Imaging of formaldehyde in plants with a ratiometric fluorescent probe

Article abstract of DOI:10.1039/c7sc00373k

The fluorescence monitoring of formaldehyde in real environmental samples and live plant tissues is of great importance for physiological and pathological studies. However, there is a lack of suitable chemical tools to directly trace and measure the formaldehyde activity in bio-systems, and developing effective and, in particular, selective sensors for mapping formaldehyde in live tissues still remains a great challenge. Here, we demonstrate for the first time that the ratiometric fluorescence monitoring of formaldehyde in live plant tissues is achieved with a newly developed ratiometric fluorescent probe, FAP, which effectively eliminated interference from other comparative analytes. Live tissue analyses reveal that FAP can potentially detect exogenous and endogenous formaldehyde in live Arabidopsis thaliana tissues, exposing a potential application for biological and pathological studies of formaldehyde.

Full text of DOI:10.1039/c7sc00373k

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Imaging of formaldehyde in plants with
a ratiometric fluorescent probe†
Cite this: DOI: 10.1039/c7sc00373k
a
a
a
ab
*
*
Zhen Li,‡ Yuqing Xu,‡ Hailiang Zhu and Yong Qian
The fluorescence monitoring of formaldehyde in real environmental samples and live plant tissues is of
great importance for physiological and pathological studies. However, there is a lack of suitable chemical
tools to directly trace and measure the formaldehyde activity in bio-systems, and developing effective
and, in particular, selective sensors for mapping formaldehyde in live tissues still remains a great
challenge. Here, we demonstrate for the first time that the ratiometric fluorescence monitoring of
formaldehyde in live plant tissues is achieved with a newly developed ratiometric fluorescent probe, FAP,
which effectively eliminated interference from other comparative analytes. Live tissue analyses reveal
that FAP can potentially detect exogenous and endogenous formaldehyde in live Arabidopsis thaliana
tissues, exposing a potential application for biological and pathological studies of formaldehyde.
Received 25th January 2017
Accepted 2nd June 2017
DOI: 10.1039/c7sc00373k
rsc.li/chemical-science
levels.8–11 For instance, a small pool of FA (0.1–10 mmol gꢀ1 fresh
weight) was identied in the intermediate of the C1 metabolism
of plants.12,13 The primary sources of FA in plants are caused by
Introduction
Formaldehyde (FA), the simplest of the aldehydes with the
formula H–CHO, has been widely used as an important
precursor to many industrial materials and chemical
compounds.1 On the other hand, it has also been classied as
a highly toxic compound to living organisms, because its strong
interactions with proteins, nucleic acids and other biomole-
cules will lead to the inactivation of their biological activities.2
For instance, excess exposure of exogenous FA to the human
body will lead to a signicant health threat and cause many
diseases,3–6 including various cancers, heart disorders, diabetes,
etc. Indeed, it has been regarded as a suspected carcinogen and
well-known human toxicant. In plants, FA pollution is a wide-
spread problem that can affect plant growth and development,
causing a signicant decrease of the productivity and quality in
agriculture.7 But, interestingly, intracellular FA can be endoge-
nously formed by normal physiological processes and sponta-
neously removed by complex metabolism using detoxication
mechanisms. It has been demonstrated that glutathione-
dependent FA dehydrogenase (FALDH), as a key enzyme of FA
metabolism, plays an essential role in the uptake and detoxi-
cation of FA in Arabidopsis plants.8 Thus, the concentration of
FA in living organisms is generally maintained in a dynamic
state of equilibrium ranging from low to high millimolar
the processes of methanol oxidation, dissociation of 5,10-
methylene-THF, glyoxylate decarboxylation and other oxidative
demethylation reactions.14 Exogenous FA can also be absorbed
and incorporated into the metabolism of plants.7,15,16 As a result,
phytoremediation has been extensively applied for the removal
of FA from the polluted environment. However, the biological
roles of FA and the mechanism of FA metabolism in plants have
not yet been clearly dened,14 and they are still challenging to
study due to the lack of tools that can map the distribution of FA
in live tissues. Thus, in order to understand the biological and
pathological roles of FA well, it is crucial to develop sensitive
methods or chemical tools for tracing and identifying FA in
environmental samples and live plant tissues.
Coupled with uorescence microscopy techniques, uores-
cent imaging using small molecular uorescent probes to study
biological species in living bio-systems has attracted great
interest owing to its high spatiotemporal resolution, non-
invasiveness, high sensitivity, and excellent selectivity,17–28
which can offer the opportunity to monitor FA directly and
easily in different biological contexts. However, the effective
and, in particular, selective mapping of FA activity in live tissues
with uorescent probes still remains
a great challenge.
Recently, only a few uorescent turn-on probes have been re-
ported for monitoring FA.17–21,29 Some could be successfully
used for imaging FA in living cells, others were primarily
applied for the in vitro determination of FA in water. These
intensity-based uorescent probes may have a number of limi-
tations imposed by the uorescent turn-on response at a single
detection window, which means it is difficult to obtain accurate
information when testing the FA concentration. Moreover,
aState Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, No. 163 Xianlin Road, Nanjing 210023, China. E-mail:
yongqian@nju.edu.cn
bCollege of Chemistry and Materials Science, Nanjing Normal University, No. 1
Wenyuan Road, Nanjing, 210046, China. E-mail: yongqian@njnu.edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7sc00373k
‡ These authors contributed equally to this work.
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these turn-on responses can also vary depending on the exper- charge transfer (ICT) effect between the N,N-dimethyl group and
imental conditions, including the probe concentration, excita- the 2-position homoallylamine of the chromophore. Upon
tion intensity, temperature, and instrument efficiency.30 To treatment with FA, however, the homoallylamino group would
minimize most of the aforementioned articial and environ- change into an aldehyde group via specically activated
mental factors, an attractive approach for the determination of processes, which would then cause an increase in the electron
FA in environmental samples and live tissues is developing withdrawing ability, thereby inducing the red shi of the uo-
ratiometric uorescent probes, due to their quantitative rescence emission spectrum of the probe and producing
tracking ability and self-calibration effect.23,31,32 So far, there is a ratiometric response for FA. Based on these design consider-
only one reported ratiometric uorescent probe for monitoring ations, we then synthesized the uorescent probe, FAP, in three
FA,33 but its relatively low excitation in the UV range and its steps starting from commercially available N1,N1-
short emission wavelength might easily be overwhelmed by the dimethylbenzene-1,4-diamine (Scheme 2), and the structures of
1
tissue’s auto-uorescence background, thereby limiting the the target compounds were fully characterized by H and 13C
probe’s further application in live tissue. In addition, uores- NMR spectroscopy and mass spectrometry (see the ESI†).
cent probes that have the ability to image inside live plant
As expected, the homoallylamino modication on FAP leads
tissues to provide relevant information on endogenous FA are to relatively short absorbance and uorescence. The free FAP
particularly attractive, but no such probe has been reported to (20 mM) exhibited two absorption peaks at 254 nm and 353 nm,
date. Therefore, developing novel ratiometric probes that can be and one broad emission peak at 495 nm. However, the incu-
applied for the in vivo imaging of FA with highly sensitive and bation of 20 mM FAP with 1 mM FA resulted in a remarkable red
selective responses is critically important and there is strong shi of the absorbance signals (23 nm and 76 nm, respectively).
demand. We herein present the development and application of As shown in Fig. 1a, two obvious absorption bands with peaks at
a FA probe, FAP (Scheme 1), a new ratiometric uorescent 277 nm and 429 nm newly emerged, and the intensities of the
sensor that can selectively report formaldehyde in real water original peaks at 254 nm and 353 nm simultaneously decreased;
samples and enable tracking of the endogenous FA activity in meanwhile, accompanying the reaction of FAP with FA, the
live tissues of Arabidopsis thaliana for the rst time.
emission wavelength was also obviously shied (75 nm),
affording an important enhancement of the uorescence
emission band at 570 nm and a signicant decrease at 495 nm
(Fig. 1b). These in vitro responses of FAP validate the previous
design strategy and conrm that FAP is a good sensor for in vitro
formaldehyde determination. In order to elaborate the reaction
mechanism, we then monitored the reactions between FAP and
FA using HPLC and analysed the reactions using MS and IR
spectroscopy. The reactions with different incubation times
showed clean conversion, and a new peak (m/z ¼ 201.1), iden-
tied as the product with the expected mass of the aromatic
aldehyde (3), began to appear aer 1 h of incubation (Fig. S1
and S2†). The reactions of different FA concentrations also
showed a gradual decrease in the probe peak and a corre-
sponding increase in the product peak with increasing FA
concentration (Fig. S2†). In addition, a signal of the freshly
formed –CHO– group was also clearly observed in the IR spec-
trum aer treatment with FA (Fig. S3†). Taken together, these
ndings show that the formation of the aldehyde is indeed
promoted by reacting with FA.
Results and discussion
To design this new FA ratiometric uorescent probe, we focus
on the 2-aza-Cope rearrangement reaction,34 as illustrated in
Scheme 1, where the starting imine intermediate can be formed
by the selective capture of FA with a homoallylamino group, and
it will further undergo a simple rearrangement and hydrolysis
process to yield an aldehyde product,17,18 potentially resulting in
obvious changes of the uorescence properties. Thus, we envi-
sioned that the homoallylamino group can possibly serve as the
FA selective response site. Subsequently, in order to obtain
a
ratiometric response, we chose
a
well-known N,N-
dimethylquinolin-6-amine as the uorescent chromophore
because of its exhibited excellent photophysical and chemical
properties,35,36 which has been used in the design of other re-
ported ratiometric uorescent probes as
a uorescence
reporter. We anticipated that the free probe would exhibit
a relatively short emission wavelength owing to the internal
Scheme 1 The structure of FAP and the proposed ratiometric sensing Scheme 2 Synthesis of the ratiometric fluorescent probe, FAP, for
mechanism for monitoring formaldehyde (FA).
monitoring formaldehyde (FA).
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Fig. 1 (a) UV-vis absorption spectra of FAP (20 mM) in the absence or presence of FA (1 mM) in PBS buffer (10 mM, pH 7.4, containing 1% MeCN) at
37 ꢁC for 120 min. (b) Fluorescence spectra of FAP (20 mM) in the absence or presence of FA (1 mM) in PBS buffer (10 mM, pH 7.4, containing 1%
MeCN) at 37 ꢁC for 120 min, with excitation at 405 nm, and excitation and emission slit widths ¼ 5 nm.
To assess the response time of FAP towards FA, a solution of capability for quantitatively monitoring FA. The limit of detec-
FAP (20 mM) was added to FA (2 mM) in PBS buffer (10 mM, pH tion (LOD) of FAP for the detection of FA was 0.5 mM in aqueous
7.4, containing 1% MeCN) for different incubation times and solution based on the signal-to-noise ratio (S/N ¼ 3).
then measured by a uorescence spectrometer (Fig. S4†).
The selectivity of the uorescent probe is an important
Importantly, a signicant ratiometric change was observed even parameter for assessing if it is suitable for biological applica-
aer 5 min, accompanying a pronounced enhancement of the tion. To determine whether FAP could detect FA specically, we
uorescence intensity at 570 nm and a distinct reduction at then examined the uorescence signal and compared the ratio
495 nm, whereas the emission band at 570 nm was gradually values of the uorescence intensity at 570 nm to 495 nm (I570
/
enhanced with the extension of the incubation time even aer I495) aer incubation with a series of comparative analytes,
180 min. Specically, the uorescence ratio I570/I495 increased including benzaldehyde, methylglyoxal, oxalaldehyde, GSH,
by 9-fold upon reaction with FA for 120 min. In order to observe H2O2, amino acids, etc. As seen in Fig. 2c and S8,† the largest
the relatively obvious uorescence changes at an appropriate change of the uorescence signal ratio was observed upon the
incubation time, we chose a reaction time of 120 min to further addition of FA, while the addition of 100 equiv. of the other
investigate the pH effect on the probe, FAP. We measured the analytes displayed a negligible response. FAP displayed a 9–13-
uorescence signal ratios of I570/I495 towards FA (2 mM) in PBS fold greater response toward FA than the other analytes
buffer with various pHs ranging from 3.0 to 11.0 and, surpris- (Fig. 2c). Additionally, exposing FAP to a mixture of FA and other
ingly, found that FAP shows a relatively wide range of pH species still yielded a signicantly increased uorescence ratio
application (Fig. S5†). Importantly, the remarkable ratiometric (I570/I495) (Fig. S9†). These results clearly indicate that FAP
uorescence response and the wide range of pH application of exhibits excellent selectivity for FA over other analytes. Collec-
FAP are advantageous to minimizing the interference of the tively, these selective and sensitive assays unambiguously
uorescence background and increasing the delity of the suggest that FAP has potential capability for tracking FA in
detection signal.
environmental and biological samples.
Encouraged by these results, we sought to investigate the
Having established FAP determination, we next moved on to
sensitivity of FAP for FA, and we assessed the uorescence evaluate the practicability of FAP. We initially carried out
response of FAP (20 mM) with different concentrations of FA (0– experiments using real-water samples from the Changjiang
3 mM) in PBS buffer at 37 ꢁC. The incubation of FAP with River, as well as so drink and rice water samples. FA (2 mM)
increasing concentrations of FA resulted in a gradual increase was previously spiked into these real-water samples and incu-
of the emission intensity at 570 nm and a corresponding bated with FAP (20 mM) for 2 hours (Fig. 2d). Obvious changes of
decrease at 495 nm (Fig. 2 and S6†). Strikingly, an excellent the uorescence signal ratio were observed aer incubation of
linear dependence (R2 ¼ 0.98827) between the uorescence FA in these buffer systems, while the so drink and rice water
signal ratios of I570/I495 and the concentrations of FA (0–200 mM) samples only showed relatively moderate changes that might be
was observed. Moreover, to further evaluate the validity and because FA was partially consumed via the interaction with
stability of the slope, different concentrations of FA (1.5, 4.5 and biomolecules. But fortunately, these changes of the uores-
9.5 equivalent) were spiked into the PBS buffer and incubated cence signal ratio were enough to help us achieve the determi-
with FAP (20 mM) for 120 min (Fig. S7†). Aer a calculation using nation of FA in the real-water samples. Moreover, we prepared
a calibration curve, seen in Fig. 2b, the concentration of form- dried FA test papers by dipping paper into a FAP solution and
aldehyde was determined, and the resultant FA recoveries further evaporating to remove the solvent (Fig. 2d inset).
ranged from 98% to 106%, revealing that FAP has potential Interestingly, using this simple FA test paper to detect FA
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Fig. 2 (a) Fluorescence spectra of FAP (20 mM) in PBS buffer (10 mM, pH 7.4, containing 1% MeCN) after treatment with FA (0–3 mM) for 120 min.
(b) The linear relationship between the fluorescence intensity ratio (I570/I495) and the concentration of FA (0–10 equiv.) in PBS buffer (10 mM, pH
7.4, containing 1% MeCN) for 120 min. (c) Fluorescence intensity ratios (I570/I495) of the probe, FAP (20 mM), in the presence of various relevant
analytes (100 equiv.) in PBS buffer (10 mM, pH 7.4, containing 1% MeCN) at 37 ꢁC. All the fluorescence data were observed after 120 min. lex
¼
405 nm, slit: 5 nm/5 nm. (d) Fluorescence intensity ratio (I570/I495) changes of FAP (20 mM) upon the addition of FA (2 mM) in different buffer
systems, including PBS, Changjiang River, soft drink, and rice water samples. Inset: the visual colors or visual fluorescence colors of the filter
paper strips loaded with FAP in the presence of different concentrations of FA (0–5 mM). All fluorescent images were captured under irradiation
by a 365 nm UV lamp.
solutions with various concentrations exhibited quick color be reected by the uorescence change of FAP. Notably, these
changes from green to yellow within several seconds, indicating FA concentrations fall well within the scope of physiological
the potential utility of FAP as a simple sensor for tracking FA in concentration, treating the condition of formaldehyde stress in
environmental samples.
plants, and are estimated to be 0.1–10 mmol gꢀ1 in the fresh
To address the capability of FAP for mapping FA in live weight of a plant,13 and up to 2–8 mM in the external treatment
tissue, we employed FAP to image FA in the live root tip tissue of of HCHO stress.14,15,37
Arabidopsis thaliana using a confocal uorescence microscope.
To further investigate whether FAP can be applied to detect
As shown in Fig. 3, the root tip tissues of Arabidopsis thaliana the change of endogenous FA levels in live plant tissues, we
were pretreated with FAP (50 mM) for 45 min, and subsequently pretreated live root tip tissues of Arabidopsis thaliana with 10%
different concentrations of exogenous FA (0 or 3 mM) were MeOH stress for 1 h to induce the production of endogenous
added and incubated for another 120 min. Gratifyingly, FAP can FA,38 then incubated with FAP (50 mM) for another 3 hours and
effectively penetrate the cell wall and cell membrane, and can imaged aer washing with PBS (Fig. 4). The endogenous FA
display a bright uorescence in the green channel and relatively freshly formed via MeOH pretreatment results in an obvious
weak uorescence in the red channel, suggesting that live Ara- change of the uorescence signal ratio in the ratiometric uo-
bidopsis thaliana tissues have a low level of intracellular FA rescence image compared with the negative control. Further-
activity.12,13 Importantly, upon incubation with a typical stress more, the stimulation of endogenous FA uptake with 1 mM o-
concentration of exogenous FA (3 mM), a signicant change of phenanthroline, a known inhibitor of formaldehyde dehydro-
the uorescence signal ratio is observed in the ratiometric genase,39 also yielded a statistically signicant increase in the
uorescence image (Fred/Fgreen) (Fig. 3). This indicates that the uorescence ratio (Fig. 4). All of these data suggest that FAP is
alteration of the intracellular FA levels in live plant tissues can capable of ratiometric monitoring of endogenous FA activity in
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Fig. 3 Confocal microscopy images of Arabidopsis thaliana incubated with FAP (50 mM) for 45 min at 37 ꢁC (a–d), followed by the addition of
3 mM HCHO (e–h) and incubation for another 2 hours. (a and e) The green channel (475–520 nm), (b and f) the red channel (545–595 nm), (c and
g) the merge images of the bright-light field, green channel and red channel, and (d and h) the ratio images of the red to green channel. lex
¼
405 nm, scale bar ¼ 50 mm. (i) Quantification of the imaging data (d and h). n ¼ 3, error bars were ꢂSD. Statistical analyses performed with a two-
tailed Student’s t-test with unequal variance, ***p value < 0.0001.
Arabidopsis thaliana in vivo. Finally, we tried to apply FAP to
measure the endogenous formaldehyde concentration in the
Conclusions
fresh root and leaf tissues of Arabidopsis thaliana. Spiked HCHO
was used as the internal standard in the prepared tissue
homogenate. The spiked samples were subsequently incubated
with the 20 mM probe for 2 hours and then monitored
(Fig. S10†). We found that the concentrations of endogenous
formaldehyde in the fresh homogenates of the Arabidopsis
In summary, we have successfully developed a ratiometric
uorescent probe, FAP, for monitoring formaldehyde based on
the N,N-dimethylquinolin-6-amine uorophore for the rst
time. This probe displayed a signicant red shi (75 nm) in the
emission proles and an obvious change of the uorescence
signal ratio for the response of FA. We have demonstrated that
this highly selective and effective probe has potential capability
for monitoring FA in real-water samples and live plant tissues.
In vivo imaging results show that FAP can not only effectively
map exogenous FA, but also can sensitively track endogenous
FA. To the best of our knowledge, this is the rst example of
a uorescent probe-based methodology for the ratiometric
imaging of endogenous FA in plant tissues. With these insights,
thaliana root and leaf tissues are 1.8 mmol gꢀ1 and 4.2 mmol gꢀ1
,
respectively. This result is consistent with the previous report
that the average endogenous FA concentration was estimated to
be 0.1–10 mmol gꢀ1 of the fresh weight of the plant.13 Taken
together, these ndings demonstrate that FAP displays the
ability of detection of the biologically relevant endogenous
formaldehyde in live plant tissues.
Fig. 4 Confocal microscopy images of endogenous FA in Arabidopsis thaliana via pretreatment with or without 10% MeOH and 1 mM o-
phenanthroline in PBS buffer for 1 h, followed by exchange into fresh PBS and further incubation with FAP (50 mM) for 3 h at 37 ꢁC before imaging.
(a, e, and i) Green channel (475–520 nm), (b, f, and j) red channel (545–595 nm), and (c, g, and k) merge images of the bright-light field, green
channel and red channel, and (d, h, l) the ratio images of the red to green channel. lex ¼ 405 nm, Scale bar ¼ 50 mm. (m) Quantification of the
imaging data (d, h, l). n ¼ 3, the error bars were ꢂSD. Statistical analyses performed with a two-tailed Student’s t-test with unequal variance, ***p
value < 0.001.
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Acknowledgements
This work is nancially supported by grants from the National
Natural Science Foundation of China (21302094), the Jiangsu
Natural Science Foundation (BK20130552), the Research Fund
for the Doctoral Program of Higher Education of China
(20130091120036), the Fundamental Research Funds for the
Central Universities (20620140214), and the Scientic Research
Foundation of Nanjing Normal University (184080H202B95).
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