Shawn Forrest
Analysis of Optical and Electrochemical Options for a
Microfabricated Potassium Sensor
Introduction
This paper summarizes a review of the various available options for fabricating a
potassium-selective sensor for use in measuring the extracellular potassium
concentrations around a single cell. The aim is to integrate this sensor into the
nanophysiometer, a device being developed to measure the coupling of metabolic and
electric activity in a single cardiac myocyte in a restricted extracellular space. A wide
variety of mechanisms are used for ion sensing with varying degrees of applicability to
microfabricated sensors. Several other relevant sensing parameters must also be
considered for appropriateness of the mechanisms to the proposed application.
Problem Statement
The integration of the potassium sensor into the nanophysiometer introduces
several requirements of varying importance. The most important requirements seem to
be:
1) Small size – the sensor will be interfacing with the extracellular fluid in a
chamber that has dimensions of 25 x 100 x 30 μm3 and therefore a volume of
75000 μm3 or 75 pL. The sensor must fit around this volume and leave room for
other sensors for oxygen, pH, and voltage-sensing electrodes. This will likely
constrain the surface area of the sensor to about 25 μm x 25 μm at the largest.
2) Good Selectivity – the aqueous solution surrounding the cell will be made up of
Tyrode’s solution. Therefore the sensor will have to be reasonably selective for
potassium over the other ions present.
3) Minimal sample perturbation – because the measurements are being taken outside
a live cell and the subsequent effects of the measured concentrations of potassium
on the cell are being studied, it is important that the sensor affect the potassium
concentration very minimally. This is an especially stringent requirement due to
the extremely small sample volume involved.
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4) Fast temporal response – this would be desirable for resolving intra-beat
fluctuations.
5) Sensitivity – this is less of an issue because the extracellular concentration of
potassium in-vivo is generally in the 10 mM range. Most ion sensors are sensitive
down to the μM range.
6) Biocompatibility – due to the simplicity of the Tyrode’s solution that will be used
as the aqueous solution, biocompatibility issues that are often confronted in the
use of ISEs should not be an issue.
General Options
The two main categories of options for ion sensing are optical and
electrochemical sensors. Both categories of sensors have been well developed over the
past few decades and have several specific mechanisms of transducing analyte
concentrations for electronic recording and analysis. I will discuss the following options.
1) Optical Sensing
a) Optical Fibers
i) Optodes
(1) Near field evanescent wave sensors
(a) Covalently linked dyes
(b) Dye-doped polymerization at tip
ii) Bulk optode spectroscopic measurements
b) Fluorescent Dyes
i) Free dyes in solution
ii) Encapsulated dyes (PEBBLEs)
2) Electrochemical Sensing
a) Potentiometric
i) Neutral-Carrier-based ISEs
(1) Liquid junction
(2) Solid state
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(a) Self-assembling-monolayer
To begin with, I list some of the pertinent details to optical measurements:
Advantages and disadvantages of Fiber-based Optical sensors: (adapted from [11])
1) They are not subject to electrical interferences.
2) No reference electrode is needed, although a reference source is useful.
3) An immobilized reagent does not have to be in contact with optical fibers.
4) Fiber-optic sensors can be miniaturized.
5) They are highly stable with respect to calibration, especially if ratiometric
intensity measurements are used.
6) They have potential for higher information content than electrical transducers.
Drawbacks:
1) They will only work if appropriate reagents can be developed.
2) They are subject to ambient light interference. This can be overcome by using a
dark environment or modulated radiation.
3) They have a limited dynamic range compared to electrochemical sensors.
4) Immobilized chemistries are subjected to problems with inadequate path lengths,
path length instability due to matrix swelling, reagent photolability, and reagent
leaching.
5) A need still exists for more selective indicators and more reproducible
immobilization procedures to enable high sensitivity and long-term stability.
6) Limited stability of the biological component immobilized onto a transducer
surface.
Near Field Optical Sensors
The original application of optical sensors to extremely small-scale (submicron)
ion sensing was reported by Kopelman and coworkers primarily for intracellular
recording of pH.[17] The details of probe construction were published separately.[15]
This method uses the covalent immobilization of reagents by photopolymerization on the
end of a fiber. The fiber is drawn to a very small tip diameter (as small as 100 nm
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diameter). Because this method uses near field optics, the diffraction limit does not apply
to limit the degree of miniaturization. In this case miniaturization is limited by the size of
the molecular probe. Because the diameter of the fiber tip is smaller than the wavelength
of excitation light used, photons cannot pass into the tip. Yet
the propagation continues as evanescent waves or excitons
traveling to the end of the tip. Only fluorescent species very
close to the end of the tip are excited, which avoids interference
signals by other fluorescent molecules in the surrounding
solution. Excitation is by argon laser and a measurement is by a
fluorescent microscope and a photomultiplier tube. Two
methods exist for the fabrication of the fibers. The first is to
pull the heated fiber in a manner similar to micropipet drawing
technique. This was the original method used. The second
involves chemical etching of the glass fibers.
This covalent immobilization of the fluorescent sensor
to the fiber surface has great advantages for the time constant of
the device. Because the molecular probe that does the actual
Figure 1 (a and b)
Examples of fiber
tips fabricated. (c)
Illustration of laserinduced
polymerization at the
fiber tip.
sensing is essentially in solution, there is no significant mass
transport event involved in binding of the analyte. The response
time using this mechanism can reach as short as 500 msec as
cited in [17]. In theory this mechanism could be used for
potassium ion determinations, but I searched the literature in vain for an actual report of
such a sensor. Such sensors have only been used for pH measurements because the
reporter involved has variable fluorescence based on protonation. The author originally
stated that “there is little difficulty in expanding these techniques into the fabrication of
other sensors.” [15]
Upon further searching I found that the reason for the lack of development of this
technique for more generalized analyte sensing is that it requires derivatizing the
fluorescent dye into a polymerizable version. [16] In the case of the pH sensitive dye
fluoresceinamine, its derivative N-acryloylfluoresceinamine has an essential double bond
that can afford polymerization. However, in many cases chemically sensitive dyes
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cannot be derivatized in the same way and therefore cannot be immobilized to the fiber
tip. One alternative to this covalent attachment of the dye is to enmesh the dye in a solgel matrix at the tip. This requires that the dye be reasonably hydrophilic and still
requires that the dye be sufficiently selective. PBFI, the standard potassium probe from
Molecular Probes, is only 1.5 times more selective for potassium than for sodium. This is
much less selective than such ionophores for other ions, particularly H+ and Ca2+.
Recently, a newly synthesized squarine dye was used in an optical potassium sensor. [3]
This dye was found to have a similar selectivity coefficient of 1.4 and binding was only
reported in the concentration range, which is much too low for the expected extracellular
Na+ levels. These facts make such covalently immobilized optical sensors unfeasible for
extracellular K+ measurements.
Bulk Optodes
As far as I have been able to find, the optical sensing of potassium and sodium
have only been accomplished by coupling of the reporter to an ion exchange event with
potassium according to the reaction
pL  HC   R  K  
C  KLp  R  H 
where L is the potassium selective ionophore, C is the chromoionophore selective for
hydrogen ions, R is the lipophilic salt incorporated into the membrane to allow
electroneutrality, and p is a stoichiometric constant. Sensors using this technique are
termed bulk optodes. It is based on the same ion binding mechanism as traditional ISEs
using a polymer matrix based ionophore to provide good selectivity, but an ion exchange
mechanism is used where protons are sensed by a chromoionophoric pH indicator.
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Figure 2 Mechanism of K+-selective optode sensing. An ion-exchange event activates a
chromoionophore to indicate the concentration of K+. M is the target ion, L is the ionophore, C is
the chromoionophore, and R is the negative-site provider. Picture taken from [7].
The method was originally demonstrated by Seitz [20] and Wolfbeis [12], and the
quantitation of the measurements is well-described by Simon [13]. The ion activities
ratio in the aqueous sample is found based on the response function
 a   eff
aK    H
 1   eff

K e  Ro  1   eff  Co 


p
  Lo  p Ro  1   eff  Co 




where Lo, Co and Ro are the analytical concentrations of ionophore, chromoionophore and
lipophilic anion, respectively and Ke is the equilibrium exchange coefficient. The
variable alpha is the normalized absorbance, which is the ratio of unprotonated form of
the chromoionophore to the total chromoionophore concentration. Several potassium
sensors based on this mechanism have been reported. The first optical K+ sensor was
introduced by Wolfbeis [19] and used valinomycin as the ionophore and rhodamine B
octadecyl ester as the reporter. Toth et. al. [18] later incorporated a plasticized polyvinyl
chloride (PVC) membrane and the ionophore BME 44, an azacrown. Improvements have
been made to the stability of these sensors by the use of ratiometric measurements, which
compare the signals at two fluorescent wavelengths. [9] A Nd:YAG laser can be used for
fluorescence measurements using Chromoionophore VI as the measuring dye and
sometimes a reference dye such as Nile red is used for ratiometric measurements. The
response time for these sensors is generally on the order of 30 sec or more.
Two disadvantages of this coupling to a pH indicator are that the sensing becomes
pH sensitive and the ion exchange process distributes protons into the sample volume,
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possibly influencing the pH of the sample significantly. This will be a disadvantage for
use in the nanophysiometer, where significant pH fluctuations are expected and
influencing the extracellular pH by the sensor will be undesirable.
A slight difficulty with the use of the fiber-optic would be the delivery of the
fiber-optic tip to the chamber. Although the tip of the fiber can have a diameter on the
order of 100 nm, the volume of the fiber increases as the cube of the distance from the tip
and most fiber optic diameters are on the order of at least 100 μm. Integration of the fiber
into the chamber would probably involve insertion of the fiber into a pre-fabricated
channel. This would be a very exacting process but I know similar fiber optics have been
inserted into channels of 40 μm.
Dyes and PEBBLEs
Another option that should at least be mentioned is the use of free dyes.
However, as explained earlier, the dyes available for K+ are probably not sufficiently
selective for extracellular measurements we are performing. PEBBLEs (Probes
Encapsulated by Biologically Localized Embedding) are based on the same ion-exchange
phenomenon as bulk optodes, but in this case they are beads in solution and their
fluorescence is excited by diffuse light of the particular wavelength. The fact that these
sensing methods are not localized is likely to interfere with the other sensors around the
chamber. Delivery of the dyes or beads would be very difficult to quantify and keep
uniform extracellularly.
Traditional Electrochemical Ion Sensors
Electrochemical ion sensors for potassium are almost exclusively in the form of
potentiometric liquid-phase electrodes. The configuration of the measurement made by
such an electrode is shown in Fig.3. Essentially the potential across the ion-selective
membrane is measured, which is proportional to the target ion concentration in the
sample solution due to the selective transport of that ion across the membrane by an
ionophore. The membrane consists of a plasticized polymer matrix with an ionophore,
and a lipophilic salt negative site provider. No ion-exchange process is necessary when a
neutral ionophore carrier (like valinomycin) is used. So the sample is generally not
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affected in the manner it is by bulk optodes, although there is often some slight leaching
of the membrane components over long periods of time.
Figure 3 Example of a liquid phase ion-selective electrode measurement. Picture taken from [10].
In turning to the more traditional electrochemical sensors, the main challenge
becomes obtaining the extremely small size necessary to fit inside the cell chamber. The
geometry involved requires a planar electrode configuration, which has been a research
focus in recent years. [14] Several examples of miniature ion sensors have been
published. Cosofret’s sensor [1] was Kapton-based
and for heart measurements (diam=1mm),
Dumschat’s [2] sensor was photolithographically
patternable acrylate (linewidth = 300μm), Uhlig’s
sensor used an underside cavity (surface area = 0.03
mm2, response time = 10-15 sec), Zielinska’s sensor
Figure 4 Schematic of
microfabricated potassium ISE by
Knoll et. al. Illustration taken
from [8].
has an area of 2.25 mm2, and Knoll’s sensor [8] had
a side length of 70 μm. The latter sensor was the
smallest and the only one close to our target size
range. It is of the cavity variety and is shown in Fig. 4. That of Uhlig was similar and
produced acceptably short response times. The ion selective membrane is placed directly
over the Ag thin film wire and interfaces with the sample solution above through the
small hole.
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One important challenge that must be faced for miniaturization of any ISE is that
of the required reference electrode. According to Suzuki, this is generally considered one
of the most difficult issues in this field. [14] There are three proposed approaches for
this. The first is a simple Ag/AgCl electrode in direct contact with the sample solution.
The second is a solid-state electrode without a liquid junction. The third is a miniaturized
version of a conventional liquid junction electrode. These options increase in stability
with complexity, but it is not clear to me yet what degree of stability will be necessary for
this application. Results vary widely between publications. I will need further study on
this issue, but one consequence is fairly certain. The space required for the K+ sensor
will be double that required for the K+ ISE because a reference electrode is necessary.
Solid-State Electrochemical Sensors
The size range makes the use of solid-state, or coated wire, ISEs desirable. These
electrodes do not require an electrolyte filling solution. While such electrodes have been
plagued in the past by extreme potential drift problems, more recent examples show very
promising improvements. While it was originally thought that these potential drifts were
due to an ill-defined redox couple, it was found in [6] that they are more likely due to
water interactions between the metal and membrane. Improvements in the stability of
solid-state electrodes has been accomplished by the use of a redox-active selfassembling-monolayer (SAM) on the metal surface. This layer successfully excludes
water infiltration and produces much more stable potentials.
Self-Assembling Monolayer ISEs
One approach to creating a solid-state ISE is to use a self-assembling monolayer
(SAM) of a redox-active compound. Successful use of this technique in producing a K+selective ISE has been reported.[4,5] There is some synthetic chemistry involved in
obtaining the chemical species that was used, but the online supplementary material
referred to in [4] concerning chemical synthesis was not available so I don’t know how
involved this synthesis was. The brief description in the text indicated that it would not
be prohibitively complex.
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The SAM has a strong affinity for the gold electrode and is hydrophobic, allowing
it to exclude water from the metal interface. It has also been shown to be redox-active,
which allows the potential change of the inner membrane surface to be communicated to
the gold electrode. The membrane composition of these ISEs is essentially the same as
previously used, so the sensing parameters will be similar to other commonly used ISEs.
The polymer matrix, however, is polyurethane rather than PVC because of the
relatively poor adhesion characteristics of PVC. This will present a trade-off because
PVC has better structural strength under high plasticizer concentration, which is generally
necessary for producing a liquid phase-like environment for the ionophore. Other macroscale ISEs have been produced with this polymer with good results though so the
polyurethane membrane should not be a concern.
Other techniques for interfacial design are the use of silane coupling, sol-gel
matrix, cellulose acetate, Nafion, poly(L-lysine), electropolymerized film, and plasmapolymerized films. I have not been able to fully research all of these methods to
determine their usefulness for nanofabrication of an electrode.
Proposed Electrode Fabrication
A reasonable proposal for an electrochemical sensor would be to use a method
similar to that of Uhlig and Knoll mentioned above by creating a silicon cavity of smaller
size to place beneath the position of the myocyte. This would provide structural stability
for the ion-selective membrane. If the electrodes were made of gold rather than silver, a
SAM could be used to stabilize the potential drift in the ISE.
The reference electrode can be fabricated in the same manner, but with a
membrane without ionophore, the silver wire chloridized electrochemically, and a
polyHEMA solution added around the Ag/AgCl wire to stabilize the reference electrode
in the face of fluctuating Cl- ion concentrations. I’m not completely certain that the
chloridization and polyHEMA addition steps will be feasible at such a small scale, but
they could likely be accomplished using microcontact printing.
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Conclusions
Near-field optical sensing of potassium is not likely to be selective enough for our
application because significant Na+ and Ca2+ concentrations are to be expected in the
extracellular media. Bulk optode sensing may interfere with ion measurements due to the
H+ ion exchange mechanism that must be used. This will likely cause some pHinfluenced shift in the potassium measurements and affect the local H+ somewhat.
However, there is a degree of flexibility in the size of the optode matrix tip. Smaller tip
sizes will have less affect on the solution and produce smaller signal, so it may be
possible to strike a balance.
It is difficult to determine what the characteristics of the SAM-based electrodes
will be because there are no current examples of electrodes in this size range, but recent
experiments are encouraging on their resulting stability are encouraging. The major
difficulty with the electrochemical method is the need for a stable reference electrode,
which has been a major obstacle for some time. I have proposed a possible solution.
The sensing parameters such as selectivity and time response have less bearing in
this case than the factors stated above. Both types of sensors provide similar responses.
The optodes have a somewhat longer response time – 30sec vs. 15 min at the fastest, but
most of the electrochemical measurements are more on the order of 1-2 min.
At this point my conclusion is that the optode method would be quicker to
develop because it has been used in this size range before and the method can be used
essentially unchanged. But the dependence of the optode response on the pH of the
environment is a fundamental drawback if significant pH shifts are expected in the
extracellular space. I assume that this will be the case with the cell in such a small
enclosed volume. The fluorescent lifetimes of often under 1 hr due to photobleaching
may be quite inconvenient as well.
Because of these considerations that the optode method is fundamentally flawed
for the application at hand because we cannot use the a pH buffered solution (as is
usually done), it seems that the best pursuit will be the pursuit of an electrode-based
solution. The electrode method considered above is my current best guess as to a useful
solution. There are several steps in this process that I am uncertain on though.
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