Bio-inspired enol-degradation for multipurpose oxygen sensing

Article abstract of DOI:10.1039/c4cc05831c

Inspired by the enol-degradation of luciferin, a new oxygen sensor with oppositely changed color and fluorescence has been designed. This new reaction-based dual mode sensor can not only be used as a highly selective instant "fluorescence on" oxygen probe, but also as a freshness indicator of food or food materials by using its property of time-adjustable color fading.

Full text of DOI:10.1039/c4cc05831c

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Bio-inspired enol-degradation for multipurpose
oxygen sensing†
Cite this: Chem. Commun., 2014,
0, 13477
5
Yu-Mo Zhang, Xiaojun Wang, Wen Li, Weiran Zhang, Minjie Li* and
Sean Xiao-An Zhang*
Received 27th July 2014,
Accepted 10th September 2014
DOI: 10.1039/c4cc05831c
www.rsc.org/chemcomm
Inspired by the enol-degradation of luciferin, a new oxygen sensor recently is a luciferin based highly sensitive disposable sensor with
with oppositely changed color and fluorescence has been designed. ‘‘turn on’’ luminescence for sensing oxygen or ATP in the presence
13,14
This new reaction-based dual mode sensor can not only be used as of luciferase.
Since luciferase is too unstable in vitro, stability
a highly selective instant ‘‘fluorescence on’’ oxygen probe, but also as of the luciferin–luciferase-mimic sensing system remains to be a
a freshness indicator of food or food materials by using its property technological challenge. Currently, there is no good way to chemi-
of time-adjustable color fading.
cally mimic the entire process of luciferin shining for oxygen
sensing as in a firefly, therefore, a highly sensitive disposable
Molecular oxygen (O
2
) is of fundamental importance for biological chemical sensor partially mimicking the bio-process might be a
1
systems and human activities. Precise sensing of O is required good choice. Herein, we present a new chemical approach of oxygen
2
2
3
in many fields, such as medicine, environmental analysis,
sensing based on a process similar to enol-degradation inspired by
4
industry, etc. Even though many admirable technologies, such luciferin. By this method, we have developed an unusual oxygen
5
6
7
8
as titration, colorimetry, electrochemistry, and luminescence,
have been developed so far to meet various O sensing needs, time, good selectivity, and an attractive prospect to meet various
intensive research has been performed for decades on redox- oxygen sensing needs.
sensor with instant fluorescence ‘‘turn-on’’, adjustable color fading
2
based fluorescent/phosphorescent O
sensitivity than dye-based color sensors. However, most of the O
2
sensors due to their better
This entire work is based on an accepted assumption that
the key function of luciferase in the bio-degradation of luciferin
9
2
sensors so far are ‘‘turn-off’’ type with the intensity of the is a-H extraction to facilitate the enolization of a luciferin–AMP
15
fluorescence/phosphorescence fading away, and the ‘‘turn-on’’ affixture, and the resulting enolate is very vulnerable to oxygen
type sensors are very rare but highly preferred due to their far attack, which subsequently leads to oxyluciferin as a product of enol-
10
better visual sensitivity. Recently, Shin et al. reported a very good degradation and further results in bioluminescence (Scheme 1a).
example of a fluorescence ‘‘turn-on’’ oxygen sensor based on a The enol-degradation process has also been speculated to be a
10
perylene diimide molecule in the reduced form. The sensor can major reaction involved in many biological processes, such as food
16
be reused several times after being reactivated electrochemically. digestion, etc. We envision that a-H extraction function of the
The reversibility of a sensor is an important property to ensure its enzyme could be replaced by a chemical base, and a pre-fluorescent
repeatable use, but a reliable process to quickly and conveniently
restore the activity of the sensor remains to be a great challenge in
11
the field. Thus, some cheap disposable sensors are also desir-
able to avoid inconvenient processes of reactivation or a waste of
6,12
not fully utilized reversible functions in many situations.
Inspired by amazing processes in Nature, several fascinating
biomimetic systems have been developed to develop turn-on
type of oxygen sensors. A successful example that appeared
State Key Laboratory of Supramolecular Structure and Materials, College of
Chemistry, Jilin University, 2699 Qianjin Street, Changchun, Jilin 130012,
P.R. China. E-mail: seanzhang@jlu.edu.cn, liminjie@jlu.edu.cn
†
Electronic supplementary information (ESI) available. CCDC 987215. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c4cc05831c
Scheme 1 Illustration of the design of oxygen sensing by bridging two
aromatic motifs with a methyl ketone group, and the color switch mechanism.
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molecule could substitute for the pre-luminous luciferin
(Scheme 1b). If we can chemically mimic the ‘‘enol-degradation’’
process to create a change in color and fluorescence, the scientific
insights obtained from this bio-inspired reaction could not only
lead to a new class of oxygen sensor, but also help to verify and
better understand the hidden mechanism of bio-degradation of
luciferin and many other biological processes.
In order to verify our assumptions, we designed a new methyl
1
7
ketone TM1 which is based on our previous work and a conjecture
Fig. 2 Fluorescence spectra (a) and absorption spectra (b) of TM1-enolate
À5
that this new nitro-containing methyl ketone molecule with a large solution (1.0 Â 10 M, acetonitrile solution, 30 eq. of t-BuOK, 25 1C, stirring)
conjugate group (or its enolate) should exhibit weak or no fluores- before and after exposure to oxygen.
cence due to some fatal nonradiative transitions of the nitro
(photon-induced electron transfer) and dangling methyl ketone
functional groups, and a strong fluorescence could be observed t-BuOK and then acidified. In addition, when the time of treat-
after removal of the nitro and dangling methyl ketone functional ment with oxygen was shortened to 5 min, nitrobenzaldehyde
18
groups by enol-degradation. We anticipate that the degradation (20%) was also isolated from the reaction. In contrast, no degra-
could occur spontaneously at a suitable temperature upon exposure dation product was detected and isolated when the same reaction
to oxygen after the molecule being treated with base. Two more solution of TM3 was treated with an inert gas (nitrogen). These
molecules (TM2 and TM3) were designed and synthesized to help results demonstrate clearly that oxygen is essential for the degra-
verify the assumption and prove the reaction mechanism (Fig. 1). dation of this methyl ketone molecule under alkaline conditions,
Their base induced enolization properties are evaluated first.
and 4-nitrobenzaldehyde and benzoate are the initial degradation
To our delight and as per our expectations, the ketone–enol products. 4-nitrobenzoic acid is presumably a further oxidation
transition of TM1 was observed in acetonitrile (Fig. S1, ESI†). product of the 4-nitrobenzaldehyde. Furthermore, a major peak
Upon addition of base to the solution of TM1, a new absorption at 674.3917 in HRMS spectrum (Fig. S4, ESI†), which is identical
peak at lmax = 585 nm was observed immediately with a notice- with the theoretical molecular weight of TMX (674.3918), is detected
able change from colorless (lmax = 376 nm) to reddish-cyan. The from the oxygen treated TM1-enolate solution. This is another strong
enolate of TM1 can be easily reverted to its original methyl evidence to further prove formation of the degradation product TMX
ketone structure by acid.
during the oxidation process. All the results prompted us to
The anticipated enol-degradation of TM1 was explored next. propose a possible mechanism to explain the entire reactions.
Much to our delight, a showy blue-fluorescence turns on instantly That is, as depicted in Fig. S3, ESI,† first the TM1-enolate is
after the non-fluorescent TM1-enolate was exposed to air at 25 1C, generated, then electrophilic oxygen reacts with the TM1-enolate
which indicates that the response time of the oxygen sensor is forming a highly unstable intermediate with a possible structure
1
8
instant. As shown in Fig. 2a, a unique emission peak emerges at of a four-membered ring, which subsequently decomposes into
50 nm and its intensity increases with time when the solution of thermodynamically more favorable 4-nitrobenzaldehyde and a
TM1-enolate is exposed to oxygen. And the intensity of its visible substituted fluorenylcarboxylate.
color with absorption peak at 585 nm decreases synchronously To evaluate its O probing sensitivity, electrochemical properties
Fig. 2b). Compared with blue-green fluorescent TM2 (Fig. S2, of the molecule were examined by using cyclic voltammetry.
4
2
(
ESI†), the oppositely changed color and emission in the solution TM1-enolate shows a very low oxidation potential (0.23 V vs. NHE,
of TM1-enolate is presumably due to an enol-degradation reaction, Fig. S5, ESI†), which indicates that it is even more sensitive than the
10
which subsequently yields a fluorescent product (TMX, as shown in latest ‘‘turn-on’’ probes that use a perylene diimide molecule. The
Fig. S3, ESI,† fluorescence lifetime, t E 1. 6 ns). turn-on response of TM1-enolate is highly selective towards oxygen.
To experimentally simplify further investigation of the reaction As shown in Fig. 3a, almost no response was observed under N
mechanism, 2-(4-nitrophenyl)-1-phenylethanone (TM3) was selected CO , H , NO , SO and NO atmospheres, and the fluorescence was
2
,
2
2
2
2
as a proof-of-principle. Both benzoic acid (60%) and 4-nitrobenzoic observed only under an oxygen environment. Most importantly, the
acid (70%) were isolated after the enol-degradation reaction in which fluorescence intensities of TM1-enolate were found in linear propor-
TM3 was first treated with oxygen in a solution containing 40 eq. of tional to the concentration of oxygen in the range of 0–100% at the
same detection time (Fig. 3b). The linear relationship between the
intensity of its fluorescence and the oxygen concentration reveals its
good potential for accurate oxygen sensing.
In order to make this new sensing method more convenient for
practical applications, a prototype of the sensor strip was developed
19
by integrating TM1 and an alkaline substrate into paper. Side-by-
side testing of the sensor strip under air or nitrogen indicates that
the originally non-fluorescent testing strip shows a beautiful blue
fluorescence instantly in air, and its fluorescence gets brighter and
brighter over time until it reaches a constant intensity (Fig. 3d).
Fig. 1 Structures of the ketone and enolate forms of TM1 and the model
molecules TM2 and TM3.
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individual sensing regions to independently and sequentially
detect oxygen (Fig. S9, ESI†).
As regards to potential application of this reaction based
oxygen sensing, we can clearly foresee that this very attractive
dual-mode feature will enable us to use the different modes for
different applications, which is usually lacking in many existing
oxygen sensors. Apart from using the unique property of instant
fluorescence for highly sensitive oxygen sensing, as a proof-of-
principle, we demonstrated another feasible use of the sensing
strip for monitoring the food freshness by taking advantage of
its another attractive property of time-adjustable color fading in
1
1
intelligent packaging. In daily life, the freshness of meat
cannot be distinguished normally when it is stored in a sealed
bag. In addition, both the quality guarantee period of fresh meat
and the fading rate of our visual testing strip are dependent on
1
1
À5
the oxygen concentration under the same testing conditions.
Fig. 3 (a) Fluorescence response of TM1-enolate (1.0 Â 10 M, 30 eq. of
t-BuOK, 25 1C, stirring) to various common environmental interferents Thus, it is easy to monitor the freshness of the meat visually,
(
CO
2
, N
2
, NO, NO
2
, SO
2
, H
2
, O
2
) in acetonitrile solution. (b) Fluorescence based on the correlation between the freshness of the meat and
À5
intensity changes in TM1 (1.0 Â 10 M, acetonitrile solution, 30 eq. of
the color fading degree of the testing strips. When the O testing
2
t-BuOK, 25 1C, stirring) with the concentration of oxygen recorded at
strips as freshness indicators are isolated and sealed with meat
in the same packet, customers or store-workers can easily deter-
À5
1
min. (c) Absorbance (at 585 nm) changes in TM1 (1.0 Â 10
M,
acetonitrile solution, 30 eq. of t-BuOK, 25 1C) in the presence of different
concentrations of oxygen (0%, 21%, 50%, 75%, 100%). The curves fitted mine the quality and freshness of the meat by observing the
with one-order reaction dynamics ln A = Àkt to calculate the degradation color of the strip against the reference provided in the package
À2
À1
À2
À1
À1
reaction rate constant: k = 4.25 Â 10 min (100%), k = 1.14 Â 10 min
(
as shown in Fig. 4a and b).
In summary, we have, for the first time, developed a novel O
À3
À1
À3
À1
(
(
75%), k = 6.43 Â 10 min (50%), k = 2.36 Â 10 min (21%), k = 0 min
2
0%). And photographs of testing paper strips showing changes in fluores-
sensor with good selectivity by bio-mimicking the enol-degradation
process. The fluorescence ‘‘turn-on’’ and color-fading dual-mode
feature of our sensor facilitates both instant (fluorescence mode)
and long-time tracking (color mode) of the oxygen concentration.
cence emission before and after 1 min (d) 21% oxygen in air; (e) 0% oxygen in
the glove box under 365 nm UV light.
In contrast, no fluorescence can be observed when the testing The sensing precursor of TM1 is stable in air, and a ketone–enol
strip is placed in a nitrogen atmosphere for the same testing isomerization, which is easily triggered by chemical base, is a
period (Fig. 3e). Preliminary results demonstrate that this new perquisite for the oxidative degradation. Therefore, such a two-step
testing strip for oxygen sensing is highly efficient with good reaction will avoid the storage problem that is usually associated
potential as a commercial portable and disposable testing strip. with other reduced dyes, and will make the sensor more practical for
In addition to its attractive property of instant fluorescence, applications. In addition, the underlying reaction mechanism was
the reddish-cyan color of TM1-enolate obviously decreases after fully proved by spectroscopy studies. Scientific insights obtained
exposure to oxygen. As illustrated in Fig. 3c and Fig. S6, ESI,† from this research will not only stimulate and accelerate further
the color fading rate of TM1-enolate in solution increased development of a new class of outstanding oxygen sensors, but will
with either the temperature or the concentration of oxygen. also help to verify and better understand the hidden mechanism of
À1
In addition, its reaction rate can be tuned from 0 min to bio-degradation of luciferin and many other biological processes,
À2
À1
4
.25 Â 10 min at 25 1C, and the relationship between the and help to better understand how the Nature works, which would
oxygen concentration and the color disappearing time of be fundamentally very important to science.
the TM1-enolate solution can be described by the function
This work was supported by the National Science Founda-
y = 233.4 À 27.7 ln(t), where y is the oxygen concentration at tion of China (Grant No. 51373068).
2
0
2
5 1C (Fig. S7, ESI†). The color fading rate of the sensing strip
was also found to be inversely related with the surrounding
vacuum level. At a fixed concentration of TM1 in the strip, the
color disappearing time of the strip varied from several hours to
several days depending on the vacuum level (Fig. S8, ESI†). Even
though this sensor is very sensitive with a very low oxidation
potential, for a strip placed in the environment with a fixed
oxygen content, the fading time of the sensor could be as short
as several minutes or as long as several days by simply adjust-
ing either the concentration of TM1-enolate or/and the oxygen
diffusion rate in the testing strip. More importantly, a reusable
Fig. 4 (a) A photograph of reference strips for judging freshness of meat.
b) Photographs of freshness monitoring TM1 visual strips before and after
(
sensing strip can be achieved when one strip contains many the expiry date of fresh meat.
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