Diverse applications of TMB-based sensing probes

Article abstract of DOI:10.1039/c8ob01364k

Extending the research on 3,3′,5,5′-tetramethylbenzidine (TMB) and its derivatives in analytical chemistry is important, considering that TMB is widely used as an enzyme catalytic substrate. In this work, two TMB derivatives, TMBS and TMBB, were synthesized via a facile and one-step condensation reaction between the -NH₂ group of TMB and the -CHO group of salicylaldehyde or benzaldehyde. Because at low pH the two Schiff base compounds can release TMB which can emit strong fluorescence, the probes could show dual-modal signal responses, fluorescence and UV-vis absorption, towards the pH. Practical applications of pH sensing in Chinese rice vinegar and lemon juice samples were successfully demonstrated. On the basis of these findings, a catalytic chromogenic reaction was developed to monitor the pH with the naked eye, too. Furthermore, considering the chemical equilibrium reaction between CO₂ and H₂O and that glucose oxidase (GOD) can catalyse the dehydrogenation and oxidation reaction of β-d-glucose to produce gluconic acid, both of which can result in lowering the pH values of the two Schiff base systems, highly sensitive and selective dual-modal sensing systems for detecting CO₂ and β-d-glucose have also been successfully established. Therefore, the two synthesized TMB derivatives can demonstrate their robust application potential.

Full text of DOI:10.1039/c8ob01364k

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Diverse applications of TMB-based sensing probes
Li Huang,a,b Ying-Juan Cao,a,b Xiang-Ying Sun,a,b Bin Liu, a,b and Jiang-Shan Shen*a,b
Received 00th January 20xx,
Accepted 00th January 20xx
Extending the research of 3,3',5,5'-tetramethylbenzidine (TMB) and its derivatives in analytical chemistry is important,
considering that TMB is widely used as the enzyme catalytic substrate. In this work, two TMB derivatives, TMBS and
TMBB, were synthesized via the facile and one-step condensation reaction between –NH2 group of TMB and –CHO group
of salicylaldehyde or benzaldehyde. Because under low pH the two Schiff base compounds can release TMB which can emit strong
fluorescence, the probes could show dual-modal signal responses, fluorescence and UV-vis absorption, towards pH. Practical
applications of pH sensing in Chinese rice vinegar and lemon juice samples were successfully demonstrated. On the basis of these
findings, a catalytic chromogenic reaction was developed to indicate pH by naked eyes, too. Furthermore, considering the chemical
equilibrium reaction between CO2 and H2O and that glucose oxidase (GOD) can catalyse the dehydrogenation and oxidation
reaction of β-D-glucose to produce gluconic acid, both which can result in lowering the pH values of the two Schiff base systems,
highly sensitive and selective dual-modal sensing systems of detecting CO2 and β-D-glucose have also been successfully established.
Therefore, the two synthesized TMB derivatives can demonstrate their robust application potential.
DOI: 10.1039/x0xx00000x
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at 450 nm, which originates from the fact that the imine-
containing compound could further be oxidized if excess H2O2
or strong acid is introduced.9, 10 Therefore, on the basis of the
Introduction
3,3',5,5'-Tetramethylbenzidine (TMB) is one of traditional
enzyme substrates, which has been widely used in the fields of
artificial enzyme mimics and natural enzymes.1-5 TMB has a
unique and symmetric molecular structure which bears two -
NH2 groups. Original TMB is a colorless compound, yet it can
produce stable and dramatic color change which is highly
sensitive to the human’s eye, when TMB is oxidized by some
oxidants in the presence of certain natural enzymes or artificial
enzyme mimics. However, even if some strong oxidants such
as H2O2 are introduced to TMB solution in the absence of any
catalysts, the oxidation reaction rate is very slow. Therefore,
the catalytic chromogenic reaction of TMB can provide very
low spectral background which is beneficial to develop spectral
sensing systems with high sensitivity. In the presence of H2O2
and certain peroxidases such as horseradish peroxidase, TMB
can be oxidized to become one-electron oxidized or even two-
electron oxidized compound.6-8 The one-electron oxidized
compound is a blue complexing product due to the charge
transfer between original TMB molecule bearing two -NH2
groups and the oxidized product bearing two imine groups,
which shows maximum UV-vis absorption wavelength at 370
nm and 652 nm. The two-electron oxidized compound is a
yellow product with maximum UV-vis absorption wavelength
catalytic chromogenic reactions of TMB, numerous natural
peroxidase/artificial peroxidase mimic-based spectral sensing
systems have been successfully established to detect a series
of targets including heavy metal ions,11-13 biomolecules,14-16
and environmental pollutants.17 In recent years, our group
developed several highly sensitive catalytic spectral sensing
systems to probe amino acids18, 19 and formaldehyde20 by using
TMB as the catalytic substrate, too.
Considering that TMB bears two -NH2 groups, it can be
easily modified by integrating some simple functional groups
onto its skeleton to prepare novel fluorogenic or chromogenic
probes, which are expected to extend the analytical
application scope of TMB. However, hitherto only several
TMB-based fluorogenic or chromogenic probes were
developed. For example, a novel fluorescent probe, 3,3’,5,5’-
tetramethyl-N-(9-anthrylmethyl) benzidine, was prepared via
the nucleophilic substituting reaction between TMB and 9-
chloromethyl- anthracene. This probe bearing two interacting
sites and two fluorescent reporters demonstrated dual
fluorescence responses towards H+ and aliphatic aldehydes,
respectively.21 Another fluorophore, N, N-dibenzyl-3,3′,5,5′-
tetramethylbenzidine, was prepared by using TMB as the reactant
and was shown to have significant fluorescence response towards
2,4-dinitrophenol or 2,4,6-trinitrophenol, when the probe was
immobilized in a plasticized poly(vinyl chloride) membrane.22
On the other hand, pH plays important roles in biological
systems and environmental sciences.23-28 For example, pH is
believed as a fundamental parameter for synthesizing energy
molecule adenosine triphosphate in life systems, driven by a
proton gradient.29 Therefore, developing facile and sensitive
sensing systems for probing pH, in particular by means of
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fluorescence spectroscopy, are very important in numerous fields. Materials. N,N-dimethylformamide (DMF, 99.5%, AR),
Many pH-sensitive materials, roughly divided into several benzaldehyde (98.5%, AR), acetic acid (99.5%, AR), methanol
31
metal (99.5%, AR), H3PO4 (85%, AR), boric acid (99.5%, AR), NaOH
categories such as organic small molecules,30,
34
complexes,32
polymers,33,
biomacromolecules,35 (96%, AR), hydrogen peroxide (30%, AR), trisodium citric acid
nanomaterials,36 and others,37-42 were employed to establish pH (Na3Cit, 99.8%, AR), citric acid (99.8%, AR), hydrochloric acid
sensing systems on the basis of various fluorescence sensing (38%, AR), and sodium acetate (99%, AR) were purchased from
mechanisms. In particular, numerous organic small molecules with GuoYao (Shanghai, China). Salicylaldehyde (99%, AR), TMB
a
wide range of fluorophore units such as xanthene,43 (99%, AR), GOD (100 U.mg-1, Aspergilus niger), and Fe(NO3)3
rhodamine,44 cyanine,45 boron dipyrromethene,46 and others,47, 48 (99.99%, AR) were obtained from Aladdin (Shanghai, China). β-
were synthesized to show pH-dependent fluorescent response. D-glucose (80%, AR) was obtained from Macklin (Shanghai,
Therefore, taken together the intriguing property of TMB widely China). Chinese rice vinegar and lemon were purchased from
used as the catalytic substrate and the importance of probing pH, Xinhuadu supermarket (Xiamen, China). Ultrapure water were
we further considered that whether TMB derivatives modified by prepared by a Milli-Q system.
simple molecules can be used as the fluorescent probes for
Instruments. Fluorescence spectra were recorded with an F-
detecting pH or pH-related targets/events? If this tentative idea
7000 fluorescence spectrometer (Hitachi, Japan). UV–vis
could be achieved, the application scope of TMB can be
absorption spectra were obtained with
a
UV-2600
substantially extended. It should be pointed out that, although
numerous works involving in pH sensing have been reported, yet
few case of TMB or its derivatives focused on sensing pH or pH-
related targets/events.
spectrometer (Shimadzu, Japan). Nuclear magnetic resonance
(NMR) experiments were conducted with a 500 MHz/AVANCE
III system (Bruker, Switzerland). The experiments of
electrospray ionization mass spectrometry (ESI-MS) were
performed by using an UHPLC–QTOF/1290–6545 system
(Agilent, China). Single-crystal X-ray diffraction (SCXRD) data
and structure chart were collected and recorded with the
Gemini E (Agilent, Poland) and WI 53711(Bruker, USA) X-ray
single crystal diffractometers.
In this work, TMB was used to react with salicylaldehyde and
benzaldehyde, respectively, to synthesize two Schiff base
compounds containing bis(imine) bonds (-C=N-), TMBS and TMBB,
via the condensation reaction between -NH2 group and -CHO
group (Scheme 1). The two compounds were found to have pH-
dependent fluorescence response, because with lowering pH
TMBS or TMBB can be gradually hydrolysed to release TMB which
emits strong fluorescence. This means that, lowering pH the
fluorescence of probes can be turned on, which is favourable in the
field of fluorescence sensing due to its low spectral background.
Similarly, this pH-dependent response in the corresponding UV-vis
absorption spectra could be observed, too. This change of
fluorescence intensity or UV-vis absorbance with changing pH can
indicate the pH value of solution system. Two practical samples,
Chinese rice vinegar and lemon juice, were successfully employed
to verify the feasibility of the practical application of the TMB
derivatives. The chromogenic reaction of the released TMB in
TMBS or TMBB case could be catalysed by introducing Fe3+-H2O2
system which was previously reported by us.18 This reaction could
be employed to facilely monitor pH by naked eyes. Therefore,
novel and feasible dual-modal sensing systems, fluorescent and
chromogenic, of probing pH, were successfully developed. On the
basis of these findings, taken together the chemical equilibrium
reaction between CO2 and H2O, a fluorescence/UV-vis absorption
sensing system of probing gaseous CO2 was further established.
Furthermore, because in the presence of O2 β-D-glucose can be
highly specific hydrolysed by glucose oxidase (GOD) to produce
gluconic acid which can lower the pH value of system, a highly
sensitive and selective dual-modal sensing system of detecting β-D-
glucose was constructed, too. Therefore, the two TMB derivatives,
TMBS and TMBB, could be successfully employed to develop dual-
modal sensing systems of detecting multi-targets.
Synthesis of TMBS and TMBB
TMB derivative, TMBS or TMBB, was synthesized via the
condensation reaction between TMB and salicylaldehyde or
benzaldehyde. The detailed synthesis procedure was followed:
0.24 g (1 mmol) TMB was dissolved in 10 mL methanol, and
320 μL (3 mmol) salicylaldehyde or 310 μL (3 mmol)
benzaldehyde was added to another 10 mL methanol. TMB
solution and salicylaldehyde or benzaldehyde solution were
mixed and put into a three-necked flask. The mixed solution
o
was refluxed for 3 h at 70 C under the protection of N2 gas.
Then, the resulting solution was cooled and filtered. The solids
were collected and washed by using methanol solvent. The
product was recrystallized by using DMF-methanol mixed
solvent (VDMF/Vmethanol = 1:3) to afford yellow crystals.
TMBS:
1H NMR (500 MHz, CDCl3): δ (ppm) = 2.30 (s, 12H), 6.98 (td,
J=7.5 Hz, J=1.0 Hz, 2H), 7.09 (d, J=8.5 Hz, 2H), 7.36–7.39 (m,
6H), 7.42-7.45 (m, 2H), 8.40 (s, 2H), and 13.12 (br, 2H).
13C NMR (126 MHz, CDCl3): δ (ppm) = 18.7, 117.3, 118.7,
119.0, 126.8, 128.7, 132.1, 133.2, 137.4, 147.2, 161.2, and
166.7.
ESI-MS+ (m/z): 449.2227 [M+H+] (the theoretical m/z value
of TMBS is calculated to 449.2224 [M+H+]).
TMBB:
1H NMR (500 MHz, CDCl3): δ (ppm) = 2.23 (s, 12H), 7.33 (s,
4H), 7.52–7.53 (m, 6H), 7.94-7.96 (m, 4H), and 8.28 (s, 2H).
13C NMR (126 MHz, CDCl3): δ (ppm) = 18.2, 126.2, 127.2,
128.2, 128.5, 131.1, 135.8, 136.2, 149.8, and 162.4.
Experimental
Materials and instruments
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ESI-MS+ (m/z): 417.2321 [M+H+] (the theoretical m/z value for accelerating the oxidation reaction catalysed by enzyme to
of TMBB is calculated to 417.2325 [M+H+]).
produce gluconic acid at room temperature.
1
All figures of H NMR, 13C NMR, and ESI-MS of TMBS and
After that, 1 mL oxidation reaction solution, 1 mL DMF, and
1 mL 0.06 mM TMBS or TMBB solution were added to a 10 mL
tested tube. After the incubation time of ca.30 min was taken,
the mixed solution was measured by using fluorescence and
UV-vis absorption spectrometer, respectively. Error bars were
TMBB were shown in Fig. S1-S6.
Sensing of pH
Non-catalytic spectral sensing of pH:
The BR buffer solutions were used to control the pH values determined by measuring three parallel solutions.
of the sensing systems varied from 1.89 to 5.02, which were
measured by a pH meter with an error less than ±0.01 unit of
Results and discussion
pH. For a typical experiment of pH sensing, the detailed
procedure was followed: 1 mL 0.06 mM TMBS or TMBB DMF
Considering TMB bearing two -NH2 groups, TMBS or TMBB
solution and 1 mL DMF solvent were added to 1 mL BR buffer
could be easily synthesized by a facile one-step condensation
solution of various pHs changed from 1.89 to 5.02. The total
reaction between TMB and salicylaldehyde or benzaldehyde in
volume of the tested solution was fixed as 3 mL. After a
methanol solvent (Scheme 1). The yellow pure Schiff base
certain incubation time (the time is pH-dependent),
compounds were finally obtained by using a recrystallization
fluorescence and UV-vis absorption spectra of TMBS or TMBB
method with DMF-methanol mixed solvent, to afford
reasonable yield of ca. 70 % for TMBS or ca. 70 % for TMBB
a
under various pHs were measured. Error bars were
determined by measuring three parallel solutions.
(both refer to TMB).
Catalytic chromogenic sensing of pH:
100 µL 6.7 mM TMBS solution and 100 µL DMF were added
to 100 µL citric acid-NaOH-HCl or Na3Cit-citric acid buffer
solutions of various pHs. After a certain incubation time (the
time is pH-dependent), 100 µL above resulting solution was
added to 900 µL 0.2 M HAc-NaAc buffer of pH 4.2 containing 4
mM H2O2 and 50 µM Fe3+ ions. After that, the mixed solutions
were placed at 50 oC water bath for 30 min, and the
corresponding UV-vis absorption spectra were measured. For
TMBB case, the experimental procedure of the catalytic
chromogenic reaction in the presence of Fe3+ ions and H2O2
was followed as that of TMBS case.
Scheme 1. The synthesis route of TMBS and TMBB
Sensing of gaseous CO2
For a typical experiment of sensing gaseous CO2, the
procedure was followed: 3 mL 0.02 mM TMBS or TMBB DMF-
H2O solutions (VDMF /VH2O of TMBS and TMBB were 0.667 and
0.5, respectively) were prepared. TMBS or TMBB solution was
bubbled by CO2 gas with a flow rate of 10 mL.min-1 through a
0.5 mm needle. The volumes of CO2 gas bubbled into the
TMBS or TMBB solutions were controlled as 0, 0.5, 1, 1.5, 2,
2.5, 3, 5, 8, 11, 14, 17, and 20 mL, respectively. After bubbling
CO2 gas was finished and ca. 30 min incubation time was
taken, the fluorescence and UV-vis absorption of the resulting
solutions were measured. Error bars were determined by
measuring three parallel solutions.
Fig. 1 Single crystal X-ray diffraction structures of (a) TMBS and (b) TMBB
Molecular structures of TMBS and TMBB were probed by
1H NMR, 13C NMR, and ESI-MS. The results of NMR and mass
spectrometry (Fig. S1-S6) clearly support the supposed
molecular structures (Scheme 1). Furthermore, the crystal
structures of TMBS and TMBB were investigated by SCXRD.
These results of SCXRD further confirm the formation of
bis(imine) bonds in TMBS and TMBB (Fig. 1). The data of
crystal structures show that the intramolecular O-H...N
hydrogen bond interactions appear in TMBS, and yet there is
no weak intermolecular or intramolecular hydrogen bonding
interactions in TMBB. Crystal stacking is likely controlled by
van der Waals forces in both Schiff base compounds. Detailed
Sensing of glucose
For a typical experiment of sensing glucose, the procedure
was followed:
0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, and 1 mL 1 M β-D-glucose solution were added to 25 mL
vials, respectively, and a certain volume of ultrapure water
was added to these vials to obtain the total volume of 9 mL.
Then, 1 mL 20 U.mL-1 glucose oxidase solution was added to
afford the final enzyme activity unit of 2 U.mL-1. Highly pure
oxygen gas was bubbled to the mixed solution for at least 8 h
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crystallographic data of TMBS and TMBB were listed in Table afforded by a pH meter (Fig. S18-19). The difference between
S1 and S2. It should be noted that the crystal structure of pH measured by using TMBS and pH measured by using a pH
TMBB was determined at first time and the results have been meter was found to be quite small.
deposited into Cambridge Crystallographic Data Centre (the
CCDC number is 1574444).
(a)
(b)
4000
3000
2000
1000
0
4000
3000
2000
1000
0
To check pH sensing performance of TMBS and TMBB, BR
buffer solutions with various pHs were used to control the pH
value of TMBS or TMBB solution. As shown in Fig. S7- S8, both
TMBS and TMBB are nonfluorescent. However, when pH of
TMBS or TMBB solution was lowered, the fluorescence with a
1.90
5.03
pH
maximum wavelength at
~390 nm was turned on and
gradually enhanced (Fig. 2 and Fig. S9). It was also found that
the incubation time for obtaining stable fluorescence intensity
at 390 nm wavelength was pH-dependent, that is, the
incubation time increased with enhancing pH, in both TMBS
and TMBB cases (Fig. S10 and S11). For most of tested pHs,
the required incubation times were found to be less than 30
min. Correspondingly, the change of UV-vis absorption spectra
of TMBS or TMBB with varied pHs from 1.90 to 5.03 were
investigated, too, and the results were shown in Fig. 3 and Fig.
S12. Two clear isosbestic points, locating at ca. 290 nm and
320 nm, could be observed in both TMBS and TMBB cases.
The appearance of the two isosbestic points was indicative of
the conversion of at least three compounds, when pH was
changed. The three compounds were likely TMB, intermediate
TMB derivative containing one –C=N– bond, and TMBS or
TMBB containing two –C=N– bonds, respectively. Obviously,
the former two compounds were the hydrolysed products of
TMBS or TMBB. This assumption could be supported by the
ESI-MS results of TMBS or TMBB solution under pH of 1.8,
which could clearly indicate the presence of TMB due to the
appearance of MS peak of m/z 241.1699 or 241.1698 which
could be ascribed to be that of [TMB+H+] (theoretical m/z of
350
400
450
2
3
4
5
pH
Wavelength / nm
Fig. 2 (a) pH-dependent fluorescence spectral change of TMBS solution with pH
ranging from 1.90 to 5.03. (b) The relationship between the fluorescence
intensity @ 390 nm wavelength and pH in TMBS solution. [TMBS] = 20 μM. The
excitation wavelength is 300 nm.
1.5
(a)
(b)
0.3
0.2
0.1
0.0
5.03
pH
1.90
1.2
0.9
0.6
0.3
0.0
200
300
400
2
3
4
5
pH
Wavelength / nm
TMB is 240.1626; Fig. S13
–
S14). Furthermore, the
390 nm
Fig. 3 (a) pH-dependent UV-vis absorption spectral change of TMBS solution with pH
ranging from 1.90 to 5.03. (b) The relationship between the absorbance @ 365 nm
wavelength and pH in TMBS solution. [TMBS] = 20 μM.
fluorescence emission with maximum wavelength at
~
can be believed to originate from the released TMB, as it is
identical to that of TMB (Fig. S15). In TMBS case a good linear
relationship between the fluorescence intensity at 390 nm
wavelength and pH value in the range of 2.09–4.01 was found
(Fig. 4), and the linear regression equation was fitted as F =
2997.94 - 715.03 pH with a good linear coefficient of 0.991 (F
represents the fluorescence intensity at 390 nm wavelength).
Similarly, TMBB also showed a similar linear relationship
between the fluorescence intensity at maximum peak of 390
nm wavelength and pH value (Fig. S16). For UV-vis absorption
spectra, the good linear relationship between the absorbance
at 365 nm wavelength and pH value could also be obtained in
both TMBS (Fig. 5) and TMBB (Fig. S17) cases. Therefore,
these results indicate that TMBS and TMBB are potentially
useful for establishing sensing systems based fluorescence or
UV-vis absorption spectra to quantitatively probe pH values.
Indeed, to verify the feasibility of practical application, TMBS
was chosen to be further employed to probe the pH value of Fig.
2000
1500
1000
500
0
2.0
2.5
3.0
pH
3.5
4.0
4
The linear response curve of the fluorescence intensity at 390 nm
wavelength towards different pHs in TMBS solution. [TMBS] = 20 μM.
two practical samples, Chinese rice vinegar and lemon juice.
The results of fluorescence responses reveal that, TMBS could
be effectively used to accurately measure the pH value of both
tested samples, which were compared with the pH values
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0.3
0.2
0.1
0.0
2.5
3.0
3.5
pH
4.0
4.5
Fig. 6 (a) The UV-vis absorption spectral change of the Fe3+ - H2O2 catalytic system in 0.2
M HAc - NaAc buffer solution of pH 4.2 with changing pH of TMBS solution which could
release TMB as the catalytic substrate. (b) The relationship between the absorbance at
652 nm and pH of TMBS solution. Insect of (a) is the photos of the color change of the
catalytic chromogenic reaction with lowering pH.
Fig. 5 The linear response curve of the absorbance at 365 nm wavelength towards
different pHs in TMBS solution. [TMBS] = 20 M. The linear regression equation was
μ
fitted as Abs @ 365 nm = -0.32 + 0.14 pH with a good linear coefficient of 0.994.
Considering that TMBS and TMBB can be hydrolysed to
release TMB under low pH, and TMB is the traditional catalytic
substrate of certain natural enzymes or artificial enzyme
mimics, the released TMB is expected to be capable of serving
as the catalytic substrate of a chromogenic reaction in the
presence of certain catalysts to indicate pH via naked eyes.
Our group recently found that Fe3+ ions have extremely high
catalytic activity towards TMB as the substrate in the presence
As mentioned in above section, in low pH, TMB can be
released from TMBS or TMBB due to the hydrolysis reaction of
–C=N– bond, which can emit strong fluorescence. Therefore,
these findings can enlighten us to have a hypothesis that CO2
gas can be detected by using the two synthesized Schiff base
compounds. This is because that dissolved CO2 gas can achieve
the following reaction equilibrium: CO2 + H2O
H2CO3
2H+ + CO32-, and its pKa1 and pKa2 are 3.6 and
H2O2 system can be employed to catalyse the chromogenic 10.329, respectively (25 oC),49,50 thereby dissolved CO2 can
-
H+ + HCO3
of H2O2.18 Therefore, it can be considered that whether Fe3+
-
reaction of released TMB to probe pH. Therefore, in our
experiments citric acid buffer solution was employed to
control the pH value of TMBS solution to release TMB. Then,
HAc - NaAc buffer solution of pH 4.2 containing 4 mM H2O2
and 50 μM Fe3+ ions was employed to perform the catalytic
chromogenic reaction when the released TMB solution was
introduced. UV-vis absorption spectra of the catalytic system
demonstrate that, a new absorption peak occurred at 652 nm
wavelength and the absorbance was gradually increased with
lowering pH of citric acid buffer, accompanied with the
appearance of blue color in the catalytic system solution (Fig.
6). The similar pH-dependent catalytic chromogenic reaction
could be observed in TMBB case in the presence of Fe3+ ions
and H2O2 (Fig. S20). It should be noted that, the catalyst role
of Fe3+ ions, oxidant role of H2O2, and its catalytic Michaelis–
Menten equation have been detailedly studied.18 Therefore,
TMBS or TMBB could be employed to establish a facile sensing
method for probing pH via naked eyes.
lower pH of the solution. In our experiments, the flow rate of
CO2 gas was set as 10 mL.min-1, and CO2 gas was bubbled into
TMBS or TMBB aqueous solution containing 50 % DMF through
a 0.5 mm needle. The fluorescence spectral change of TMBS
or TMBB reveal that, with increasing CO2 volume the new
fluorescence peak at
~390 nm occurred and gradually
increased (Fig. 7 and Fig. S21), and these observations were
likely similar to those of pH responses. Similarly, the CO2-
dependent change of UV-vis absorption spectra of TMBS or
TMBB were found, too (Fig. 8 and Fig. S20). With increasing
CO2 volume, two clear isosbestic points appeared at ca. 290
nm and 320 nm wavelength (Fig. 8a and Fig. S22a),
respectively, indicative of the conversion of at least three
compounds. This observation was similar to that of pH
response. The linear relationship between the fluorescence
intensity at 390 nm wavelength and CO2 volume bubbled
through TMBS solution could be obtained, and the linear
regression equation was fitted as F = 7.87 + 340.48 VCO2 with a
good linear coefficient of 0.999 (Fig. 9). A similar linear
relationship could also be observed in the TMBB case (Fig.
S23). Correspondingly, the linear relationships between Abs
@365 nm and CO2 volume bubbled through TMBS or TMBB
solution could also be established in UV-vis absorption spectra
of both TMBS and TMBB cases (Fig. 8b and Fig. S22b). Other
common gases such as O2 and N2 were chosen to investigate
the selectivity of the fluorescence and UV-vis absorption
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response of TMBS or TMBB under similar experimental
conditions. These results were shown in Fig. 10 - 11 and Fig.
S24 - S25. It was found that both tested O2 and N2 gases did
not show any significant effect on the fluorescence and UV-vis
absorption spectra of the two Schiff base compounds. These
observations indicate that the two synthesized TMB–based
Schiff base compounds can be considered to be potential
probes for reliably detecting CO2 gas. For the fluorescence
response, the limit of detection (LOD) of detecting CO2 gas
(3S/K, n=11) were estimated to 110 μM (for TMBS) and 132
μM (for TMBB), respectively.
Fig. 9 The linear response curve of the fluorescence intensity at 390 nm wavelength
towards different CO2 volumes bubbled through TMBS solution. [TMBS] = 20 μM.
Fig. 7 (a) CO2-dependent fluorescence spectral change of TMBS solution with CO2
volume changing from 0 to 20 mL. (b) The relationship between the fluorescence
intensity @ 390 nm wavelength and CO2 volume bubbled through TMBS solution.
Fig. 10 The response of the fluorescence intensity at 390 nm wavelength of TMBS
solution under different volumes of CO2, N2, and O2 gas. [TMBS] = 20 M.
[TMBS] = 20 μM. The excitation wavelength is 300 nm.
μ
Fig. 8 (a) CO2-dependent UV-vis absorption spectral change of TMBS solution
with CO2 volume changing from 0 to 20 mL. (b) The relationship between the
absorbance @ 365 nm wavelength and CO2 volume bubbled through TMBS
Fig. 11 The response of the UV-vis absorbance at 365 nm wavelength of TMBS solution
under different volumes of CO2, N2, and O2 gas. [TMBS] = 20 M.
μ
solution. [TMBS] = 20 μM. The linear regression equation could be obtained as
Abs @ 365 nm = 0.33 - 0.1 VCO2 (R2=0.995, the CO2 volume ranges from 0 to 2.0
On the basis of these findings, it can be considered that, if
N2 gas is bubbled through the TMBS or TMBB solution which
has been bubbled by CO2 gas in prior, the fluorescence peaked
at 390 nm is expected to be decreased, because the dissolved
CO2 can be drove out by bubbled N2 gas, which can lead to
increasing pH and thus regenerating Schiff base compounds in
operated solution. The experimental results could support this
mL).
assumption. When
a certain amount of CO2 gas was
introduced into TMBS solution, the fluorescence intensity at
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390 nm wavelength was enhanced. Then, a certain amount of expected that TMBS or TMBB could be employed to establish a
N2 gas was bubbled through the above solution for 1 h, and fluorescence sensing system for detecting β-D-glucose. Nearly
then the solution bubbled by N2 gas was heated at 60 oC water 100 % of tested glucose can be converted to gluconic acid in
bath for another 1 h. The fluorescence intensity at 390 nm was this dehydrogenation and oxidation reaction under the
found to be significantly decreased. However, when a certain appropriate experimental conditions. 2 U.mL-1 GOD and β-D-
amount of CO2 gas was introduced into the operated TMBS glucose with various concentrations ranging from 2 mM to 100
solution again, the fluorescence intensity peaked at 390 nm mM were mixed, and the mixed solution was bubbled by O2
wavelength was enhanced. The fluorescence change was gas with the flow rate of 10 mL.min-1 for accelerating the
reversible, and this reversible process could be roughly oxidation reaction to fully convert β-D-glucose to gluconic acid.
repeated at least 10 times (Fig. 12). Similar repeatability of the Then,
a certain volume of the resulting solution was
fluorescence intensity at 390 nm wavelength of the TMBB transferred into TMBS or TMBB solution to hydrolyse the Schiff
solution alternately bubbled by CO2 and N2 gas could also be base compounds, and the hydrolysed solution was monitored
observed (Fig. S26). The results of ESI-MS experiments can by using fluorescence and UV-vis absorption spectrometers.
confirm the regeneration of Schiff compounds after the CO2 The fluorescence and UV-vis absorption spectra reveal that,
gas-bubbled TMBS or TMBB solution was bubbled by N2 gas with increasing β-D-glucose concentration, the fluorescence
(Fig. 13 and Fig. S27), because the recorded MS peak of m/z with maximum peak at 390 nm wavelength was turned on and
449.2222 and 417.2323 could be attributed to those of TMBS gradually increased (Fig. 14 and Fig. S28), and two isosbestic
and TMBB, respectively.
points clearly appeared at ca. 290 nm and 320 nm wavelength,
respectively (Fig. 15 and Fig. S29). These observations were
similar to those of the responses of the fluorescence and UV-
vis absorption spectra towards pH and CO2 gas in TMBS or
TMBB case.
Fig. 12 The fluorescence intensity at 390 nm wavelength reversible change of TMBS
solution alternately bubbled by CO2 and N2 gas for 10 cycles. [TMBS] = 20 μM.
Fig. 14 (a) The change of the fluorescence spectra of TMBS under different β-D-glucose
concentrations. (b) The relationship between the fluorescence intensity @ 390 nm
wavelength and β-D-glucose concentration. [TMBS]
= 20 μM. The excitation
wavelength is 300 nm.
Fig. 13 ESI-MS experimental result of TMBS after the CO2 gas-bubbled solution was
bubbled by N2 gas.
Fig. 15 (a) The change of the UV-vis absorption spectra of TMBS under different β-D-
glucose concentrations. (b) The relationship between the absorbance 365 nm
wavelength and β-D-glucose concentration. [TMBS] = 20 M.
Furthermore, considering that GOD can catalyse the
dehydrogenation and oxidation reaction of β-D-glucose with a
highly specific recognition ability to produce gluconic acid with
a pKa of 3.651 which can lower the pH value of the system, it is
@
μ
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It should be pointed out that the relationship between the pH-dependent dual-modal signal responses, fluorescence and
fluorescence intensity at 390 nm wavelength and β-D-glucose UV-vis absorption, because TMB can be released via the
concentration was found to have two linear curves. For TMBS hydrolysis reaction of –C=N- bonds under low pH value.
case, the ranges of β-D-glucose concentration were from 4 Therefore, the dual-modal sensing systems for probing pH
mM to 10 mM and from 20 mM to 100 mM, respectively (Fig. were successfully established. By introducing the Fe3+ - H2O2
S30). Yet for TMBB case, the ranges of β-D-glucose system, the oxidation reaction of the pH-dependently released
concentration were from 4 mM to 8 mM and from 10 mM to TMB in TMBS or TMBB case can be catalysed to produce a blue
100 mM, respectively (Fig. S31). The two observed linear color product, resulting in conveniently probing pH via naked
response curves in TMBS or TMBB case indicate that, with eyes. On the basis of CO2 gas dissolved reaction equilibrium in
increasing β-D-glucose concentration, there were two kinds of aqueous solution and GOD can catalyse the dehydrogenation
reaction. One could produce an intermediate TMB derivative and oxidation reaction of β-D-glucose, dual-modal sensing
containing one –C=N– bond, and another could form final systems of detecting gaseous CO2 and β-D-glucose could also
TMB. The LOD of detecting β-D-glucose could be estimated to be successfully developed, respectively. All in all, TMB-based
0.7 μM (3S/K, n=11), according to the first linear curve of F = - derivatives used as dual-modal sensing probes of detecting
582.54 + 300.47 Cβ-D-glucose (R2 = 0.994) in TMBS case.
In order to confirm that whether the oxidation reaction of work have several intriguing enlightenments, for example, (i)
β-D-glucose with tested concentration could be catalysed by 2 By ingeniously modifying catalytic substrate molecule,
multi-targets including pH, gaseous CO2, and glucose in this
a
U.mL-1 GOD to reach an equilibrium of converting into gluconic detected target can trigger the substrate derivative to release
acid within 8 h of reaction time, different concentrations of β- the substrate which can be catalysed by the catalyst to
D-glucose including 2, 10, 50, and 100 mM were chosen to produce colored or fluorescent species. The chromogenic or
investigate the effect of reaction time in TMBS case. fluorogenic reaction can sensitively indicate the target.
Experimental results show that, with increasing the reaction Therefore, this proposed mechanism can afford a new entry
time, the fluorescence intensity at 390 nm wavelength for other traditional enzyme substrates like o-
gradually increased in all tested β-D-glucose concentration phenylenediamine bearing two neighboring –NH2 groups. (ii)
cases (Fig. 16), and when the reaction time was longer than 6 The application scope of TMB and its derivatives can be
h, the fluorescence intensity at 390 nm wavelength nearly substantially extended. (iii) Compared to previously reported
reached a platform. These observations substantially support cases of pH sensing which only involved in single target, the
that the oxidation reaction of β-D-glucose catalysed by GOD two TMB derivatives in this work have demonstrated the
could reach an equilibrium within 8 h, which was employed to versatility of their application potential.
study the effect of β-D-glucose concentration on the catalytic
oxidation reaction.
Conflicts of interest
There are no conflicts to declare.
4500
C
/ mM
glucose
Acknowledgements
100
50
10
2
This work was supported by the National Natural Science
Foundation of China (No. 21377124 and 21575044), the
Natural Science Foundation of Fujian Province (No.
2017J01015), Promotion Program for Young and Middle-aged
Teacher in Science and Technology Research (No. ZQN-YX303),
the Project of New Faculty (No. 15BS202) of Huaqiao
University, and Graphene Powder & Composite Research
Center of Fujian Province (2017H2001).
3000
1500
0
0
100
200
300
400
500
600
Time / min
Notes and references
Fig. 16 The time-dependent fluorescence intensity
@ 390 nm wavelength under
different β-D-glucose concentrations in TMBS case. [TMBS] = 20
μM. β-D-glucose
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Diverse applications of TMB-based sensing probes
Li Huang, Ying-Juan Cao, Xiang-Ying Sun, Bin Liu, and Jiang-Shan
Shen*
Two TMB derivatives, TMBS and TMBB, have been successfully synthesized and
employed to develop dual-modal spectral sensing systems of detecting multi-targets
including pH, CO2 gas and glucose.
X
N
N
[H+
[CΟ2]
-D-glucose]
]
X
H+
[
β
or CO2
or β-D-glucose-GOD
X
CHO
+
H2N
NH2
X = - OH
H
350
400
450
500
Wavelength / nm