Rapid and Visual Detection and Quantitation of Ethylene Released from Ripening Fruits: The New Use of Grubbs Catalyst

Article abstract of DOI:10.1021/acs.jafc.8b05874

Herein, we report on fluorophore-tagged Grubbs catalysts as turn-on fluorescent probes for the sensitive detection and quantitation of ethylene, a plant hormone that plays a critical role in many phases of plant growth and fruit ripening. The ruthenium-based weak fluorescent probes were prepared handily through the metathesis reaction between the first-generation Grubbs catalyst and selected fluorophores that have high quantum yields and contain terminal vinyl groups. Upon exposure to ethylene, fluorescence enhancement was observed via the release of fluorophore from the probe. Our probe shows an excellent limit of detection for ethylene at 0.9 ppm in air and was successfully applied for monitoring ethylene released during the fruit-ripening process. Our work opens up a new avenue of application of Grubbs catalysts for bioanalytical chemistry of ethylene, which is critically important in plant biology, agriculture, and food industry.

Full text of DOI:10.1021/acs.jafc.8b05874

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New Analytical Methods
Rapid and visual detection and quantification of ethylene
released from ripening fruits: the new use of Grubbs catalyst
Mingtai Sun, Xin Yang, Yuannian Zhang, Suhua Wang, Ming Wah Wong, Runyan Ni, and Dejian Huang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05874 • Publication Date (Web): 03 Dec 2018
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Page 1 of 26
Journal of Agricultural and Food Chemistry
1 Rapid and Visual Detection and Quantitation of Ethylene
2 Released from Ripening Fruits: the New Use of Grubbs Catalyst
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Mingtai Sun,†, ‡ Xin Yang,† Yuannian Zhang,† Suhua Wang,‡ Ming Wah Wong,† Runyan Ni§,
Dejian Huang*,†,§
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†Food Science and Technology Program, Department of Chemistry, National University of
Singapore, 3 Science Drive 3, 117543, Singapore
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‡School of Environment and Chemical Engineering, North China Electric Power University,
Beijing 102206, China.
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§National University of Singapore (Suzhou) Research Institute, 377 Linquan Street, Suzhou,
Jiangsu, 215123, China.
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*Corresponding author
Tel: 65-6516-8821. Fax: 65-6775-7895. Email: chmhdj@nus.edu.sg
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ABSTRACT:
Herein we report on fluorophore-tagged Grubbs catalysts as turn-on fluorescent probes for the
sensitive detection and quantitation of ethylene, a plant hormone that plays a critical role in many
phases of plant growth and fruit ripening. The ruthenium (Ru) based weakly fluorescent probes
were prepared handily through metathesis reaction between the first generation Grubbs catalyst
and selected fluorophores that have high quantum yields and contain terminal vinyl groups. Upon
exposure to ethylene, fluorescence enhancement was observed via the release of fluorophore from
the probe. Our probe shows an excellent limit of detection (LOD) for ethylene at 0.9 ppm in air
and was successfully applied for monitoring ethylene released during the fruit ripening process.
Our work opens up a new avenue of application of Grubbs catalysts for bioanalytical chemistry of
ethylene, which is critically important in plant biology, agriculture and food industry.
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KEYWORDS: Grubbs catalyst, ethylene, fluorescent probes, olefin metathesis reaction, fruit
ripening
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INTRODUCTION
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Ethylene is a gaseous plant hormone that plays critical roles in many phases of plant biology
including plant growth, development, response to environmental stresses and pathogen infection1
as well as seed germination, and fruit ripening.2-4 In food and agriculture, ethylene concentration
has been used as a maturity index to determine the time of harvest.5 Knowing the concentration of
ethylene is critical since ethylene activity varies with the fruit maturity and is dependent on the
type of fruits.6 In storage room and transportation chains, the control of ethylene concentration is
necessary to avoid the deterioration of produce that are sensitive to ethylene.7 Ethylene
concentrations of less than 1 ppm could be effective for the ripening of certain fruits. However,
this concentration is usually held between 10 to 200 ppm in ripening rooms depending on the type
of fruits.1 Thus, knowing the relationship between ethylene concentration and the fruit ripening
process is important in order to manage the harvesting, storage and transportation processes yet
there is no convenient way to rapidly quantitate ethylene concentrations.
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Currently, quantitation of ethylene relies heavily on time-consuming and sophisticated
traditional methods such as GC (GC-MS), photoacoustic spectroscopy, and electrochemical
methods. Hence, real-time measurements are highly desired to control fruit ripening.5, 8-11 In the
past decade, great progress has been made in the chemical biology field with the help of fluorescent
probes that are designed to be sensitive and selective towards specific biological targets including
small molecules of importance such as gluthathione, ATP, nitric oxide, nitrogen dioxide, singlet
oxygen, hypochlorous acid, and hydrogen sulfide.12-19 However, only limited work has been
reported on the fluorescent probes for ethylene detection. The reported methods took advantage of
the ability of ethylene as a ligand that could reversibly coordinate to metals particular copper (Cu(I))
and silver (Ag(I)).20-24 However, these methods suffer from low sensitivity because ethylene is a
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rather weak ligand and require high concentration in order to effectively undergo ligand
substitution reaction that releases the metal bound fluorophores. Therefore, a new approach is
needed to achieve probes with high sensitivity.25 To this end, we were intrigued by the potential
of using alkene metathesis catalyst based on ruthenium carbene complex, PhCH=RuCl2(PCy3)2
(Ph = phenyl, Cy = cyclohexyl) the first generation Grubbs catalyst, which has found broad
application in organic and polymer synthesis.26, 27 We envisioned that the high tolerance of Grubbs
catalyst towards other functional groups, good air and water stability would be of potential to
explore its application for detection of ethylene if we could design and prepare fluorophore-tagged
Grubbs catalysts that are responsive, selective, and sensitive to ethylene.
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MATERIALS AND METHODS
Reagents and apparatus. Chemical reagents were purchased from the commercial sources
66 (Sigma-Aldrich Chemical Co., Singapore) and used directly without further purification unless
67 specified. The solvents dichloromethane and THF were further treated before use by distillation
68 and dried over molecular sieves before use. 1-Vinylpyrene, 5, was synthesized according to a
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literature report.28 Compounds 1 and 3 (Figure 1) were prepared in our lab. Aqueous solutions
were all prepared using ultrapure water (18.2 MΩ·cm) from a Millipore water purification system,
and all glassware was cleaned with ultrapure water and then dried before use. Fluorescence
measurement was recorded on a Fluoromax-4 spectrofluorometer (HORIBA Jobin Yvon,
Piscataway, NJ) equipped with a plotter unit and a quartz cell (1 cm × 1 cm). UV/Vis absorption
was recorded on a UV-1601 UV-visible spectrophotometer (Shimadzu, Kyoto, Japan) fitted with
a quartz cell. The FTIR spectra were obtained with a Nicolet iS10 spectrometer (Thermo Scientific,
Madison, WI). 1H and 13C NMR spectra were recorded with an AC300 spectrometer at 300 MHz
or an AV500 spectrometer (Bruker, Karlsruhe, Germany) at 500 MHz. The electrospray ionization
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mass spectra were obtained from an LCQ ion trap mass spectrometer (Finnigan/MAT, San Jose,
CA) equipped with electrospray ionization (ESI) source. Thin-layer chromatography (TLC) was
performed by using F254 silica gel 60 plates (Merck, Singapore).
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Synthesis of compound 2. To a solution of 1st generation Grubbs catalyst (39 mg, 0.046 mmol,
1 equiv) in dry CH2Cl2 (5 mL), compound 1 (149 mg, 0.368 mmol, 8 equiv) was added at room
temperature. The solution was stirred for another 15 min. The solvent was removed under vacuum,
and the residue was repeatedly washed with acetone (3 mL) and dried in vacuo for several hours.
Compound 2 was obtained as an orange solid. Yield: 48 mg (92%). 1H NMR (500 MHz, CDCl3)
δ 20.17 (s, 1H), 8.66 (s, 2H), 7.32 (d, J = 8.5 Hz, 2H), 2.69 (m, 6H), 2.57 (s, 6H), 2.35 (q, J = 7.5
Hz, 4H), 1.93–1.70 (m, 30H), 1.49 (m, 12H), 1.35 (s, 6H), 1.31–1.18 (m, 18H), 1.02 (t, J = 7.5 Hz,
6H). 31P NMR (202 MHz, CDCl3) δ 35.58. 13C NMR (126 MHz, CDCl3) δ 153.95, 151.95, 140.31,
137.99, 136.46, 132.93, 131.81, 129.65, 129.47, 32.42, 32.35, 32.28, 29.67, 27.81, 27.76, 27.73,
26.50, 26.36, 17.10, 14.64, 12.53, 11.76. Anal. Calcd for C60H93BCl2F2N2P2Ru: C, 64.05; H, 8.33;
N, 2.49. Found: C, 64.48; H, 8.27; N, 2.42. HR-MS (ESI positive): m/z calculated for
[C60H93BCl2F2N2P2Ru + H]+ 1124.5308; found 1124.5318.
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Synthesis of compound 4. To a solution of 1st generation Grubbs catalyst (25 mg, 0.03 mmol,
1 equiv) in dry CH2Cl2 (5 mL), compound 3 (96 mg, 0.18 mmol, 6 equiv) was added at room
temperature. The solution was allowed to stir for another 15 min. The solvent was removed under
vacuum, and the residue was repeatedly washed with acetone (3 mL) and dried under vacuum for
several hours. Compound 4 was obtained as a red solid. Yield: 33 mg (91%). 1H NMR (300 MHz,
CD2Cl2) δ 19.91 (s, 1H), 8.38 (d, J = 6.8 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 3.76 (s, 2H), 3.26 (s,
2H), 2.72–2.35 (m, 24H), 2.12 (s, 6H), 1.84–1.60 (m, 30H), 1.42 (m, 12H), 1.20 (m, 18H), 1.13–
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0.97 (m, 6H). P NMR (202 MHz, CD2Cl2) δ 36.33. C NMR (126 MHz, CD2Cl2) δ 206.23,
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150.00, 130.92, 129.32, 35.55, 35.06, 32.11, 32.04, 31.96, 30.52, 29.61, 27.83, 27.79, 27.75, 26.97,
26.88, 26.53, 26.35, 26.33, 26.20, 16.98, 14.51, 14.01. Anal. Calcd for C66H105BCl2F2N4P2Ru: C,
64.07; H, 8.05; N, 4.53. Found: C, 64.11; H, 8.09; N, 4.52. HR-MS (ESI positive): m/z calculated
for [C66H105BCl2F2N4P2Ru + H]+ 1237.6388; found 1237.6415.
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Synthesis of compound 6. To a solution of 1st generation Grubbs catalyst (PhCH=Ru(PCy3)2Cl2,
59 mg, 0.072 mmol, 1 equiv.) in dry CH2Cl2 (5.0 mL), 1-vinylpyrene, 5 (165 mg, 0.72 mmol, 10
equiv) was added at room temperature. The purple solution turned to yellow immediately and was
stirred for another 10 min. The solvent was removed under vacuum, and the residue was repeatedly
washed with acetone (3 mL) and dried under vacuum for several hours. Compound 6 was obtained
as a khaki solid. Yield: 57 mg (84%). 1H NMR (500 MHz, CDCl3) δ 21.85 (s, 1H), 9.89 (d, J =
8.4 Hz, 1H), 8.96 (d, J = 9.5 Hz, 1H), 8.43 (d, J = 7.7 Hz, 1H), 8.37 (t, J = 8.6 Hz, 3H), 8.09 (d, J
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= 8.4 Hz, 1H), 8.06–7.97 (m, 2H), 2.72 (m, 6H), 1.91–1.63 (m, CyH), 1.52–1.10 (m, CyH). C
NMR (126 MHz, CDCl3) δ 144.89, 132.82, 132.18, 131.47, 131.18, 128.89, 128.37, 127.04,
126.77, 126.21, 125.75, 125.63, 125.60, 124.59, 122.54, 32.44, 32.37, 32.30, 29.81, 27.89, 26.51.
31P NMR (202 MHz, CDCl3) δ 36.09. FT-IR (KBr, cm-1): 2925, 2849, 1582, 1500, 1445, 1216,
1174, 1005, 916, 848, 732, 714, 510. Anal. Calcd for C53H76Cl2P2Ru: C, 67.21; H, 8.09. Found:
C, 67.05; H, 8.05. ESI 911.1(100%) (M-Cl)+, 913.1(62%), 907.1(60%), 906.1 (35%).
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Reaction of 1-vinylpyrene with 2nd generation Grubbs catalyst. To a solution of 2nd
generation Grubbs catalyst (52 mg, 0.055 mmol, 1 equiv) in dry CH2Cl2 (5 mL), 1-vinylpyrene
(125 mg,0.55 mmol, 10 equiv) was added at room temperature and was allowed to stir for another
10 min. The solvent was removed under vacuum, and the residue was repeatedly washed with
acetone (5 mL) and dried under vacuum for several hours. An orange solid with strong green
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fluorescence was obtained. This compound is slightly soluble in common solvents such as hexane,
acetone, CHCl3, toluene, CH2Cl2, and DMSO.
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Determination of ethylene concentration by NMR spectroscopic analysis. To an NMR tube,
126 5 μL of methyltriethoxysilane and 0.6 mL of CD2Cl2 were added. Ethylene gas was bubbled to the
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solution for 5 min carefully using a long syringe needle. The 1H NMR spectrum of the resulting
solution was measured. The concentration of ethylene dissolved in CD2Cl2 (nethylene) was calculated
using the following equation:
퐼푒푡ℎ푦푙푒푛푒 × 푛푀푇퐸푆 × 6
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푛푒푡ℎ푦푙푒푛푒
=
퐼푀푇퐸푆(푎) × 4
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where Iethylene represents the integration of ethylene peak at δ 5.43; IMTES represents the integration
of methyltriethoxysilane peak at δ 3.80; nMTES represents the concentration of
methyltriethoxysilane in CD2Cl2.
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Procedures for ethylene gas sensing and other species. Generally, probe 6 was dissolved in
CH2Cl2 to get a stock solution with the concentration at 1×10-3 M. Ethylene gas at concentrations
of 50 ppm, 100 ppm, and 200 ppm were obtained from commercial ethylene gas cylinder (Chem-
Gas Pte Ltd, Singapore). Then the ethylene gas was diluted to 25, 12.5, 6.25, and 2.5 ppm by
adding corresponding ethylene gas to a round bottom flask with a certain volume, respectively. 2
mL of these gases were injected to 2 mL of CH2Cl2 solvent in a small sealed bottle with a plastic
gap using a syringe slowly before 10 μL of probe 6 stock solution was added. The fluorescence
intensity of the solution was measured after 3 min by recording the fluorescence spectra in the
range 380-600 nm using a 365-nm excitation wavelength and a 500 nm/min scan rate. For the
control, the probe solution was prepared by adding the stock solution of the probes into 2 mL of
CH2Cl2 solvent, and then the fluorescence was measured. SO2, NO2, H2S, CO2, and NH3 gases
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were prepared according to previous methods.29 Generally, 200 μL of these gas samples were
bubbled into the probe solution in CH2Cl2 before 10 μL of the probe stock solution was added and
the fluorescence spectra were recorded. For the selectivity of the probe towards ethylene solution
in CH2Cl2, the known concentration of ethylene in CD2Cl2 was used to prepare the desired ethylene
solution at concentrations of 5 and 10 μM. Other species including acetonitrile, ethanol, ethyl
acetate, water, toluene, THF, 1-propanol, and 1-butanol were calculated and taken using a pipette
to 2 mL of CH2Cl2 solvent to give the desired solution at concentrations of 5 and 10 μM.
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Procedures on the detection of ethylene released during fruit ripening. Passion fruits (346
g) were loaded in a capped gas-tight jar (2.25 L), and then the outlet was closed. After incubating
the jar with a given time (2, 4, 6, and 8 h), sample gas (2.0 mL) was taken from the headspace of
the jar using a gas-tight syringe with a long needle. The sample gas was bubbled slowly to the
CH2Cl2 solution (2.0 mL) in a capped cuvette to dissolve the ethylene. To this solution, the probe
stock solution (10 µL) was added and the mixture was shaken for 3 min. The fluorescence intensity
of the solution was recorded from the fluorescence spectra in the range 380-600 nm using a 365
nm excitation wavelength and a 500 nm/min scan rate. The ethylene released from the apple (366
g), banana (360 g), and avocados (375 g) were determined using the same procedure as that of the
passion fruit.
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The concentration of ethylene in the jar (Cppm) was measured using the following equation:
I
퐼0
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퐶푝푝푚 = ( ― 1.042)/0.044
The ethylene releasing rate of these fruits was calculated using the following equation:
∆
푝푝푚 × 푉푗푎푟
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S =
푊푓푟푢푖푡 × ∆ℎ
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Where S represents speed rate of ethylene release, Δppm represents the ethylene concentration
change in the jar, Vjar represents the volume of the jar, Wfruit represents the weight of selected fruit,
Δh represents the incubation time of selected fruit.
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Statistical Analysis: One-way analysis of variance (ANOVA) and Tukey’s test using SPSS 17.0
software for Windows (SPSS Inc., Chicago, IL) were applied to evaluate the significant differences
among different fermentations (P < 0.05).
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RESULTS AND DISCUSSION
Design and synthesis of the fluorophore-tagged Grubbs catalysts-based probes. We
174 designed and prepared three fluorophore-tagged Grubbs catalysts that were responsive to ethylene
175 sensitively and their application for sensitive detection of ethylene released during the fruit
176 ripening process. Our design strategy to the fluorophore-tagged Grubbs catalysts based probes
177 were illustrated in Figure 1. To obtain the ideal probe with optimal sensitivity, the fluorophores
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with high fluorescence quantum yields were chosen and attached to the Grubbs catalyst through
the vinyl group by metathesis reaction. Moreover, the fluorescence of the resulted complex should
be weak if there were energy or electron transfer between fluorophores and Ru center.
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Taking these factors into consideration, we first prepared compound 2 (Figure 1), a BODIPY
tagged Ru complex via the metathesis reaction between the first generation Grubbs catalyst and
the fluorophore 1. The design and preparation of these probes were reasonable and easy, with high
yield. The maximal absorbance and emission wavelengths of probe 2 were centered at 528 and
545 nm, respectively. The probe itself showed relatively weak fluorescence suggesting the
effective fluorescence quench by energy transfer between the fluorophore and Ru center. However,
when the dichloromethane solution of probe 2 was irradiated at 500 nm for 20 min, the
fluorescence steadily increased over time, indicating that probe 2 was unstable under the assay
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conditions that may be needed for ethylene monitoring. Furthermore, H NMR spectra of
compound 2 showed that it was oxidized over time to fluorescent BODIPY-CHO. We speculate
that BODIPY group coupled directly to the Ru metal center may act as a photosensitizer30, 31 which
may induce the decomposition of the probe under irradiation.
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In the hope to reduce the photosensitivity and increase the stability of the fluorophore-tagged
Grubbs catalyst, we prepared probe 4, in which the BODIPY group is separated from the Ru center
through pyrazine ring. Dichloromethane solution of compound 4 showed weak fluorescence likely
due to efficient quench effect of the FRET mechanism. Moreover, upon treatment of probe 4 with
ethylene gas, 8-fold fluorescence enhancement accompanied by bright green fluorescence color
was obtained, suggesting a potential detection system for ethylene. However, we found that
compound 4 was somewhat unstable after 20 min irradiation. Therefore, although probe 4
exhibited increased stability compared with compound 2, it is still not an ideal fluorescent probe
for ethylene analysis in assay conditions.
We suspected that, although BODIPY has high quantum yields, its property as photosensitizer
could compromise the stability of the probes. We then turned to pyrene, which has high
fluorescence quantum yield but is a poor photosensitizer. The pyrene-based probe 6 was
synthesized readily in high yield by attaching the pyrene group to Ru center by metathesis reaction
between the first generation Grubbs catalyst with an excess amount of 1-vinylpyrene, 5, which
showed the fluorescence maximum at 394 nm and a shoulder peak at 414 nm exhibiting high bluish
violet fluorescence. The dichloromethane solution of 6 showed weak aquamarine fluorescence
color with maximum emission at 468 nm and a shoulder peak at 500 nm, indicating a
bathochromic-shift effect when compound 5 was bound to Ru metal center. This result was in
agreement with those observed with pyrene functionalized Ru nanoparticles having Ru carbene π
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bonds, suggesting an extended conjugation of metal-carbene π bond which may lead to the
appearance of new emission peaks in the lower energy region, allowing the pyrene moieties to
behave analogously to their dimeric counterparts32. Attempts to synthesize the second generation
Grubbs catalyst based probe using similar procedure failed while only the coupling product of 1-
vinylpyrene was observed. Somehow the second generation Grubbs catalyst acted as a metathesis
reaction catalyst was likely due to its higher catalytic activity comparing to the first generation
one. UV/Vis absorption spectra of the reaction mixture showed that absorption peak at 441 nm for
the probe 6 had disappeared and new absorption bands emerged at 495 nm, 360 nm, and 285 nm,
which can be attributed to the reaction product and the released of compound 5. Consistently, the
1H NMR spectra of the reaction mixture showed that compound 5 was released (Figure 2A). When
the probe solution was bubbled with 500 ppm ethylene for one min, the intensity of the signals
belonging to the probe disappeared gradually, while several new peaks appeared which can be
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assigned to compound 7 and compound 5. The P NMR also shows the transformation of the
probe 6 to compound 7 (Figure S7). These results suggested that 6 reacted with ethylene via alkene
metathesis to give compound 7 and the highly fluorescent species 1-vinylpyrene in quantitative
yield (Figure 2B).
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When the probe 6 was treated with ethylene gas at an ambient temperature in dichloromethane,
the fluorescence intensity of the solution was turned on significantly and reached its maximum
within three minutes. Without ethylene, no significant fluorescence enhancement was observed
when probe 6 was irradiated with excitation light at 365 nm for 25 min, indicating relatively good
photostability. In addition, compound 6 in the solid state was not sensitive to air or humidity and
could be kept in the refrigerator at 4 °C for 3 months without any decomposition. In brief, among
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the three synthesized probes, compound 6 showed the best sensing performance with high signal-
to-noise ratio and good stability (Figure 3).
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Fluorescence responses of the probe toward ethylene. To test the sensitivity, an ethylene stock
237 solution was prepared by bubbling ethylene gas to CD2Cl2 to give the NMR spectrum of the
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resulting solution. The ethylene concentration was calculated from the integration of the ethylene
peak and compared with the added internal standard (methyltriethoxysilane). Upon addition of an
increased amount of ethylene, the emission intensity of probe 6 (5.0 μM) that was dissolved in
CH2Cl2 at 468 nm decreased gradually, whereas the emission at 394 nm increased significantly,
resulting in fluorescence enhancement (Figure 4A) by ca 20-fold when the concentration of
ethylene reached 20 μM. Moreover, the fluorescence enhancement has a good dose-response
relationship (R2 = 0.992) with the concentration of ethylene in a wide concentration range (Figure
4A inset). In addition, this fluorescence enhancement could be easily visualized with the probe
solution changing in color from green to intense bluish violet under a 365 nm UV lamp (Figure
4A inset image).
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For detection of gaseous ethylene, various concentrations of ethylene gas in the air were first
prepared and added to the solutions of 6 in dichloromethane with a gas-tight syringe. The
fluorescence intensity of the solution was measured after three minutes. A good ethylene dose
response was observed for the fluorescence intensity at 394 nm, with linear dependence in a wide
range of 0-200 ppm (R2 = 0.990), which could be used for the quantification of ethylene gas (Figure
4B). The limit of detection (LOD) for ethylene was determined at 0.9 ± 0.016 ppm in the air, which
is sufficiently sensitive to test the ethylene gas concentrations released by the fruits. Although the
LOD is much lower than that of GC, which has been developed for decades, it is sufficiently
sensitive to test the ethylene gas concentrations released by the fruits. The probe also has other
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advantages including low cost, mobility, visual detection, and real-time detection. Our probe has
the best sensitivity among the developed fluorescent probes reported up till now. Our work opened
up a new avenue of application of fluorescent probes for bioanalytical chemistry of ethylene. We
believe more fluorescent probes with comparable sensitivity to GC can be developed.
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Selectivity. The probe 6 not only exhibited good sensitivity for the detection of ethylene but also
showed good selectivity (Figure 5A). Other possible coexisting species such as acetonitrile,
ethanol, ethyl acetate, water, toluene, THF, 1-propanol, and 1-butanol could not turn on the
fluorescence at the same concentration as ethylene, which gave a remarkable fluorescence
enhancement. In addition, the response of the probe to other gaseous species including CO2, NO2,
SO2, NH3, and H2S were also examined under the same conditions as ethylene (50 ppm). CO2,
SO2, and H2S only enhanced the fluorescence intensity slightly, while NO2 and NH3 only induced
slight fluorescence quench, since these species may induce the decomposition of Grubbs catalyst
slowly.27 It is worth mentioning that no apparent interference was obtained in fluorescence
intensity of solution 6 in the presence of other potential coexisting species at the concentration of
50 ppm (Figure 5B). The good selectivity could be attributed to the high tolerance of Grubbs
catalyst for other functional group and reaction priority to vinyl group.
273
Fluorescence determination of ethylene released from ripening fruits. Climacteric fruits
274 increase respiration and ethylene biosynthesis rates during fruit ripening. Ethylene formation
275 during fruits ripening has been used as a maturity index to determine the time of harvest. The
276 control of ethylene concentration is also of great importance for the ripening and storage of fruits.
277 Thus, it would be very useful to be able to monitor the ethylene release conveniently in ambient
278 air around the fruits. To demonstrate the utility of 6 in such application, we developed a simple
279
method for determining the ethylene released during fruits ripening process. As shown in Figure
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6A, weighed fruits were enclosed in an airtight jar. At a given time, 2 mL of gas sample was taken
from the headspace of the jar by an air-tight syringe and bubbled slowly into the dichloromethane
solvent in a cuvette with a rubber cap. Probe 6 solution was then added to the same cuvette
immediately. Then the fluorescence intensity of the solution was measured after three minutes.
The ethylene releasing dynamics of the different fruits were determined by monitoring the ethylene
concentrations over time. The response curves of the different fruits were shown in Figure 6B. For
the four fruits measured, there was a linear increase of ethylene concentrations with increased
storage time. Passionfruit gave the most remarkable response followed by avocado, banana, and
apple. From the slopes of the curves, the rates of ethylene released for different fruits were
determined in a range of 5-80 μL/kg/h (Figure 6C). The result was consistent with previous
reports.8
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In summary, we have demonstrated that the fluorophore-tagged first generation Grubbs catalysts
are promising for rapid and sensitive visual detection of ethylene as well as quantitation of ethylene
released during fruits ripening. In particular, probe 6 showed the best ethylene sensing
performance with good stability. Our finding would open up a new avenue of application of Grubbs
catalysts for bioanalytical chemistry of alkenes that are of critical importance for plant science.
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ASSOCIATED CONTENT
Supporting Information
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Synthesis of the three probes 2, 4, and 6, decomposition route of compound 2 under light, the
reaction of 1-vinylpyrene with 2nd generation Grubbs catalyst, ANOVA data regarding selectivity
of probe 6, characterization of the synthesized probes 2, 4, and 6, fluorescence and absorption
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responses of probe 6 toward ethylene, the images of weighted fruits which are enclosed in the gas-
tight jar. This material is available free of charge via the Internet at http://pubs.acs.org.
303
FUNDING SOURCES
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305
306
The work was supported by Singapore Ministry of Education for financial support (grant no:
MOE2014-T2-1-134), the National Natural Science Foundation of China (grant no: 21475134,
21775042, 2160151), and Natural Science Foundation of Jiangsu, China (grant no: BK20141219).
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FIGURE CAPTIONS
Figure 1. Illustration of fluorescence turn-on detection of ethylene and preparation of the fluorophore-Ru
based probes.
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Figure 2. The 1H NMR spectra of A) probe 6; (B) probe 6 bubbled with 500 ppm ethylene; (C) probe 6
bubbled with excess ethylene. (D) Mechanism of ethylene sensing using pyrene tagged Grubbs catalyst
probe 6.
Figure 3. Normalized fluorescence enhancement of the three probes, 2, 4, and 6, treated with excess
ethylene gas and control. All data were taken 20 min after treatment.
Figure 4. (A) The emission spectra of the probe 6 (5 μM) exposed to different concentrations of ethylene
(0-30 μM). Inset shows the linearity relationship of I/I0 as a function of the concentration of ethylene,
where I0 and I represent the fluorescence intensity at 394 nm (λex = 365 nm) before and after the addition
of ethylene 3 min, respectively. (B) Linearity relationship of fluorescence enhancement as a function of
the ethylene gas concentration in the range of 0-200 ppm.
Figure 5. (A) Selectivity of the probe 6 (5 μM) for ethylene, determined as the fluorescence response
after addition of these species at 5 and 10 μM. (B) Selectivity and interference of the probe 6 (5 μM) for
ethylene in the presence of other gaseous species at 50 ppm. I0 and I represent the fluorescence intensity
at 394 nm (λex = 365 nm) before and after the addition of ethylene, respectively.
Figure 6. (A) Procedures for determining the ethylene released from ripening fruits using probe 6. (B)
Fluorescence intensity changes of the probe exposed to ethylene released from different fruits at different
incubation time (2-8 h). (C) Ethylene releasing rates of different fruits.
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1st
generation
PCy3
Ru
FL
Grubbs
catalyst
Cl
Cl
C2H4
FL
FL
H
H
ET
Metathesis
reaction
PCy3
FL = Fluorophore
Highly Fluorescent
Weakly Fluorescent
N
N
N
B
N
F
N
N
B
F
F
F
3
5
1
Grubbs
catalyst 1st
Grubbs
catalyst 1st
Grubbs
catalyst 1st
PCy3
PCy3
PCy3
Cl
Ru
Cl
Cl
Cl
Cl
Ru
Ru
Cl
PCy3
PCy3
PCy3
N
N
N
B
N
F
F
N
N
B
F
F
4
6
2
Figure 1
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