Indicator Dyes and Catalytic Nanoparticles for Irreversible Visual Hydrogen Sensing

Article abstract of DOI:10.1021/acs.analchem.0c01769

Using ultraviolet-visible (UV-vis) absorption spectroscopy, we have tested the reactivity of various indicator molecules combined with catalytic bimetallic gold-palladium nanoparticles (Au-Pd NPs) in solution for an irreversible and visual response to H2. Our aim was to identify the most suitable indicator/Au-Pd NP system for the future development of a thin, wearable, and visual H2 sensor for noninvasive monitoring of in vivo Mg-implant biodegradation in research and clinical settings with fast response time. The indicators studied were bromothymol blue, methyl red, and resazurin, and the reactions of each system with H2 in the presence of Au-Pd NPs caused visual and irreversible color changes that were concluded to proceed via redox processes. The resazurin/Au-Pd NP system was deemed best-suited for our research objectives because (1) this system had the fastest color change response to H2 at levels relevant to in vivo Mg-implant biodegradation compared to the other indicator/Au-Pd NP systems tested, (2) the observed redox chemistry with H2 followed well-understood reaction pathways reported in the literature, and (3) the redox products were nontoxic and appropriate for medical applications. Studying the effects of the concentrations of H2, Au-Pd NPs, and resazurin on the color change response time within the resazurin/Au-Pd NP system revealed that the H2-sensing elements can be optimized to achieve a faster or slower color change with H2 by varying the relative amounts of resazurin and Au-Pd NPs in solution. The results from this study are significant for future optical H2 sensor design.

Full text of DOI:10.1021/acs.analchem.0c01769

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Article
Indicator Dyes and Catalytic Nanoparticles
for Irreversible Visual Hydrogen Sensing
Michael E Smith, Angela L Stastny, John A Lynch, Zhao Yu, Peng Zhang, and William R. Heineman
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.0c01769 • Publication Date (Web): 06 Jul 2020
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Analytical Chemistry
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Indicator Dyes and Catalytic Nanoparticles for Irreversible Visual
Hydrogen Sensing
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Michael E. Smith, Angela L. Stastny, John A. Lynch, Zhao Yu, Peng Zhang*, William R. Heineman*
University of Cincinnati, Department of Chemistry, Cincinnati, OH 45221-0172 USA
ABSTRACT: Using ultraviolet-visible (UV-Vis) absorption spectroscopy, we have tested the reactivity of various indicator
molecules combined with catalytic bimetallic gold-palladium nanoparticles (Au-Pd NPs) in solution for irreversible, and visual
response to H2 with the aim to develop the most suitable indicator/Au-Pd NP system into a thin, wearable, and visual H2 sensor for
non-invasive monitoring of in vivo Mg-implant biodegradation in research and clinical settings with fast response time. The indicators
studied were bromothymol blue, methyl red, and resazurin, and the reactions of each system with H2 in the presence of Au-Pd NPs
caused visual and irreversible color changes that were concluded to proceed via redox processes. The Resazurin/Au-Pd NP system
was deemed best suited for our research objectives because (1) this system had the fastest color change response to H2 at levels
relevant to in vivo Mg-implant biodegradation compared to the other indicator/Au-Pd NP systems tested, (2) the observed redox
chemistry with H2 followed well-understood reaction pathways reported in the literature, and (3) the redox products were non-toxic
and appropriate for medical applications. Studying the effects of the concentrations of H2, Au-Pd NPs, and resazurin on the color
change response time within the Resazurin/Au-Pd NP system revealed that the H2 sensing elements can be optimized to achieve a
faster or slower color change with H2 by varying the relative amounts of resazurin and Au-Pd NPs in solution. The results from this
study are significant for future optical H2 sensor design.
Magnesium (Mg) and its alloys have the capability of
outperforming traditionally used materials for orthopedic
applications such as bone repair because they can act as
load-bearing implants that safely biodegrade when no longer
needed.1–11 We have previously shown that an appreciable
fraction of the hydrogen gas (H2) liberated as a corrosion
product from Mg-implant biodegradation can be detected
transdermally and non-invasively at levels ranging from ~30 to
~700 µM by placing electrochemical or visual H2 sensors on
mouse skin directly above implants as they biodegraded in
vivo.2,5,6,12 These non-invasive H2 sensing methods are
considered to be fast, reliable, and accurate representations of
the extent of in vivo Mg-implant biodegradation since the
measured H2 levels correlated nicely with the biodegradation
rates measured via the highly accurate mass loss method of
explanted implants.2,5 These significant discoveries opened the
door to using H2 sensing as a platform for monitoring the in vivo
biodegradation of Mg-implants by a simple, non-invasive
procedure without exposure to X-rays. Furthermore, this
sensing platform enables in vivo biodegradation to be measured
in real-time. These features are desirable in medical research
settings in which new biodegradable materials and devices are
developed and in clinical settings for the routine evaluation of
patients with Mg-implants. However, the sensors used in the
aforementioned studies are limited for monitoring H2 in such
settings. For example, the electrochemical H2 sensor offers
remarkable sensitivity, yet the sensor itself and the associated
instrumentation are costly, complex, and require a reasonable
skill level for proper use. This sensor also requires regular
calibration and tedious multiple measurements if mapping of H2
levels over an area above an implant is desired since the sensor
surface is ~50 µm in diameter. The visual H2 sensor of ~10 µm
thickness and ~1 cm2 surface area afforded detailed mapping of
H2 levels above implants, which is a very useful tool for
monitoring larger implants such as plates, and the H2 detection
could be made easily using a cell phone.5,12 However, the
exposure times required to induce detectable color changes
were on the order of hours, which limit its practical use. To
circumvent these problems, the objective of this research was to
test the reactivity of various indicator molecules combined with
catalytic bimetallic gold-palladium nanoparticles (Au-Pd NPs)
in solution for fast, irreversible, and visual response to H2 with
the aim to later develop the most suitable indicator/Au-Pd NP
system into a thin, wearable, and optical H2 sensing device for
non-invasive monitoring of in vivo Mg-implant biodegradation
in medical research and clinical settings with fast response time.
Nanoparticles (NPs) such as those of platinum (Pt)13–17
palladium (Pd)5,11,18–20 and Au-Pd21–25 have attracted
,
,
considerable interest for use in a variety of H2 sensing platforms
and applications due to their unique catalytic abilities. Our basis
for choosing Au-Pd NPs for our research objectives stems from
reports describing how alloys containing Au and Pd are
particularly advantageous for H2 sensing by exhibiting
enhanced H2 adsorption and solubility.26,27 With respect to
signal transduction for H2 sensing, catalytic NPs have been
combined with different indicators for the development of
optical H2 sensors in which the sensing mechanism relies on the
dissociation of H2 at the NP surface to form highly reactive
hydrogen atoms that subsequently undergo a redox reaction
with an indicator to cause a visual color change.5,13 For
example, Seo et al.13 used a reagent containing methylene blue
and Pt NPs to quantify the amount of H2 dissolved in water as
the reagent underwent a blue to colorless transition upon
reaction with H2. Although this simple method was shown to be
as effective as an electrochemical sensor for H2 quantification,13
the use of methylene blue as a H2 sensing molecule is
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Analytical Chemistry
disadvantageous for certain applications because the reduction
Page 2 of 8
In this work, we tested the reactivity of H2 with bromothymol
blue, methyl red, and resazurin indicators combined with Au-
Pd NP catalysts by preparing buffered solutions (pH~7.5) and
exposing each to H2 at a known flow rate and concentration
while ultraviolet-visible (UV-Vis) absorption spectra were
monitored over time. The indicator/Au-Pd NP system most
suitable for our target application for future optical H2 sensor
development was identified by comparing the changes in the
characteristic UV-Vis absorption maxima of each indicator/Au-
Pd NP system as a function of H2 exposure time. The most
suitable indicator/Au-Pd NP system was further studied via
UV-Vis spectroscopy to examine the effects of varying the
concentrations of H2, Au-Pd NPs, and indicator on the color
change response time.
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of methylene blue and subsequent color change is known to be
reversible in the presence of oxygen (O2).28 Regarding our goal
of future optical H2 sensor development for monitoring Mg-
implant biodegradation in vivo, the sensing mechanism should
ideally be an irreversible process such that the sensor color
change resulting from redox reactions between hydrogen atoms,
formed at the NP catalyst surface, and indicator molecules
cannot be reverted back to the original sensor color by O2. An
optical H2 sensor with irreversible capabilities would likely
achieve the lower limits of detection needed for medical
applications since O2 would not affect the overall color change
response time of the sensor. From a practical perspective, our
development of an optical H2 sensor should also exhibit a color
change response on the order of ~10 minutes. Therefore, the
primary goal of this work was to investigate various indicators
that could undergo irreversible color changes upon reaction
with H2 at levels relevant to in vivo Mg-implant biodegradation
(e.g. ~30 to ~700 µM) in the presence of Au-Pd NP catalysts,
and to identify the most suitable indicator/Au-Pd NP system for
future optical H2 sensor development by comparing the color
change response time of each system.
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EXPERIMENTAL SECTION
Chemicals, Materials, and Instrumentation. All chemicals
and materials were used as received. Resazurin sodium salt
(C12H6NNaO4, ˃85.0% by HPLC) was purchased from TCI
America Inc. Sodium salts of methyl red (C15H14N3NaO2) and
bromothymol blue (C27H27Br2NaO5S) were acquired from
ACROS Organics (USA). Gold (III) chloride trihydrate
The specific indicators investigated in this study were those
shown in Figure 1 and included bromothymol blue, methyl red,
and resazurin. Bromothymol blue (Figure 1A) is a well-known
acid/base indicator that exhibits a blue to yellow color change
upon protonation.29 Methyl red (Figure 1B) is another
well-known acid/base indicator that has yellow and red colors
in the deprotonated and protonated forms, respectively.30 Due
to the reported electrochemical reduction pathways in the
literature for these molecules,31–33 it was hypothesized that
bromothymol blue and methyl red could undergo rapid, and
irreversible reduction reactions with H2 in the presence of Au-
Pd NP catalysts that would result in visual color changes
through a similar mechanism described by Seo et al.13 for the
methylene blue/Pt NP system. Resazurin (Figure 1C), a
common redox indicator that exhibits a blue color at neutral pH,
can undergo an irreversible reduction to lose water to form a
≥
(HAuCl4·3H2O,
hexachloropalladate (IV) (K2PdCl6, 99%) were purchased from
Sigma-Aldrich (MO). Trisodium citrate dihydrate
99.9% trace metal basis), and potassium
(Na3C6H5O7·2H2O), phosphate buffered saline (PBS, 10X
solution, pH 7.4), and a 3-way valve of polytetrafluoroethylene
(PTFE) were obtained from Fisher Scientific (NJ). Hydrion pH
paper (0–13 range) was obtained from Grainger (IL). All
≥
aqueous solutions were prepared using de-ionized water ( 18
MΩ·cm, referred to as DI water herein). Ultra-high purity
grades of H2 and N2 gases were obtained from Wright Brothers
Inc. (OH). A gas proportioning rotameter (#GMR2-010343)
equipped with two flow tubes (FL-2GP-61C-61C) was
purchased from Omega Engineering (CT). A rotameter with a
single flow tube (#FP 1/8-038-G-6) was acquired from
Lab-Crest, Fisher & Porter Co. (PA). Tygon tubing (¼” ID, ½”
OD) was purchased from McMaster-Carr (OH). Hypodermic
needles (305120-23G) were acquired from BD Co. (NJ).
Stainless-steel tubing (1/8” OD) was purchased from Supelco
(PA). An amperometric H2 sensor (H2-NP-705273) and
multimeter were obtained from Unisense (Denmark). A DryCal
DC-Lite flow calibrator was acquired from Brandt Instruments,
Inc. (LA). Microcentrifuge tubes were purchased from Avant
(MO). NPs were centrifuged using a Sorvall, Legend Micro 21
(MA). UV-Vis spectra of all samples were collected using a
USB4000-UV-VIS spectrophotometer (Ocean Optics, FL) by
pink-colored, and highly fluorescent resorufin product.34–38
A
subsequent reduction of resorufin to the colorless
dihydroresorufin can occur, but the process is reversible in the
presence of O2.35 Despite reversibility of this subsequent
reaction, resazurin was hypothesized to be a good candidate for
our research objectives since the initial irreversible reduction
that forms the highly luminescent resorufin product would
likely be the only color transition observed at low levels of H2
exposure, and this product could serve nicely as the analytical
signal. Furthermore, previous work has shown that resazurin
can be converted to resorufin upon reaction with H2 in the
presence of Pt NP catalysts.39
×
loading samples in a 1 cm
1 cm quartz cuvette. For all time-
based UV-Vis spectra, data shown at t ≥ 0 seconds correspond
to gas being delivered into samples while data at t < 0 seconds
correspond to no gas being delivered into samples.
Transmission electron microscopy (TEM) measurements of NP
samples were performed on a Biotwin 12 TEM (FEI) by
depositing a drop of solution on a formvar-covered, carbon-
coated copper grid (Electron Microscopy Sciences, PA) and
letting the solvent dry at room temperature. Size distributions,
푑
휁
average hydrodynamic diameters ( ), and zeta potentials ( ) of
NP samples were measured using dynamic light scattering
(DLS) instrumentation (Microtrac, USA).
Figure 1. Anions of the sodium salts of (A) bromothymol blue,
(B) methyl red, and (C) resazurin.
Synthesis of Au-Pd Bimetallic Nanoparticle Master
Solution. Au-Pd NPs were synthesized by the co-reduction of
2
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Analytical Chemistry
[AuCl4]— and [PdCl6]2– ions via trisodium citrate as described
flow calibrator for pure N2 and measuring the flow rate at
various scale readings. It was deemed unsafe to measure H2
flow in either pure or mixed forms using the DryCal DC-Lite
flow calibrator due to potential sparking hazards. Thus, flow
rates of pure H2 coming from the single flow tube rotameter at
various scale readings were calculated theoretically using
previously with slight modifications.40 A batch of Au-Pd NP
solution was prepared by gently boiling and vigorously stirring
a solution containing 312 µL of 20 mM HAuCl4·3H2O, and 312
µL of 20 mM K2PdCl6 at a total volume of 50.00 mL in DI
water. Three mL of 2% (m/V) trisodium citrate was added
quickly, and the solution was left to boil under vigorous stirring
for 15 minutes. The solution was then cooled to room
temperature before being stored in the dark at ~4°C until further
use. A total of five batches of Au-Pd NPs were prepared and
combined in the following way to make a highly concentrated
Au-Pd NP master solution. Firstly, the contents of five batches
were transferred to one borosilicate Erlenmeyer flask and
boiled, uncovered, under vigorous stirring until the solvent level
reached ~25 mL. This solution was left to cool to room
temperature and the contents were transferred into several
microcentrifuge tubes and centrifuged at 14,000 rpm for 20
minutes. The supernatants were discarded to waste while the
pellets of Au-Pd NPs remained in the bottom of each tube. All
pellets were sonicated, creating highly concentrated micro
volumes of Au-Pd NPs, and combined into a borosilicate glass
container thereby creating a highly concentrated solution of
Au-Pd NPs with a volume of ~5.00 mL in DI water. This
solution will be referred to as the Au-Pd NP master solution
herein.
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푅 푄 휌
, , and represent the rotameter scale
Equation S2 where
reading (mm), volumetric flow rate (mL/min), and gas density
(g/L).41 The gas densities of pure N2 and H2 used were 1.25 g/L
and 0.09 g/L, respectively, and were determined using the ideal
gas law at standard temperature and pressure. When comparing
flow rates for two gases at the same rotameter scale reading,
(i.e., where
The rotameter calibration for N2 could therefore be applied to
pure H2 using Equation S3.41 The measured flow rates for pure
N2 and calculated flow rates of pure H2 at various scale readings
are summarized in Table S2.
For flow rate determinations of H2/N2 gas mixtures, it was
necessary to measure the concentration of H2 within the mixture
using an amperometric H2 sensor to calculate theoretical gas
densities. To do so, an amperometric H2 sensor was calibrated
as described previously.1,5,6 The sensor was connected to a
multimeter and polarized at +1000 mV for at least one hour
before use. Pure H2 was delivered into ~20 mL of DI water at
257 mL/min for 30 minutes to form a 100% saturated H2
solution constituting an 800 µM concentration at room
temperature.1,2,5,6 From this stock solution, standards of 0, 8, 40,
100, 200, 400, and 600 µM were prepared via dilution with DI
water. The amperometric H2 sensor tip was immersed in each
standard, and 6 measurements of the steady state current were
taken after at least 2 minutes to generate a current vs. H2
concentration calibration curve (Figure S3). After calibrating
the amperometric H2 sensor, the scale readings of GPR-H2 and
GPR-N2 flow tubes were set to desired levels to form a H2/N2
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41
푅 = 푅
2), Equation S2 reduces to Equation S3.
1
Gas Flow Setups and Determining the Concentration and
Flow Rate of H2 Delivered to Samples. The general gas flow
setup for various experiments is depicted in Figure S1. Using
appropriate fittings and stainless-steel tubing, H2 and N2 gas
tanks were fitted with regulators then connected to the inlets of
a gas proportioning rotameter (GPR) containing two separate
flow tubes for H2 and N2 (labeled “GPR-H2” and “GPR-N2” in
Figure S1). Varying the flow rates of H2 and N2 in the GPR flow
tubes permitted the ability to make a sample mixture of H2/N2
gas at a particular volume to volume ratio (v/v) based on the
respective GPR flow tube scale readings. The outlet of the gas
proportioning rotameter was connected to a rotameter equipped
with a single flow tube to allow controlled flow of sample gas.
The outlet of this flow tube was then connected to a 3-way valve
via stainless-steel and Tygon tubings. Lastly, stainless-steel
tubing was connected to the remaining outlets of the 3-way
valve to allow directional flow of sample gas to vent or to an
experiment at a controlled rate. Flow rate data of N2 and H2
gases from the GPR flow tubes at various scale readings were
provided by Omega Engineering (Table S1), and plots of gas
flow rate (mL/min) vs. rotameter scale reading (mm) were made
for each gas and fit to quadratic equations (Figure S2). To create
a H2/N2 gas mixture with a desired volume percentage of H2 (
%푉
gas mixture with defined volume percentages of H2 ( 퐻 ) and
2
%푉
N2 ( 푁 ). The H2 concentration of a H2/N2 gas mixture was
measured using the apparatus in Figure S4. Two mL of DI water
2
×
was placed in a 1 cm
1 cm glass vessel and clamped to a
support stand. A needle connected to the gas flow apparatus was
inserted into the water. H2/N2 gas was delivered into the water
for 20 minutes by setting the single flow tube rotameter scale
reading to 15.00 mm to saturate the solution. The gas flow to
the sample was cut off and the H2 concentration, in µM, was
measured by inserting the calibrated amperometric H2 sensor
into the sample and covering the glass vessel with a Teflon cap
that had the corner sectioned off. The volume percentage of H2
%푉
(
퐻 ) in the H2/N2 mixture was calculated according to
was the electrochemically
measured µM concentration of H2 in the H2/N2 mixture, and
2
[
]
퐻2
푚푒푎푠푢푟푒푑
Equation S4 where
%푉퐻 ,푡ℎ푒표푟푒푡푖푐푎푙
)
, a reasonable flow rate of H2 at the GPR flow
(퐺푃푅:퐻 푓푙표푤 푟푎푡푒)
2
tube
was chosen. Equation S1 was then
2
[
]
퐻2
푠푎푡푢푟푎푡푒푑
was the saturated concentration of H2 in water,
used to calculate the necessary flow rate of N2 at the GPR flow
which is 800 µM at room temperature and standard
퐺푃푅:푁 푓푙표푤 푟푎푡푒)
tube (
of
that would achieve the desired value
퐻 ,푡ℎ푒표푟푒푡푖푐푎푙. To determine the precise GPR flow tube
2
pressure.1,6,13 The volume percentage of N2 ( 푁 ) in the H2/N2
%푉
2
%푉
2
mixture was calculated according to Equation S5. Next, the
theoretical density of the H2/N2 mixture ( 푚푖푥) was calculated
푀푊퐻 푀푊푁
are the molecular
2
weights of H2 and N2, respectively. Once 푚푖푥 was known, this
value was plugged into Equation S7 to calculate the flow rate
of the H2/N2 mixture 푚푖푥) at a particular flow tube rotameter
scale readings for H2 and N2 that would achieve the desired
%푉
휌
퐻 ,푡ℎ푒표푟푒푡푖푐푎푙, the flow rates (y-values) for the respective
2
via Equation S6 where
and
2
gases were plugged into the corresponding quadratic equations
from Figure S2 to solve for the scale readings (x-values).
To deliver gas at a known rate to an experiment from the
single flow tube rotameter, this flow tube needed to be
calibrated. This was accomplished using a DryCal DC-Lite
휌
(푄
푄
scale reading using the calibrated flow of N2 ( 푁 ) from Table
S2. Three different H2/N2 mixtures were made according to the
2
3
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data shown in Table S3. From the data in Table S3, the flow
Page 4 of 8
NP solutions of Au-Pd and Au exhibited bands at ~520 and
~525 nm, respectively (Figure S7A). These bands were
attributed to the localized surface plasmon resonance of the Au
material within the nanostructures.43 NPs are known to
aggregate in high salt concentrations,44 and this phenomenon
can be seen when samples at 0.8% v/v concentrations were
dispersed in buffer (Figure S7A, dashed lines). The average
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rates of these sample gases were calculated using Equation S7
at various single flow tube rotameter scale readings, and the
results are summarized in Table S4. Plots of flow rate vs. scale
reading were made for each gas listed in Table S4, and the
results are shown in Figure S5. Quadratic functions were fit to
the data in Figure S5 and were then used to calculate the
necessary single flow tube rotameter scale reading to deliver
sample gas at a 23.40 mL/min flow rate during an experiment.
This flow rate was used for all experiments described herein.
Testing Various Indicator/Au-Pd NP Systems for Visual
Color Change Upon Reaction with H2. Aqueous solutions of
bromothymol blue, methyl red, and resazurin indicators were
prepared in volumetric flasks in 1X PBS buffer, had an indicator
concentration of 20 µM, and had Au-Pd NPs present at 0.8%
(v/v). The samples were then exposed to 800 µM H2 using the
apparatus shown in Figure S6 as UV-Vis absorption spectra of
all samples were collected over time.
푑
hydrodynamic diameters ( ) of the Au-Pd and Au NPs were
18.4 and 24.9 nm, respectively (Figure S7D and S7E).
Comparisons of the Visual Color Changes of Various
Indicator/Au-Pd NP Systems Upon Reaction with H2. All
indicator/Au-Pd NP systems underwent irreversible and visual
color changes upon reaction with H2 (Figure 2A-C). One
reaction pathway involving a blue to yellow color transition was
observed for the Bromothymol Blue/Au-Pd NP system upon H2
exposure for 20 minutes (inset photographs in Figure 2A).
UV-Vis absorption spectra of this sample before (navy
blue-trace) and 20 minutes after (yellow-trace) H2 exposure
revealed maxima at ~615 and ~437 nm, respectively (Figure
2A). These values agree nicely with those reported in literature
for the protonated and deprotonated forms of pure bromothymol
blue.29 However, the observed color changes in Figure 2A are
attributed to a redox reaction rather than an acid/base reaction
since the solution pH was maintained at neutral throughout the
experiment.
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Resazurin/Au-Pd NP System: Effects of the
Concentrations of H2, Au-Pd NPs, and Resazurin on the
Color Change Response Time. The effects of the
concentrations of H2, Au-Pd NPs, and resazurin on the response
time of observed color changes within Resazurin/Au-Pd NP
systems were studied using the apparatus in Figure S6. In all
experiments, 1X PBS buffer was used as the solvent. To study
H2 concentration effects, three identical samples were prepared
and had 20 µM resazurin concentrations and Au-Pd NPs present
at 0.8% (v/v). These samples were then exposed to H2 at 800,
558, and 169 µM concentrations. The effects of Au-Pd NP
concentration were studied by preparing two samples with
resazurin concentrations of 20 µM. Each sample differed in the
v/v percentage of Au-Pd NPs present in solution, and the
amounts used were 2% and 0.8%. Each sample was then
exposed to 800 µM H2. The effects of resazurin concentration
were studied by preparing three samples with Au-Pd NPs at
0.8% (v/v), varying the resazurin concentrations at 5, 20, and
40 µM, then exposing each sample to 800 µM H2.
Resazurin/Au-Pd NP System: Control Experiments. To
elucidate the cause of observed color changes in the
Resazurin/Au-Pd NP system, a series of control experiments
were performed by flowing pure H2 or N2 gases into various
sample combinations (Table S5) using the apparatus described
in Figure S6. To ensure the UV-Vis absorption signal remained
stable during the data collection interval, a sample of
Resazurin/Au-Pd NPs was placed in a cuvette and monitored
without any gas flow (Experiment 1, Table S5). Gold colloidal
nanoparticles (Au NPs) of ~25 nm size were synthesized as
described previously42 to examine if Au had any catalytic effect
on the reduction of resazurin by H2. A master solution of Au
NPs was prepared in a similar fashion as described for the Au-
Pd NP master solution, except the centrifugation conditions
were 7,000 rpm for 10 minutes. A series of controls using N2
(Experiments 2–7) and H2 (Experiments 8–12) were then
conducted (Table S5). Samples containing resazurin had 20 µM
concentrations. Samples containing NPs had at 0.8% (v/v)
concentrations.
For the Methyl Red/Au-Pd NP system, three colors were
observed (inset photographs of Figure 2B) and had reaction
pathways that proceeded from yellow to colorless during 2
minutes of H2 exposure, then colorless to faint red after
exposure to ambient air for 10 minutes. Similarly, the yellow to
colorless transition of the Methyl Red/Au-Pd NP system upon
reaction with H2 is attributed to a redox reaction rather than an
acid/base reaction since the solution pH did not change
throughout H2 exposure. UV-Vis absorption spectra of the
various sample colors observed for the Methyl Red/Au-Pd NP
system before (brownish yellow-trace), and 2 minutes after
(black-trace) H2 exposure revealed maxima at ~430 and ~305
nm, respectively, while the sample after 10 minutes of air
exposure had maxima at ~316 nm with additional bands at
~514, ~552, and ~700 nm (Figure 2B).
The Resazurin/Au-Pd NP system also displayed visual color
changes upon reaction with H2, and these data are displayed in
Figure 2C. Three colors were observed during the experiment
(inset photographs of Figure 2C) and included reaction
pathways that went from blue to pink after 1 minute of H2
exposure, then pink to colorless after additional H2 exposure for
2 minutes, then colorless to pink after exposure to ambient air.
These color changes were attributed to the redox reaction
described in Figure 3, in which resazurin (blue color) underwent
an irreversible reduction in the presence of H2 and Au-Pd NP
catalysts to form resorufin (pink color) with a subsequent
reduction to dihydroresorufin (colorless) that was reversible in
the presence of ambient O2.34–36,38 These observations were
supported by the spectral data in Figure 2C. For example, the
UV-Vis absorption maxima for the Resazurin/Au-Pd NP
sample before (blue-trace), and after (pink-trace) H2 exposure
were ~601 and ~571 nm, respectively (Figure 2C). These
maxima agree nicely with those reported in the literature for
pure resazurin and resorufin at comparable pH.37 The redox
reaction reversibility of dihydroresorufin back to resorufin in
the presence of ambient O2 is well known35 and can be seen
RESULTS AND DISCUSSION
Characterization of Au-Pd and Au NPs. UUV-Vis, TEM,
and DLS instrumentation were used to characterize the Au-Pd
and Au NPs (Figure S7). UV-Vis spectra of the as-synthesized
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visually in the third photograph in the inset of Figure 2C. This
photograph was taken after converting the sample to the
colorless form via H2 exposure, then exposing it to ambient air
for ~2 minutes. A small layer of pink color, corresponding to
resorufin, formed at the solution/air interface shortly after
exposure to ambient air
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Figure 2. Comparison of the color changes observed for various indicator/Au-Pd NP systems upon reaction with 800 µM H2. Raw
UV-Vis spectra are shown in (A), (B), and (C). The Bromothymol Blue/Au-Pd NP system before (A, navy blue-trace) and after 20
minutes of H2 exposure (A, yellow-trace). The Methyl Red/Au-Pd NP system before (B, brownish yellow-trace) and after 2 minutes
of H2 exposure (B, black-trace) followed by exposure to air for 10 minutes (B, red-trace). The Resazurin/Au-Pd NP system before
(C, blue-trace) and 1 minute after exposure to H2 (C, pink-trace), and after additional H2 exposure for 2 minutes (C, black-trace).
Inset photographs in (A), (B), and (C) are the sample solution colors observed during the experiment. (D) Comparison of the real-
time changes in the characteristic UV-Vis absorption bands for each indicator/Au-Pd NP system as a function of H2 exposure time.
demonstrating that dihydroresorufin is very sensitive to O2. The
colorless solution eventually reverted back to a full pink color
after ~30 minutes of air exposure. Like the other indicator/Au-
Pd NP systems investigated, the pH of the Resazurin/Au-Pd NP
system did not change throughout experimentation with H2.
seconds of H2 exposure. These results suggest that much longer
exposure times would be required to induce a detectable color
change. Therefore, bromothymol blue would not be a practical
indicator for future H2 sensor development for our target
application. The Methyl Red/Au-Pd NP system had relatively
fast color change response to H2 since the UV-Vis absorption
maxima corresponding to the yellow form of methyl red
(brownish-yellow trace in Figure 2D) decreased quickly during
H2 exposure. With respect to redox reactions, researchers have
shown that methyl red can electrochemically undergo an
irreversible reduction to form anthranilic acid and
N,N-dimethyl-p-phenylenediamine as products,32,33 of which
the latter product is known to exhibit acute toxicity.45
Consequently, methyl red was not used as an indicator for our
research objectives in order to avoid toxic chemicals that would
be problematic for medical applications. The Resazurin/Au-Pd
NP system also displayed fast color change response to H2 since
the resazurin band (blue-trace in Figure 2D) sharply declined
while the resorufin band (pink-trace in Figure 2D) increased to
a maximum and eventually flatlined as H2 was further
introduced into the system and all molecules were converted to
dihydroresorufin.
Figure 3. Redox reactions of resazurin upon reaction with H2
in the presence of Au-Pd NP catalysts at pH ~7.5.
The characteristic UV-Vis absorption maxima of all
indicator/Au-Pd NP samples as a function of H2 exposure time
are displayed in Figure 2D. It is evident from these data that the
Bromothymol Blue/Au-Pd NP system had a much slower color
change response to H2 compared to the other systems
investigated since the disappearance of the blue color and
formation of the yellow color (navy-blue and yellow traces in
Figure 2D, respectively) did not change significantly after 100
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Of the three systems we examined, the Resazurin/Au-Pd NP
slower color change response to H2 by varying the relative
amounts of resazurin and Au-Pd NPs.
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system was best suited for our research objective of future
optical H2 sensor development because (1) this system had fast
color change response to H2 compared to the other indicator/NP
systems tested, (2) resazurin has well-studied reduction
pathways that involve an initial irreversible reduction to a
highly luminescent resorufin product, and (3) the reactants and
products involved in the overall redox processes are non-toxic
and appropriate for medical applications. For these reasons, the
effects of the concentrations of H2, Au-Pd NPs, and resazurin
on the response time of observed color changes within the
Resazurin/Au-Pd NP system were studied.
Resazurin/Au-Pd NP System: Effects of the
Concentrations of H2, Au-Pd NPs, and Resazurin on the
Color Change Response Time. The effects of H2 are shown in
the time-based UV-Vis absorption spectra of Resazurin/Au-Pd
NP systems exposed to H2 at 169, 558, and 800 µM
concentrations (Figure 4A). These data illustrate how
decreasing the H2 concentration exposed to a Resazurin/Au-Pd
NP system decreased the color change response time. For
example, when monitoring the resazurin bands over time
Resazurin/Au-Pd NP System: Control Experiments.
From the set of control experiments described in Table S5, it
was observed that the UV-Vis absorption bands in Experiment
1 were very stable and remained nearly constant across the
visible wavelength range during measurement with no gas flow
(Figure S8). Similar results were observed in Experiments 2–7
as N2 was delivered into solutions (Figure S9). By comparison,
flowing H2 in Experiments 8-11 gave the same lack of spectral
change until both resazurin and AuNPs were present as shown
in Figure S10. These observations confirm that N2 had no effect
on the observed color changes for the Resazurin/Au-Pd NP
system and that color changes only arose when H2 reacted with
resazurin in the presence of Au-Pd and Au NP catalysts. For
example, exposure of H2 in Experiment 12 (Table S5) to the
sample containing resazurin and Au NPs led to dramatic UV-
Vis spectral changes as the peaks corresponding to resazurin
and resorufin increased and decreased, respectively (Figures
S10E and S11). These results show that the Au NPs catalyzed
the reduction of resazurin by H2 to form resorufin, and it has
been shown in the literature that Au can catalyze the
dissociation of molecular H2 into hydrogen atoms.46,47
Comparing the color change response times of the
Resazurin/Au NP (Figure S11) and the Resazurin/Au-Pd NP
systems (Figure 2D), the system containing Au-Pd NPs
underwent faster color changes. This result was attributed to the
presence of Pd in the nanostructures. Pd NPs have been shown
to be more effective catalysts for H2 sensing than those of Au.48
Also, Au-Pd alloys have been shown to exhibit accelerated H2
adsorption compared to Pd.26 The Resazurin/Au NP sample
could not be converted to the colorless form, even after 10
minutes of H2 exposure (data not shown) while the
Resazurin/Au-Pd NP system was fully converted to the
colorless form after ~65 seconds of H2 exposure (Figure 2D).
These results highlight the importance of Pd on the overall color
change response time of the Resazurin/Au-Pd system upon
reaction with H2.
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~
(dashed-line traces in Figure 4A), these bands flatlined after
65, 125, and 1000 seconds of H2 exposure for the experiments
at 800, 558, and 169 µM H2 concentrations, respectively.
Similarly, the peaks corresponding to resorufin (solid-line
traces in Figure 4A) reached maxima after ~50, 100, and 800
seconds of H2 exposure for the experiments at 800, 558, and 169
µM H2 concentrations, respectively.
The effects of the concentration of Au-Pd NPs on the
Resazurin/Au-Pd NP system are shown in Figure 4B.
Comparing the resazurin bands (dashed-line traces in Figure
4B), the sample containing 2% (v/v) Au-Pd NPs flatlined at ~70
seconds while the sample containing 0.8% (v/v) Au-Pd NPs
flatlined at ~90 seconds. Comparing the resorufin bands (solid-
line traces in Figure 4B), the sample containing a larger amount
of Au-Pd NPs reached a maximum at ~54 seconds while the
sample containing a lower amount of Au-Pd NPs reached a
maximum at ~58 seconds. These data show how increasing the
concentration of Au-Pd NPs within the Resazurin/Au-Pd NP
system decreased the color change response time upon reaction
with H2.
The effects of the concentration of resazurin on the
Resazurin/Au-Pd NP system are shown in Figure 4C. It is
evident from these data that the sample containing the lower
concentration of resazurin (blue traces in Figure 4C) underwent
a faster color change since the resazurin band (blue dashed-line
trace in Figure 4C) flatlined much earlier compared to other
samples and the resorufin band (blue solid-line trace in Figure
4C) reached a maximum earlier than the other samples. The data
obtained in this section demonstrate that the Resazurin/Au-Pd
NP system is viable for the future development of optical H2
sensors for our target application because the system quickly
changed color upon reaction with H2 at levels relevant to in vivo
Mg-implant biodegradation on a practical time scale, and the
H2 sensing elements can be optimized to achieve a faster or
CONCLUSIONS
We have studied various indicator/Au-Pd NP systems that are
potentially useful for a variety of optical H2 sensing applications
since each system underwent visible and irreversible color
changes upon reaction with H2 in the presence of Au-Pd NP
catalysts in solution. The indicators studied were bromothymol
blue, methyl red, and resazurin, and the reactions of each system
with H2 in presence of Au-Pd NPs were concluded to proceed
via redox processes. The Resazurin/Au-Pd NP system was
deemed best suited for our target application of future optical
H2 sensor development for non-invasive monitoring of in vivo
Mg-implant biodegradation in medical research and clinical
settings because (1) this system had a fast color change response
to H2 compared to the other indicator/Au-Pd NP systems tested,
(2) resazurin has well-studied reduction pathways that involve
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Figure 4. Effects of the concentrations of (A) H , (B) Au-Pd NPs, and (C) resazurin on the color change response time of the
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Resazurin/Au-Pd NP system. The bands corresponding to resazurin (dashed lines) and resorufin (solid lines) were monitored
over time during H2 exposure.

Page 7 of 8
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an initial irreversible reduction to a highly luminescent
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
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Figures, tables, and equations describing various gas flow setups
and determinations of flow rates of N2, H2, and H2/N2 gases; TEM
characterizations of Au-Pd and Au NPs; and UV-Vis spectra of
control experiments (PDF).
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AUTHOR INFORMATION
Corresponding Author
* Email: zhangph@ucmail.uc.edu. Phone: 513/556-9222.
* Email: heinemwr@ucmail.uc.edu. Phone: 513/556-9210.
ORCID
Michael E. Smith: 0000-0002-4295-8917
Peng Zhang: 0000-0003-3902-6876
William R. Heineman: 0000-0003-2428-5445
Author Contributions
M.E.S, W.R.H., and P.Z. designed the research project. M.E.S.
wrote the manuscript, and designed/carried out all experiments and
measurements, except for TEM measurements. A.L.S. critically
revised the manuscript and provided a program to analyze UV-Vis
spectra. Z.Y. acquired TEM images of nanoparticle samples. J.A.L.
provided technical assistance with the amperometric H2 sensor.
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Phase Semicrystalline Microstructures Drive Exciton
Dissociation in Neat Plastic Semiconductors. J. Mater. Chem. C
2015, 3, 10715–10722.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
The authors gratefully acknowledge the National Science
Foundation for financial support (NSF ERC 0812348). Dr. Patrick
Slonecker is acknowledged for his assistance with gas flow setups.
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