear effect on the viability of the cultured cells could be observed. A design rule of 1 cm salt bridge per C of stimulation charge transferred was however sufficient to ensure separation between cells and silver at all times.
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