In Situ Electrochemical Monitoring of Caged Compound Photochemistry: An Internal Actinometer for Substrate Release

Article abstract of DOI:10.1021/acs.analchem.0c03452

Caged compounds are molecules that release a protective substrate to free a biologically active substrate upon treatment with light of sufficient energy and duration. A notable limitation of this approach is difficulty in determining the degree of photoactivation in tissues or opaque solutions because light reaching the desired location is obstructed. Here, we have addressed this issue by developing an in situ electrochemical method in which the amount of caged molecule photorelease is determined by fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes. Using p-hydroxyphenyl glutamate (pHP-Glu) as our model system, we generated a linear calibration curve for oxidation of 4-hydroxyphenylacetic acid (4HPAA), the group from which the glutamate molecule leaves, up to a concentration of 1000 μM. Moreover, we are able to correct for the presence of residual pHP-Glu in solution as well as the light artifact that is produced. A corrected calibration curve was constructed by photoactivation of pHP-Glu in a 3 μL photoreaction vessel and subsequent analysis by high-performance liquid chromatography. This approach has yielded a linear relationship between 4HPAA concentration and oxidation current, allowing the determination of released glutamate independent of the amount of light reaching the chromophore. Moreover, we have successfully validated the newly developed method by in situ measurement in a whole, intact zebrafish brain. This work demonstrates for the first time the in situ electrochemical monitoring of caged compound photochemistry in brain tissue with FSCV, thus facilitating analyses of neuronal function.

Full text of DOI:10.1021/acs.analchem.0c03452

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Article
In Situ Electrochemical Monitoring of Caged Compound
Photochemistry: An Internal Actinometer for Substrate Release
Romana Jarosova, Sam V. Kaplan, Thomas M. Field, Richard S. Givens, Sanjeewa N. Senadheera,
and Michael A. Johnson*
Cite This: Anal. Chem. 2021, 93, 2776−2784
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ABSTRACT: Caged compounds are molecules that release a
protective substrate to free a biologically active substrate upon
treatment with light of sufficient energy and duration. A notable
limitation of this approach is difficulty in determining the degree of
photoactivation in tissues or opaque solutions because light
reaching the desired location is obstructed. Here, we have addressed
this issue by developing an in situ electrochemical method in which
the amount of caged molecule photorelease is determined by fast-
scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes.
Using p-hydroxyphenyl glutamate (pHP-Glu) as our model system,
we generated a linear calibration curve for oxidation of 4-
hydroxyphenylacetic acid (4HPAA), the group from which the
glutamate molecule leaves, up to a concentration of 1000 μM.
Moreover, we are able to correct for the presence of residual pHP-
Glu in solution as well as the light artifact that is produced. A corrected calibration curve was constructed by photoactivation of pHP-
Glu in a 3 μL photoreaction vessel and subsequent analysis by high-performance liquid chromatography. This approach has yielded a
linear relationship between 4HPAA concentration and oxidation current, allowing the determination of released glutamate
independent of the amount of light reaching the chromophore. Moreover, we have successfully validated the newly developed
method by in situ measurement in a whole, intact zebrafish brain. This work demonstrates for the first time the in situ
electrochemical monitoring of caged compound photochemistry in brain tissue with FSCV, thus facilitating analyses of neuronal
function.
he use of light-driven approaches to manipulate biological
systems has grown dramatically over the past decade.
antagonists for neuronal function.2,3,5,8−13 The function of
dopaminergic neurons and circuits can be explored using
recently developed caged dopamine and its caged receptor
antagonists.14,15
A major challenge of this approach, as well as others that
employ photoactivation strategies, is quantitation of a
photoreleased substrate when sample variability is not
controlled. Traditionally, the amount of a photoreleased
substrate has been determined indirectly by measuring the
photon flux impinging on the sample using actinometry
methods, e.g., potassium ferrioxalate solutions16 and the
quantum yield (the ratio of yield to photons absorbed) for
substrate release upon exposure to the light source. By
T
One such approach, caged compound photoactivation, allows
the application of biologically active molecules with micro-
meter spatial and sub-second temporal resolution. When
treated with light of sufficient energy, the photoremovable
protecting group on a caged compound separates from the
biologically active form of the molecule.1−6 This photo-
chemical delivery method has been used to activate biologically
dormant or inactive biosystems such as neurons and sensory
cells in ex vivo and in vivo tissue platforms as well as reactions
at the molecular level such as enzymatic reactions and protein
folding. Moreover, the protecting group’s rapid release occurs
within nanoseconds (ns) to microseconds (μs) depending on
the protecting group and the substrate,7 making this
photoactivation process compatible for probing temporal and
spatial parameters by electrophysiological and electrochemical
detection methods. For example, caged substrate photo-
activation combines the photorelease process with patch
clamp detection to examine the influence of the amino acids
glutamate and γ-aminobutyric acid (GABA) as agonists and
Received: August 13, 2020
Accepted: January 11, 2021
Published: January 25, 2021
https://dx.doi.org/10.1021/acs.analchem.0c03452
© 2021 American Chemical Society
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knowing the quantum yield, one can determine the number of
moles released when the number of photons absorbed by the
sample is known. This approach is reliable as long as the
photon delivery system and the substrate photolysis reaction
are identical for both the actinometry and substrate release
measurements.
Determination of yields of the photoreleased substrate in
complex biological and tissue entrainments present additional
confounding variables. Such complications arise because
actinometer solutions are homogeneous and contain only the
reactive chromophore in physiological media. Conversely, in
actual physiological experiments, there may be several
competing chromophores as well as opaque materials that
competitively absorb light or reflect it away from the caged
material. This interference diminishes the amount of light
reaching the caged compound, thereby resulting in an
overestimation of substrate release.
A direct, in situ measurement of photorelease of the
substrate, which is independent of interferences of the incident
light and the nature of the biological sample, would mitigate
many of the inherent complications associated with traditional
actinometer methods. We propose a remedy for quantifying
the photoreaction by employing a doubly responsive photo-
reaction that couples substrate release with the generation of a
separate electroactive byproduct. This concept parallels the
photogeneration of active fluorophores with substrate release
that uses fluorescence intensities to quantify the release.3
Fast-scan cyclic voltammetry (FSCV) is a technique of
choice for measuring chemicals secreted from individual
cells,16,17 in acutely dissociated brain sections,12,16,18−22 and
in whole animals.23−28 This method is compatible with caged
compound photoactivation studies because it has good
temporal resolution (sub-second to millisecond), selectivity
(a characteristic cyclic voltammogram, or CV, is formed), and
spatial resolution (nanometer to micrometer). Here, we report
a new synergistic approach by FSCV to quantify photo-
activation of substrate release and caged compound decom-
position simultaneously with an in situ single probe. The
concept requires that the byproduct of the caging chromo-
phore be electroactive, but preferably not the caged compound
itself. However, as demonstrated here, the starting material
signal can be subtracted to remove it from interfering with the
product analyses.
In this work, we use a p-hydroxyphenacyl cage to
demonstrate proof of concept for the electrochemical
monitoring of photoactivation. The use of this cage for a
wide variety of substrates has several inherent advantages for
FSCV-based analyses. The pHP class of caged compounds
offers high quantum yields, nanosecond timescales of release,
and deep-seated rearrangement of the caging chromophore
into biologically benign 4-hydroxyphenylacetic acid (4HPAA;
Figure 1). This molecule can be measured electrochemically
and distinguished from other biologically active compounds
such as monoamine neurotransmitters.29
The method described here uses a microliter photoreaction
vessel to generate a calibration curve that relates the
electrochemical oxidation signal of photoreleased 4HPAA
measured by FSCV to the concentration of 4HPAA obtained
by HPLC with ultraviolet−visible detection. We have
previously demonstrated the simultaneous, electrochemical
quantitation of 4HPAA vs dopamine by FSCV.30 Thus, the
method proposed here extends our capability to include the
Figure 1. Reaction mechanism of uncaging p-hydroxyphenacyl-based
compounds.
direct measurement of caged compound degradation in
conjunction with dopamine release.
EXPERIMENTAL SECTION
■
Reagents. A stock solution (0.1 mmol L−1) of 4HPAA
(CAS no. 156-38-7, 98%, Sigma-Aldrich, St Louis, MO) and
pHP-Glu (synthesized by the Synthetic Chemical Biology Core
of the Center for the Molecular Analysis of Disease Pathways
at The University of Kansas, Lawrence) were prepared by
dissolving the appropriate analyte mass in artificial cerebrospi-
nal fluid (aCSF). The aCSF consisted of 126 mM NaCl, 2.5
mM KCl, 1.2 mM NaH2PO4, 2.4 mM CaCl2, 1.2 mM MgCl2,
25 mM NaHCO3, and 20 mM HEPES, adjusted to a pH of 7.4.
Solutions used for generating the calibration curves were
prepared by serial dilution of the stock solution. A stock
solution of 4HPAA was refrigerated in glass vials in the dark
when not in use. A stock solution of pHP-Glu was prepared
daily. All chemicals for high-performance liquid chromatog-
raphy (HPLC) were obtained from Sigma-Aldrich (St Louis,
MO). Ultrapure water (∼18.2 MOhm-cm) was used to
prepare all aqueous solutions.
Animals. Adult Danio rerio (zebrafish, AB wild-type strain)
were purchased from Zebrafish International Resource Center
(ZIRC, University of Oregon, Eugene, OR) and housed in the
Shankel Structural Biology Center at the University of Kansas.
The animals were housed in three-liter tanks (15−20 fish per
three-liter system rack tank) that were connected to a
recirculation filtration system. All tanks were maintained
under constant chemical, biological, and mechanical filtration,
as well as the UV sterilizing unit to ensure adequate conditions.
Conductivity (∼800 μS cm−1) and pH (7.2) of the reverse
osmosis purified system water (maintained at 28 °C) was
controlled and adjusted using a Multiparameter Monitoring
and Control Instrument 5200A (YSI, Yellow Springs, OH).
Fish were fed twice a day and kept in a light/dark cycle (16 h/
8 h). All procedures involving zebrafish were approved by the
Animal Care and Use Committee of the University of Kansas.
Uncaging Apparatus. The experimental uncaging appa-
ratus was adapted from an approach used previously for
electrophysiology.31 The output from a mercury lamp, directed
through a 280 nm cut-off high-pass filter and gated with a
shutter, was delivered to the sample through a fiber-optic cable
(PolyMicro Technologies, Inc., Phoenix, AZ). A micro-
manipulator was used to position the fiber-optic cable near
the carbon-fiber microelectrode.
Fast-Scan Cyclic Voltammetry. A ChemClamp potentio-
stat (Dagan, Minneapolis, MN, USA), modified to enhance the
range of available gain settings, was used. Data were collected
and analyzed using TarHeel CV software (R.M. Wightman and
M.L.A.V. Heien, University of North Carolina, Chapel Hill,
NC, USA). Carbon-fiber working electrodes were constructed
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using previously published methods25 and were trimmed to an
exposed length of 40 μm. Electrodes were used in the flow
injection analysis apparatus, the microliter reaction vessel, and
in whole zebrafish brains. Flow injection analysis was carried
out by directing the solution flow with a six-port sample
injector through a custom designed flow cell. For the detection
of 4HPAA, a triangular waveform was used in which potential
(vs Ag/AgCl electrode) was linearly scanned from −0.4 V to
+1.3 V to −0.4 V. The potential between scans was held at
−0.4 V. The scan rate was 600 V/s and the update rate was 10
Hz.30
bottom of the tubing, while the pHp-glutamate solution was
injected from the top of the tubing. The carbon-fiber working
electrode and a Ag/AgCl reference electrode were micro-
manipulated into the solution from the top of the reaction
vessel. Once the photo-uncaging occurred and the photo-
released 4HPAA was measured by FSCV, the electrodes were
removed, and the sample was carefully transferred from the
reaction vessel to a vial for a further HPLC analysis. To ensure
sufficient amount of material for an HPLC analysis, three
uncaging samples (i.e., 3 × 3 μL) were consolidated into a vial
for an HPLC analysis for each of the three electrodes.
Measurements performed to quantify the photoelectric effect
that were carried out in solutions in which pHP-Glu was not
present.
Electrochemical Measurement of pHP-Glutamate
Photoactivation. A microliter reaction vessel (Figure 2)
Electrochemical Detection of pHP-Glutamate Photo-
activation in a Zebrafish Brain. Zebrafish were euthanized
by hypothermic shock followed by decapitation. Whole brains
were harvested as described previously.32 Briefly, the
decapitated zebrafish head was immobilized in a Petri dish
filled with 2% agarose (BioReagent grade, Sigma-Aldrich, St.
Louis, MO, USA) and oxygenated (95% O2 and 5% CO2)
aCSF. After removal from the skull, the fish brain was carefully
lifted and transferred to a perfusion chamber. The brain was
kept viable by a continuous flow of oxygenated aCSF heated at
a physiological temperature of 28 °C. Prior to any measure-
ment, the fish brain was first kept in the perfusion chamber for
40 min to equilibrate. The carbon-fiber working electrode was
positioned into the ventral telencephalon of the zebrafish with
a micropositioner. A 50 μm optic fiber (Polymicro
Technologies, Phoenix, Arizona) was further micromanipu-
lated into a close proximity of the tip of the carbon-fiber
microelectrode. The photoelectric effect was examined in a
zebrafish brain perfused by only aCSF (no pHP-glutamate
presented). Finally, the pHp-glutamate (c = 1000 μM) in aCSF
was perfused through the brain for 30 min and the
electrochemical detection of pHP-glutamate (4HPAA, respec-
tively) was carried out. Both the photoelectric effect and the
uncaging of pHP-glutamate in a zebrafish brain were
performed using 1000 ms duration of light exposure.
Figure 2. Microliter-scale reaction vessel used to determine the
degree of photo-uncaging with FSCV. Left, without light application,
and right, with light application through the optical fiber.
was fabricated with 1 mm plastic tubing (Cole Parmer, Vernon
Hills, Illinois) that allowed a 3 μL volume of aqueous pHP-
glutamate solution to be held in place by surface tension. A 1
mm diameter optical fiber (Polymicro Technologies, Phoenix,
Arizona) was press fitted into the plastic tubing from the
HPLC Analysis. An HPLC system (Shimadzu Scientific
Corp) is consisted of a CBA-20A system controller, a DGV-
Figure 3. (A) Fast-scan cyclic voltammetric i−E curve for 100 μM 4HPAA in aCSF measured at a carbon-fiber microelectrode in flow injection
arrangement. Two oxidation peaks occur at 1.23 and 1.12 V on the forward and backward sweep, respectively. Direction of the forward (black) and
the reverse (red) potential sweep is indicated by arrows. (B) Response curves for 4HPAA in aCSF for injected concentrations from 10 to 1000 μM.
○
△
Currents were evaluated at +1.23 V, open circles ( ), and + 1.12 V , open triangles ( ). Data are presented as mean std. dev. (N = 3).
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20A3 degassing unit, an LC-20 AD high pressure pump, an
SIL-20 AC autosampler, and an SDP-20 AV UV−Vis detector.
An Ascentis C18 (15 cm × 4.6 mm × 5 μm) column
(Millipore-Sigma, Inc., Burlington, MA) was used. The sample
volume injected was 5 μL. The mobile phase consisted of (A)
99% H2O, 1% MeOH, and 0.06% formic acid and (B) 99%
MeOH, 1% H2O, and 0.06% formic acid. Analytes were eluted
isocratically at A:B = 70:30 (v/v %) at a flow rate of 1 mL
min−1. The spectrophotometric detector was set at 220 nm.
The quantification of 4HPAA was performed by generation of
an external calibration curve. The calibration curve was
constructed from the height of the peak, as a function of
4HPAA concentration, with a linear dynamic range from 1 to
1000 μM (R2 = 0.9996, N = 3).
Statistics. Statistical analysis and graphical presentation
were carried out using OriginPro software, version 2020b
(OriginLab Corp., Northampton, MA). A p-value of 0.05 or
less was considered as significant.
RESULTS AND DISCUSSION
Figure 4. Flow injection analysis of pHP-Glu and 4HPAA. Solutions
of 100 μM pHP-Glu and 100 μM 4HPAA in aCSF were injected
separately. Parameters: scan rate, 600 V/s, update rate, 10 CVs/s,
holding potential, −0.4 V.
■
Measuring 4HPAA with FSCV. We have previously
shown that 4HPAA can readily be measured by FSCV.30
Thus, we sought here to demonstrate that the liberation of
4HPAA from p-hydroxyphenacyl-based caged compounds,
such as p-hydroxyphenacyl glutamate, could be used to
quantify the formation of the resulting bioactive molecule.
Our approach quantifies glutamate photorelease by measuring
differences between currents produced by the liberation of
4HPAA after light application and currents present prior to
light application. Therefore, it is essential that the current
associated with 4HPAA increase linearly within a useful
concentration range. Figure 3A shows a CV of 4HPAA (0.1
mM) obtained by flow injection analysis and FSCV. As
described by Shin et al.,30 we observed two well-resolved
peaks, suggesting that two distinct electrochemical processes
occur. The first peak occurred at +1.23 V as the potential
swept toward the most positive value, while the second peak
occurred at +1.12 V as the potential swept toward the most
negative value. Figure 3B shows that the electrochemical
response to 4HPAA, evaluated from peaks at +1.23 V (forward
sweep) and +1.12 V (backward sweep), was linear from 10 μM
up to at least 1000 μM (higher concentrations were not
investigated). This result indicates that it is possible to quantify
the amount of 4HPAA photoreleased even at high concen-
trations.
Another concern was that the electroactivity of the caged
form of the compound would complicate the interpretation of
the electrochemical data because the CV is similar to that of
4HPAA. Despite the similarities, however, it is still possible to
distinguish between the two voltammograms. For example,
shown in Figure 4 are CVs of 4HPAA and pHP-Glu, obtained
by flow injection analysis. The red trace is the result of
subtracting the CV of pHP-Glu from that of 4HPAA. These
CVs are unfolded at a switching potential of +1.3 V so that the
direction of the potential sweep, which occurs from left to right
in the figure, is easier to visualize. The oxidation peak for pHP-
Glu appears at a similar position compared to the first
oxidation peak of 4HPAA (about +1.23 V on the forward scan)
and a small second pHP-Glu oxidation peak appears at +1.05 V
on the backward scan. As the subtraction of the CVs reveals, a
measurable signal proportional to the amount of 4HPAA
formed by photo-uncaging can be derived by subtracting the
two CVs. Even though in an actual uncaging experiment the
concentrations of 4HPAA and pHP-Glu may not be equal due
to the formation of species other than 4HPAA, the
measurements in Figure 4 demonstrate that the CVs for the
two species are qualitatively different and result in a
characteristic CV upon subtraction.
Removing the Artifactual Light Signal from the
4HPAA Signal. Currents produced by photons striking the
surface of the carbon-fiber microelectrode complicate the
measurement of the current produced by 4HPAA because the
CVs are similar. Therefore, we explored the effects of
subtracting this signal from the overall electrochemical signal
produced by photoactivation of pHP-Glu. Samples of aCSF
with different concentrations of pHP-Glu, ranging from 0 to
1000 μM, were placed in the reaction vessel described in
Figure 2. The solutions were exposed to light, gated by a
shutter of selected durations ranging from 200 to 1000 ms.
Even in the absence of pHP-Glu, the application of light
produced a large photoinduced current that gradually decayed
(Figure 5). The current response appeared to increase in linear
fashion with increasing exposure time up to about 600 ms
(Figure 6A). Beyond this time, the current deviated from
linearity. Measurements obtained in the presence of
successively increasing concentrations of pHP-Glu resulted in
stepwise increase in measured current for all examined
solutions (concentration of pHP-Glu from 400 to 1000 μM).
The curvature similar to that found in the absence of pHP-Glu
was evident in measurements obtained with successively
increasing concentrations of pHP-Glu present in the solution;
however, increasing the concentration tended to remove the
curvature, particularly above concentrations of 600 μM.
The generation of currents upon exposure of electrode
surfaces to visible and ultraviolet light has been docu-
mented.33,34 Although these currents reach peak values over
extended continuous exposure times, their measurement at
carbon-fiber microelectrodes with FSCV has not been
published in the literature, to our knowledge. We found that,
in our particular case, this photoinduced current could be
subtracted from the currents produced in the presence of pHP-
Glu to yield current time points that fall into a linear
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Figure 5. Current response measured in a solution containing 0 μM of pHP-Glu, and therefore, attributed to a light artifact. A representative
current vs time plot (top), color plot (bottom), and FSCV (right) are shown. Direction of the potential sweep is indicated by arrows. Duration of
light exposure: 1000 ms.
Figure 6. Light artifact removal. (A) Plot of the oxidation current vs the duration of light exposure. (B) Current response after the subtraction of
the light artifact. Data are presented for three measurements at three different carbon-fiber microelectrodes as mean std. dev.
relationship with increasing time of light exposure (Figure 6B;
linear regression analysis: R2 = 0.9998 (1000 μM), 0.9987 (800
μM), 0.9942 (600 μM), and 0.9690 (400 μM), N = 3
electrodes). The linear character of these curves suggests that
the light artifact can be removed from the overall electro-
chemical signal and the remaining currents can be used to
monitor the formation of 4HPAA.
Generation of a Calibration Curve. Our ability to isolate
currents produced by photolysis indicates that quantitation of
4HPAA in the solution is feasible. However, a critical aspect of
this quantitation is the correction for other electrochemically
active species present. Therefore, we designed a microliter
reaction vessel (Figure 2) that allows direct comparison
between oxidation currents that occur during photoreaction,
obtained by FSCV, and the actual concentration of 4HPAA
photoreleased, determined by high-performance liquid chro-
matography (HPLC). The reaction vessel was fabricated with
an ∼3 cm long segment of plastic tubing that allowed small
volumes (∼3 μL) of pHP-glutamate solution to be
immobilized due to the surface tension of water. A 1 mm
fiber-optic cable was positioned in the tube, but below the
solution, so that the entire solution could be irradiated. After
adding the solution, the carbon-fiber and Ag/AgCl electrodes
were inserted, thereby allowing the measurement of photo-
uncaging events with electrochemistry.
We measured how 4HPAA concentration changes at
different UV exposure times by combining FSCV and HPLC
analyses. The process of generating a light intensity
independent calibration curve is illustrated in Figures 7A−C.
The typical recording of a photo-uncaging measurement
involved the background subtraction of an averaged group of
reference CVs, collected prior to light application, from the
entire set of CVs obtained in the file. This operation removes
current contributions from un-photolyzed pHP-Glu and
generates a CV similar to that shown in Figure 4. The
photo-uncaging of pHP-Glu, present in the vessel at an initial
concentration of 1000 μM, was measured with FSCV as a
function of exposure time. After removing the electrodes, the
sample solution was analyzed by HPLC. Currents generated at
each time point were also obtained in the absence of pHP-Glu
and were subtracted from the measurements in which pHP-
Glu was present. This operation removes the artifactual current
generated by light application, similar to the subtraction
process in Figure 6.
From this analysis, we constructed two linear graphs that
plotted current (Figure 7A, R2 = 0.9985) and 4HPAA
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Figure 7. Generation of a calibration curve independent of the light source characteristics for an electrode. (A) Light artifact-subtracted current
response (for 1000 μM pHP-glutamate) vs duration of light exposure. (B) Concentration of photoreleased 4HPAA determined via HPLC vs
duration of light exposure. (C) Calibration curve obtained by combining (A, B). Data are reported as mean
electrodes.
std. dev for N = 3 separate
concentration (Figure 7B, R2 = 0.9986) against exposure time.
Combining these graphs provided a third linear plot that
directly related current to 4HPAA concentration (Figure 7C,
R2 = 0.9980). By direct quantification of 4HPAA and knowing
that 0.95 molecules of 4HPAA are released for every molecule
of glutamate in aqueous solutions at physiological pH,35,36 we
are able to quantify glutamate generated by photoactivation of
pHP-Glu. Therefore, this method represents the means for
quantifying the released, biologically active glutamate that is
more direct than actinometry. Furthermore, knowing the
relative yield and the quantum efficiency for 4HPAA from the
photorelease of any pHP leaving group (whether electroactive
or not), one can temporally track the concentration of the
leaving group on a real-time basis in a microliter-sized vessel.
Electrochemical Detection of pHP-Glutamate Photo-
activation in a Zebrafish Brain. We next sought to validate
our newly developed method for in situ electrochemical
detection of photoreleased 4HPAA in a living, whole zebrafish
brain, which is opaque. After brain dissection, the carbon-fiber
microelectrode and optic fiber were positioned in thetelence-
phalon, in close proximity to each other (Figure 8), and the
brain was perfused by oxygenated aCSF for 40 min. Upon
stabilization of the background signal, 1000 ms light pulses
were applied to quantify the photoelectric effect in brain tissue
responsible for the above discussed light artifact. A current of
0.52 0.04 nA (N = 3) was recorded.
The uncaging of pHP-Glu was photochemically evoked after
30 min of brain perfusion with 1000 μM pHp-glutamate in
aCSF. The light was applied with a duration of 1000 ms.
Representative data documenting the successful uncaging
process in whole brain and subsequent detection of 4HPAA
are shown in Figure 8. A cyclic voltammogram sampled from
the color plot at the time of light exposure (5 s) exhibits
characteristics reflecting the oxidation of 4HPAA, including a
prominent peak in current on the reverse scan at +1.24 V. The
examination of the photoreleased current vs time plot (Figure
8, above color plot) shows a large increase in current at the
time of light exposure. The measured current is significantly
higher compared to the one recorded during light artifact
measurement (Figure 9, one-way ANOVA with Tukey test, p <
0.0001), and therefore, can be assigned primarily as a
voltammetric response to the appearance of photoreleased
4HPAA.
We observed a prolonged current in the I−t profile. It is
possible that this current arises from adsorption of 4HPAA or
another uncaging product to the electrode surface, resulting in
either a faradaic current or alteration of the electrode double
layer capacitance. Another contribution to this current might
be slower diffusion of 4HPAA in the brain tissue. More work is
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Figure 8. Representative data of 4HPAA detection in a zebrafish whole brain after pHp-glutamate photo-uncaging. Current vs time plot with
corresponding color plot (A) and CV (B) are shown. pHP-Glu (1000 μM) in aCSF was perfused through the brain for 30 min. Direction of the
potential sweep is indicated by arrows. Duration of light exposure: 1000 ms. The placement of the carbon-fiber working electrode and optic fiber in
the telencephalon of a zebrafish brain (C, D).
Figure 9. Representative current vs time (A) and CV (B) data for light artifact (LA) before the pHp glutamate administration (black),
photoactivation of pHP Glu (red), and the light artifact recorded after 30 min post washout (post washout light artifact, PWLA) with pHp-Glu free
aCSF (blue). Inset: photoinduced 4HPAA release measured after 30 min of pHp Glu perfusion through the brain was significantly increased in
comparison with light artifact (*p < 0.0001, one way ANOVA, Tukey post hoc test, N = 3). The light artifact recorded after caged compound
washout was significantly different from the 4HPAA measurement (**p < 0.0001), but not significantly different from the signal recorded before
the caged compound administration (p = 0.3462). Duration of light exposure: 1000 ms.
needed to characterize each of these contributions. After
subtraction of the photoinduced current, the resulting current
was 1.23 0.06 nA (N = 3 different electrodes and locations),
(calculated from the concentration vs current plot, Figure 7C)
and 22.2 1.0 μM glutamate. A significantly lower signal was
obtained for the experiments carried out in a whole brain in
comparison with the one performed in the reaction vessel (for
which corresponds to formation of 21.1
0.9 μM 4HPAA
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the same exposure time of 1000 ms), suggesting lower
uncaging power of the system in an opaque brain tissue.
Despite this fact, we have successfully provided proof of
concept for our newly developed method for the in situ
monitoring of caged compound photochemistry. Importantly,
the uncaging capacity is powerful enough to photorelease an
amount of glutamate in excess of its estimated extracellular
concentration range (0.02 to 20 μM).37 Additionally, the
amount of the photoreleased glutamate can be tailored based
on individual needs, as the released concentration can be
altered by the length of the uncaging time and light intensity.
Finally, to ensure that the recorded signal arises from the
oxidation of 4HPAA, we conducted an experiment in which we
applied a pulse of light to the zebrafish brain in the region
where the electrode was located and measured the current
response with FSCV. Prior to addition of pHP-Glu, a small
artifactual current signal occurred that aligned with the light
application. Upon application of pHP-Glu for 30 min, the
magnitude of this light-induced signal increased several folds.
After washing out pHP-Glu (and the products of the uncaging
process) for at least 30 min, the signal returned to a level
similar to that noted prior to adding pHP-Glu (Figure 9, one-
way ANOVA with Tukey test, p = 0.3462, N = 3 different
locations). These data indicate that the observed current arises
from 4HPAA formation and possibly other electrochemically
active species formed from the uncaging process and not
simply the light artifact.
5078-9896; Phone: 785-864-4269; Email: johnsonm@
ku.edu
Authors
Romana Jarosova − Department of Chemistry and R.N.
Adams Institute for Bioanalytical Chemistry, University of
Kansas, Lawrence, Kansas 66045, United States; Department
of Analytical Chemistry, UNESCO Laboratory of
Environmental Electrochemistry, Charles University, Prague 2
12843, Czech Republic
Sam V. Kaplan − Department of Chemistry and R.N. Adams
Institute for Bioanalytical Chemistry, University of Kansas,
Lawrence, Kansas 66045, United States
Thomas M. Field − Department of Chemistry and R.N.
Adams Institute for Bioanalytical Chemistry, University of
Kansas, Lawrence, Kansas 66045, United States;
orcid.org/0000-0002-9536-6317
Richard S. Givens − Department of Chemistry and R.N.
Adams Institute for Bioanalytical Chemistry, University of
Kansas, Lawrence, Kansas 66045, United States
Sanjeewa N. Senadheera − Department of Pharmaceutical
Chemistry, University of Kansas, Lawrence, Kansas 66045,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.0c03452
Author Contributions
M.A.J. conceived the work. This manuscript was written by
M.A.J., S.V.K., R.J., and R.S.G. Experiments were carried out by
R.J., M.A.J., S.V.K., and T.M.F. Compounds were synthesized
by S.N.S. and the Synthetic Chemical Biology Core.
CONCLUSIONS
■
Our newly developed method can be used to quantify the
degree of caged compound photolysis in situ with FSCV, a
well-characterized electrochemical method. This approach is
largely independent of the characteristics of the light source
because light artifacts are subtracted out and the electro-
chemical signal is related directly to the concentration. Thus,
our method depends only upon the electroactivity of the
photoreleased cage, in this case, 4HPAA. Importantly, we have
shown that 4HPAA, the product of photoreleased pHP-
glutamate, can be easily detected and quantified in the
telencephalon of a zebrafish whole brain. Thus, our work
demonstrates the feasibility of photodelivering biologically
active compounds in living tissues. In future studies, it will be
important to validate this method in higher species such as
mice and rats. This point is especially important since the
brains in these species may have a more complex chemical
makeup and opacity, resulting in problems such as increased
electrode fouling or impairment of light access. Nevertheless,
we expect this approach to have broad applicability, but it will
be particularly well suited for cases that make quantitation of
photons reaching the sample difficult, such as tissues or opaque
liquids. Moreover, this method should also work when using
caged compounds other than those based on pHP that release
electroactive photocages, such as coumarins. Ultimately,
adapting this method for in vivo application with a combined
electrode, light guide, and caged compound delivery method
will maximize its utility.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support for this research was provided by grants from the
National Institute of Neurological Disorders and Stroke of the
National Institutes of Health under award nos. R21NS109659
(M.A.J.) and R21NS077485 (M.A.J.). Additionally, M.A.J.
received support from the Center for Molecular Analysis of
Disease Pathways and the Synthetic Chemical Biology Core,
an NIH Center of Biomedical Research Excellence (COBRE),
supported by an Institutional Development Award (IDeA)
from the National Institute of General Medical Sciences of the
National Institutes of Health under award no. P20GM103638.
R.J. received financial support from Charles University in
Prague under award no. SVV260560.
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AUTHOR INFORMATION
Corresponding Author
Michael A. Johnson − Department of Chemistry and R.N.
Adams Institute for Bioanalytical Chemistry and Graduate
Program in Neuroscience, University of Kansas, Lawrence,
Kansas 66045, United States; orcid.org/0000-0001-
■
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