Cascade Chromogenic System with Exponential Signal Amplification for Visual Colorimetric Detection of Acetone

Article abstract of DOI:10.1021/acs.analchem.0c00149

The signal of the traditional chromogenic systems is directly proportional to analyte concentration, leading to an unsatisfactory sensitivity. Herein, we report a cascade chromogenic system to realize exponential amplification of colorimetric signal through coupling chemical oxidation with photoinduced radical chain reaction. The chemical oxidation of o-phenylenediamine (OPD) by Fe³⁺ generates Fe²⁺ and photoactive 2,3-diaminophenazine (DAP). Under blue-light irradiation, DAP initiates the formation of holes and H₂O₂ that reacts with Fe²⁺ to hydroxyl radicals (·OH) and Fe³⁺ via an intersystem crossing (ISC) process. Moreover, the holes oxidize water to yield ·OH as well. The resulting ·OH and regenerated Fe³⁺ in turn oxidize OPD to yield more DAP, leading to a self-propagating reaction cycle that continues to proceed until all the OPD molecules are consumed, along with a distinct color change from colorless to yellow. Through the generation of the complex between DAP and acetone that limits the ISC process, and therefore quenches the colorimetric signal, the highly sensitive and selective naked-eye detection of acetone is achieved from 50 μM to 3 mM, with a limit of detection of 35 μM. Additionally, the feasibility of this colorimetric assay to detect acetone in real water samples is also demonstrated.

Full text of DOI:10.1021/acs.analchem.0c00149

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Article
A Cascade Chromogenic System with Exponential Signal
Amplification for Visual Colorimetric Detection of Acetone
Jingkai Liang, Hua Li, Junren Wang, Haili Yu, and Yi He
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Apr 2020
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A Cascade Chromogenic System with Exponential
Signal Amplification for Visual Colorimetric Detection
of Acetone
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Jingkai Liang+ a, Hua Li+b, Junren Wang a, Haili Yu a, Yi He* a
a National Collaborative Innovation Center for Nuclear Waste and Environmental
Safety, School of National Defence Science & Technology, Southwest University of
Science and Technology, Mianyang 621010, P. R. China.
b SUSTech Core Research Facilities, Southern University of Science and Technology,
Shenzhen 518055, China.
+These authors contributed equally.
*Corresponding author: Prof. Dr. Yi He, Tel: +86-816-6089885, Fax: +86-816-
6089889, Email: yhe2014@126.com.
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ABSTRACT: The signal of the traditional chromogenic systems is directly
proportional to analyte concentration, leading to an unsatisfactory sensitivity. Herein,
we report a cascade chromogenic system to realizing exponential amplification of
colorimetric signal through coupling chemical oxidation with photo-induced radical
chain reaction. The chemical oxidation of o-phenylenediamine (OPD) by Fe3+ generates
Fe2+ and photoactive 2,3-diaminophenazine (DAP), Under blue light irradiation, DAP
initiates the formation of holes and H2O2 that reacts with Fe2+ to hydroxyl radicals (.OH)
and Fe3+ via an intersystem crossing (ISC) process. Moreover, the holes oxidize water
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.
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to yield OH as well. The resulting OH and re-generated Fe3+ in turn oxidize OPD to
yield more DAP, leading to a self-propagating reaction cycle that continues to proceed
until all the OPD molecules are consumed, along with a distinct color change from
colorless to yellow. Through the generation of the complex between DAP and acetone
that limits the ISC process and therefore quenches the colorimetric signal, the highly
sensitive and selective naked-eye detection of acetone is achieved from 50 μM to 3
mM, with a limit of detection of 35 μM. Additionally, the feasibility of this colorimetric
assay to detect acetone in real water samples is also demonstrated.
KEYWORDS: Cascade chromogenic system, exponential amplification, colorimetric
detection, acetone, o-phenylenediamine
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INTRODUCTION
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Colorimetric assays have been received a great research interest because of their simple
operation, portable device, low cost, and convenient readout with the naked eyes1-3.
These characteristics enable various colorimetric assays extremely for in-field detection
of several target analytes, especially in resource-limited region4-6. To date, various
chromogenic systems such as enzyme-catalyzed chromogenic reactions and plasmonic
nanomaterials (gold, silver and highly doped semiconductors) have been employed for
visual colorimetric analysis6-11. However, the absorbance signal of these systems is
directly proportional to the concentration of analytes, resulting in a relatively low
sensitivity and narrow dynamic range12, 13. In order to increase the colorimetric response
to an analyte, exponential amplification methods have been utilized, including
polymerase chain reaction (PCR) and dendritic chain reaction (DCR)13-16. Nevertheless,
the reported exponential amplification protocols rely on enzymes or auto-inductive
molecular receptors which require tedious and laborious multiple organic preparation
procedure.
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On the other hand, the reported exponential amplification methods are limited to the
detection of fluoride ions, hydrogen peroxide, Pd(II), and nerve agents17-23. Other
analytes such as acetone is not realized. As a common organic solvent, acetone is highly
volatile, and it may damage human brain, kidney, and liver24. Traditional analytical
techniques for detection of acetone, such as electrochemistry, chromatography, mass
spectroscopy, are expensive, time-consuming or high detection temperature25. On the
contrary, visual colorimetric methods are facile, rapid and cost-effective. Accordingly,
it is vital to develop reliable, highly selective and sensitive colorimetric assays for
acetone.
In this work, we propose a cascade chromogenic system based on coupling chemical
oxidation with photo-induced radical chain reaction for highly sensitive and selective
detection of acetone. Figure 1 shows the principle of the colorimetric assay for acetone.
The oxidation-sensitive o-phenylenediamine (OPD) is selected as the chromogenic
substrate. When the ferric ion (Fe3+) as oxidant is introduced to the OPD solution, the
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redox reaction between Fe3+ and OPD occurs to yield Fe2+ and 2,3-diaminophenazine
(DAP) (Figure 1a).
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Figure 1. Schematic illustration of the cascade chromogenic system based on coupling
chemical oxidation with photo-induced radical chain reaction for visual colorimetric
detection of acetone.
Under blue light irradiation, DAP is excited after absorption of photons. The
electrons are transited to higher-energy singlet orbitals (S1) that are further transited to
lower-energy triplet excited states (T1) via an intersystem crossing (ISC) process due
to the presence of a small singlet-triplet energy gap (△EST), along with the formation
of the holes (Figure 1a). After that, the excited electrons react with dissolved oxygen to
produce H2O2 in an acidic medium. Subsequently, the Fenton reaction of Fe2+ and H2O2
proceeds to yield hydroxyl radicals (.OH) and Fe3+ (Figure 1a). Moreover, the holes can
.
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directly oxidize water to generate OH as well. The re-generated Fe3+ and OH can
oxidize OPD to create more DAP. The oxidation process of OPD progresses
exponentially until all of the OPD are oxidized under an ideal condition. The cascade
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chromogenic system is able to be monitored by the UV-vis absorption spectra of yellow
DAP solution. In contrast, when acetone is added to the cascade chromogenic system,
the complex of DAP and acetone with a molar ratio of 1:1 is engendered (Figure 1b).
The resulting molecular complex gives a large increase in the value of △EST, which
restrains ISC process and the photo-induced radical chain reaction and thus less DAP
is produced. Through the change of the UV-vis absorption spectra of this chromogenic
system, visual and quantitative analysis of acetone can be performed in water.
Meanwhile, the colorimetric assay is further applied to analyze diluted real water
samples.
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EXPERIMENTAL SECTION
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Materials.OPD, ferrous(III) chloride hexahydrate (FeCl3 6H2O), ferrous chloride
.
tetrahydrate (FeCl2 4H2O), acetone, 1,10-phenanthroline, sulphuric acid (H2SO4), and
methanol were purchased from Aladdin Chemical Reagent Co., Ltd (Shanghai, China).
5,5-dimethyl-1-pyrroline N-oxide (DMPO), sodium hydroxide (NaOH), ethanol,
isopropanol, glycerol, ethyl acetate, ethylenediamine, dimethyl sulfoxide, methyl
methacrylate, N-methyl pyrrolidone, diethylene glycol, ethylene acetate, sodium
carbonate (Na2CO3), and other inorganic salts were obtained from ChengDu KeLong
Chemical Co., Ltd (Chendu, China). All the reagents were of analytical grade and used
as received. Deionized water was applied to prepare various solutions.
Procedure for Monitoring the Chromogenic System. Briefly, 0.3 mL of 1 mM
OPD aqueous solution was added to a 0.25 mL of 0.6 mM FeCl3 solution. Subsequently,
the pH of the mixture solution was adjusted to 4.5 by using 0.1 M H2SO4. Then, the
resulting solution was further diluted with deionized water, and a final reaction volume
was maintained at 3 mL, followed by illumination with blue light-emitting diode (LED,
12 W) for 15 min at room temperature (approximatively 25°C ). The UV-vis absorption
spectra were acquired by a UV-vis photometer, and the corresponding optical images
were collected by a digital camera. In order to evaluate the related reaction conditions,
including pH, Fe3+ concentration, and LEDs of various colors, a similar experimental
operation was carried out.
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Procedure for Visual Colorimetric Determination of Acetone. To a 5 mL of glass
bottle, 0.3 mL of 1 mM OPD aqueous solution, 0.25 mL of 0.6 mM Fe3+, and 0.3 mL
acetone with different concentrations were sequentially added; the pH of this mixture
was adjusted to 4.5, followed by dilution to 3 mL with deionized water. After
illumination with blue light-LED for 15 min at room temperature, the UV-vis
absorption spectra of the mixture solution and optical images were collected. In order
to investigate the selectivity of this colorimetric assay, acetone was replaced by other
common solvents, and the same experimental operations were conducted.
Procedure for Acetone in Spiked Real Water. Three types of real water such as
lake water, river water, and pond water were collected from Mianyang (China). These
real water samples were filtered and diluted 50 times with deionized water. Then, the
diluted water samples were spiked with 0.05, 0.4, and 2 mM of acetone, and the spiked
samples were determined by the present colorimetric assay.
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Characterization. Ultraviolet-visible (UV-vis) absorption spectra were acquired by
an UV-1800 spectrophotometer. Electron paramagnetic resonance (EPR) spectra were
performed at 298 K on a Bruker I200 EPR spectrometer equipped with a blue-light LED.
Mass spectra were collected from a Q-Exactive high-resolution mass spectrometer.
Fluorescence measurements were performed with the F-7000 fluorescent
spectrophotometer.
RESULTS AND DISCUSSION
Exponential Amplification Chromogenic System. In order to examine the cascade
chromogenic system based on coupling chemical oxidation with photo-induced radical
chain reaction, 0.1 mM OPD solution is incubated with 50 μM Fe3+ under blue-light
LED illumination for 15 min. As we expected, the chromogenic system with a yellow
color shows a strong optical absorption in the visible region, and the
maximum absorption peak is found at 446 nm (Figure 2a, curve iv), which is the
characteristic absorption band of DAP26-29. Moreover, the production of DAP is further
demonstrated by mass spectroscopy, and its molecular ion peak at 211. 0972 is clearly
seen in the Figure S1. In contrast, the mixture of 50 μM Fe3+ and 0.1 mM OPD without
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blue-light LED illumination displays a weak absorption (Figure 2a, curve iii),
suggesting that the chemical oxidation reaction between Fe3+ and OPD can not
effectively generate optical absorption because of the lack of enough DAP molecules
in the solution.
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Figure 2. OPD oxidation-involved chromogenic reaction. (a) UV-vis absorption
spectra of 0.1 mM OPD solution under different reaction conditions: i) in the dark, ii)
blue-light LED illumination for 15 min, iii) reaction with 0.05 mM Fe3+ in the dark and
iv) under blue-light LED illumination. (b) Time-dependent UV-vis absorption spectra
for the chromogenic system. (c) Exponential relationship between reaction time and
absorbance values of the chromogenic system at 446 nm. (d) Effect of several metal
ions on the chromogenic system. The concentration of all the metal ions is 50 μM.
Similarly, direct blue-light illumination of OPD solution at a low concentration (0.1
mM OPD) is unable to induce the chromogenic phenomenon (Figure 2a, curve ii). The
corresponding absorbance value of the cascade chromogenic system at 446 nm is 5.9
times and 13 times higher than those of Fe3+-triggered chemical oxidation and direct
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blue light-induced oxidation of OPD, respectively. It should be noted that when the
OPD concentration is higher than 5 mM, direct blue-light illumination can induce the
OPD oxidation because partial OPD is oxidized by dissolved oxygen to yield DAP that
autocatalytically oxidize OPD30.
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To confirm the exponential amplification of colorimetric signal, the kinetic curves of
the chromogenic system are collected as shown in Figure 2b. It can be seen that the
DAP is formed with an exponentially increasing rate under illumination with blue light.
The absorbance data in Figure 2c fit well to the exponential progress curve,
demonstrating the exponential signal behavior of this chromogenic system. In addition,
some experimental conditions such as pH and Fe3+ concentration are capable of
influencing this chromogenic system. As shown in Figure S2, the optimized pH is found
to be 4.5. The reason is that Fe3+ is easily hydrolyzed at a high pH, and OPD will be
completely protonated under a strong acidic medium, which is adverse to the
photochemical oxidation of OPD. Figure S3 shows the effect of Fe3+ concentration on
the absorbance value of this chromogenic reaction. When the concentration of Fe3+ is
more than 50 μM, the absorbance tends to reach a saturation value. Therefore, 50 μM
Fe3+ is utilized in the exponential amplification chromogenic system. Except for Fe3+,
other common metal ions such as Cd2+, Co2+, Cu2+, Mg2+, Mn2+, Ni2+, K+, Na+, Zn2+,
and Ce3+ can not exhibit an obvious signal enhancement (Figure 2d). Although Ce3+
has a certain capacity for oxidation of OPD, the reduction product (Ce2+) may not be
able to initiate the Fenton reaction. Accordingly, the cascade reaction between Fe3+-
induced chemical oxidation and photo-induced radical chain produces an exponential
amplification chromogenic system.
Identification of Chromogenic Reaction Mechanism. To thoroughly identify the
reaction mechanism of the cascade chromogenic system, we firstly illuminate the
mixture of Fe3+ and OPD with red, green, and blue LED. We find the generation of the
strong absorbance signal only under blue-light illumination (λmax= 454 nm) (Figure 3a
and Figure S4), which overlaps well with the absorption of DAP (λmax= 446 nm). To
confirm the fact that DAP can be photo-excited, the fluorescent emission spectra of
DAP solution is collected with a 445 nm-excitation wavelength. A significant emission
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peak is observed at 564 nm (Figure S5), demonstrating that the excited-state DAP
(DAP*) will be created under blue-light LED illumination.
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Figure 3. Reaction mechanism. (a) UV-vis absorption spectra of the exponential
amplification chromogenic system under irritation with different colors of LED for 15
min. The light power density of blue, green, and red LED is about 48ꢀmWꢁcm−2. (b)
Effect of reaction atmosphere on the chromogenic system. (c) Electron paramagnetic
resonance spectra of DMPO and the mixture of DMPO, Fe3+ and OPD under irradiation
with blue-light LED. (d) UV-vis absorption spectra of 0.5 mM 1,10-phenanthroline and
the mixture of 0.5 mM 1,10-phenanthroline (phen) and OPD in the absence and
presence of 0.4 mM Fe3+. The inset shows the coordination reaction of phen with Fe2+.
According to the reaction principle of the cascade chromogenic system (Figure 1),
the production of H2O2 as an important transient intermediate is directly dependent on
the concentration of dissolved oxygen. To confirm this reaction pathway, the role of
dissolved oxygen is examined. When the nitrogen gas is bubbled into the chromogenic
system, the absorbance value at 446 nm remarkably decreases up to 92% (Figure 3b),
indirectly support the formation of transient H2O2.
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After affirming the role of dissolve oxygen, we then investigate the reactive oxygen
species (ROS) with ROS scavengers, including ascorbic acid for all the ROS31, thiourea,
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NaN3 as well as Na2CO3 that are served as scavengers of OH, O2, and O2
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respectively (Figure S6-Figure S9). Only ascorbic acid and thiourea are in a position to
quench the cascade chromogenic reaction. Besides, the type of ROS is also studied by
EPR spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the ROS probe.
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The characteristic quadruplet peak from the spin-adduct of DMPO and OH with a
1:2:2:1 intensity is obtained as illustrated in Figure 3c. These results clearly
demonstrate that the .OH is the key intermediate in the cascade chromogenic system36.
Additionally, the existence of Fe2+ is verified by introducing 1,10-phenanthroline (phen)
as the ligand for Fe2+. As indicated in Figure 3d, there is an absorption peak at 510 nm,
which is attributed to the absorption feature of Fe(phen)32+ complex37, 38. Based on these
results, the chromogenic reaction mechanism in Figure 1 is well certified.
Acetone-Response Behavior and Visual Quantitative Detection of Acetone. Next,
the acetone-response behavior of this cascade chromogenic system is further explored.
As displayed in Figure 4a, the addition of acetone results in a distinct quenching for the
absorbance in the visible region, and only background signal from the optical
absorption of Fe3+ is gained, accompanying a color change from yellow to colorless. In
order to appraise the reaction product in the presence of acetone, a high-resolution mass
spectrometer is conducted. The molecular ion peak at 269.13876 is ascribed to the
complex of DAP and acetone with a molar ratio of 1:1 (Figure S10), revealing that the
generation of DAP . acetone complex inhibits the chromogenic reaction. Furthermore,
the interaction of DAP and acetone in the complex is calculated according to the
B3LYP with 6-31G basis set based on the time-dependent density functional theory
(TD-DFT) approach, which is analyzed by Multiwfn software39. The calculated
interaction force between DAP and acetone is mainly consisting of hydrogen bonds and
Van der Waals interaction (Figure 4b).
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Figure 4. (a) Analytical performance for detection of acetone. UV-vis absorption
spectra and the corresponding optical images of the exponential amplification
chromogenic system in the absence and presence of 13 mM acetone. (b) Calculated
interaction force between DAP and acetone in the complex of DAP . acetone. (c) Linear
calibration curve for colorimetric detection of acetone. The inset shows the optical
images of chromogenic system upon addition of different concentrations of acetone. (d)
Selectivity evaluation for acetone detection. The concentration of acetone and all other
common solvents is kept at 3 mM.
From the above-mentioned chromogenic reaction mechanism, the ISC process from
.
S1 to T1 is the key step in the production of OH and Fe3+ cycle, and it is largely
influenced by △EST. Generally, a small △EST value is favorable for the ISC process40-
42. By calculating the energy levels of the singlet and triplet states, we can assess the
△EST values, namely, 2.76 eV and 1.96 eV for DAP. acetone complex and DAP (Figure
S11), respectively. Such an increase of △EST by approximately 41% should be
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associated with the interaction between acetone and DAP, suppressing the ISC process
and therefore leading to an extremely inefficient .OH formation.
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Benefiting from its acetone-response behavior, the cascade chromogenic system is
applied for visual colorimetric detection of acetone. Varying concentrations of acetone
from 0 to 3 mM were injected into the cascade chromogenic system, and the UV-vis
absorption spectra are recorded at 15 min under blue-light LED illumination (Figure
S12). With the increase of acetone concentration, the optical absorption of this cascade
chromogenic system becomes gradually weak. A linear calibration curve is acquired
from the absorbance values at 446 nm as a function of acetone concentration in the
range of 50 μM-3 mM (Figure 4c). The limit of detection (LOD) is calculated to be 35
μM based on 3 σ/k, where σ and k are the standard deviation of the absorbance without
acetone and slope of the linear calibration curve, respectively. This LOD is lower two
to three order of magnitudes than that of the reported fluorescent assays43-48. The high
sensitivity of this colorimetric assay is ascribed to the fact that the production of .OH
with high reaction activity is obviously restrained when the 1:1 DAP . acetone complex
is generated (Figure S11). Fascinatingly more, the presence of acetone at a
concentration of 1 mM can be observed with the naked eye due to the distinct color
fading (the inset of Figure 4c). Likewise, the selectivity of this colorimetric assay is
estimated with common solvents each at a concentration of 3 mM. It is clear that only
acetone leads to a notable absorbance quenching, and colorimetric signal produced by
other solvents is close to the background signal (Figure 4d), illustrating a good
selectivity of the present colorimetric method in differentiating acetone from non-target
analytes. The generation of 1:1 DAP . acetone complex with unique energy levels are
responsible for the high selectivity of the present system.
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Finally, to demonstrate its practical potential, the performance of this colorimetric
assay in diluted real water samples is also tested. The 50-fold diluted real water samples,
including lake water, pond water, and river water, are spiked with acetone at different
concentrations (50 μM, 0.4 mM, and 2 mM). The spiked samples are analyzed by the
present colorimetric assay, and the results are listed in Table 1. The scope of the
recoveries for real water samples is from 90.6% to 100.7%, and the calculated relative
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standard deviation is not more than 9%. The three concentrations of acetone are lower
than the maximum limit level for environmental water set by U.S. Environmental
Protection Agency (3.79 mM)49. These results prove the present colorimetric assay as
a potential analytical technique for practical environmental monitoring. It should also
be noted that this colorimetric assay can not be applied for direct detection of acetone
in highly polluted water because the organic pollutants can not only influence the
dissolved oxygen concentration, but also consume the .OH. Some sample pretreatment
techniques have to be performed before colorimetric detection of acetone using this
colorimetric assay.
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Table 1. Colorimetric detection results of acetone in real water samples
Samples
Added (mM)
Found (mM)
0.048
0.38
Recovery (%)
96.1
RSD (%)
2.7
0.05
0.4
2
94.6
8.1
Lake water
2.01
100.7
94.9
5.1
0.05
0.4
2
0.047
0.37
1.8
93.1
3
Pond water
River water
1.99
99.3
7.2
0.05
0.4
2
0.049
0.39
98.5
2.6
99
2.8
1.81
90.6
5.7
CONCLUSIONS
In conclusion, we have developed a cascade chromogenic system based on coupling
chemical oxidation with photo-induced radical chain reaction. The high efficiency of
Fe3+-induced chemical oxidation and photo-induced Fenton-like reactions trigger an
amplification process and generates an exponential colorimetric signal growth. By
relying on the complex formation of DAP and acetone, this cascade chromogenic
system is successfully utilized to visually determine acetone with a good selectivity
over ten common solvents. The obtained sensitivity is two to three order of magnitudes
better than that of the reported fluorescent assays. Of special note, the present
colorimetric assay can be performed even in diluted real water samples. The resulting
cascade chromogenic system features exceptional merits such as good accuracy, low
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cost, easy operation, high portability, inexpensive instrumentation as well as high
controllability, without the participation of enzymes or auto-inductive molecular
receptors, which offers great prospects as a simple and reliable analytical method for
in-field environmental monitoring. Meanwhile, by further coupling this cascade system
with specific reactions or Fe3+-involved nanomaterials, in principle, it is most likely to
extend this chromogenic system to detect various desired analytes.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website
at DOI:.
Calculation details, Mass spectrum, fluorescent emission spectrum, UV-vis absorption
spectra and Energy level diagram (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: yhe2014@126.com
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
This work is supported by the Longshan Scholars Programme of Southwest University
of Science and Technology (18LZX204) and China Aerodynamics Research and
Development Center Foundation (Grant No. JPD20170142).
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