Transmissive pH
Sensing in Microtiter Wells
Introduction
Our newest transducer materials are designed to
encapsulate pH dyes for optical pH sensing.
These materials can be applied to various
sampling devices and combined with fiber optic
spectrometers and accessories for convenient,
non-intrusive monitoring. The pH sensor has been
developed for monitoring samples within the pH
range of 5-9 (biological range). The proprietary
sol-gel matrix doped with bromocresol green dye
(BCG) serves as an excellent material for
optical pH measurements.
Thru the use of a mathematical
algorithm the sensor can detect pH changes down
to 0.01 pH units. Smart pH Cuvettes are
semi-disposable and require very little
maintenance. The Sol-Gel BCG has been coated
inside microtiter plates for the detection and
continuous monitoring of pH changes over the
biological range as a proof of concept to be
used for the monitoring of cellular pH
environments.
Case Study
Transparent plastic
microtiter wells were affixed with a thin
adhesive PMMA material coated earlier with Smart
pH sol-gel formulation at the bottom of the
wells. This allows for transmissive pH sensing
of the analyte solutions placed into the wells.
These wells come together as a plate of many,
and are moved via a robotic positioning system
to align them with the light source and
recovery. Our customer's current configuration
uses photodiode detectors and light filters to
observe absorbance at the necessary wavelengths.
As a quick feasibility study, a
brief titration was performed using two buffers
only: pH 1 (absorbance reference) and pH 11
(observed). The pH 11 buffer produced a strong,
though distorted, absorbance curve very similar
to what is seen for the Smart pH Cuvettes, which
showed that these wells had the potential to
work as hoped:
This was performed using a
standard cuvette holder turned vertically, with
collimating lenses on each side. Light was fed
to the top of the cuvette holder from an LS-1
light source with a blue filter via a P200
UV/VIS fiber. The signal was then sent through a
P300 UV/SR fiber to a USB2000 with grating #2,
200um slit, and no lens installed. The
experimental setup is shown below:
However, when our customer
carried out their own set of titrations, the
data did not adhere to the pH algorithm
whatsoever; everything appeared very randomized:
As a result, a full titration was
performed here at Ocean Optics to determine the
cause of this significant error. The same
fibers, light source, spectrometer, and general
setup were used as previously described. Two
issues were determined to be the most probable
causes of the poor data seen by our customer;
these included the lack of collimating lenses
being used at the facility, as well as an
incorrect baseline correction wavelength. They
were previously instructed to use 750nm as the
baseline correction found in the algorithm,
though they used 880nm due to availability of
that filter. The tests performed here showed
that neither 750nm or 880nm were acceptable
baseline wavelengths, rather the ideal
wavelength to use was 509nm. Results from the
full titration are shown below:
This has shown that the pH sensor
affixed to the bottom of microtiter wells can
accurately monitor and report the pH of
transmissive analyte solutions.
Algorithm used:
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