2
M. Yang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 245 (2021) 118899
samples and apply to fluorescence imaging for ClO− in live cells. Detection
processes of Fe3+/2+ and ClO− by HPHN were illustrated by using diverse
analytical methods and DFT calculations.
make 3 mL. The solutions were stirred for 7 s before getting UV–
visible spectra.
For ClO−, HPHN (0.31 mg, 1 × 10−6 mol) was dissolved in MeOH
(1 × 103 μL). 54–6 μL of the HPHN was transferred to vials, which
were diluted with buffer to give 2.940 mL. 25.6 μL of NaClO solution
(1.95 M) was diluted with 4.974 mL of distilled water to make
1 × 10−3 M. 6–54 μL of the ClO− was added to each diluted HPHN.
Each vial was filled with buffer to make 3 mL. The solutions were stirred
for 7 s before getting UV–visible spectra.
2. Experimental section
2.1. Materials and equipment
Acros Organics provided di-2-pyridiyl ketone, and Sigma-Aldrich did
other chemicals. 1H and 13C NMR and absorption spectra were gained
with Varian and Perkin Elmer UV–visible spectrometers. A Thermo
Finnigan machine was employed for ESI-MS.
2.6. Competition experiments
For Fe3+/2+, HPHN (3.1 mg, 1 × 10−5 mol) was dissolved in
DMSO (1 × 103 μL). 2 × 10−5 mol of AgNO3, Al(NO3)3, NaNO3, Fe
(ClO4)2, Cr(NO3)3, Co(NO3)2, Hg(NO3)2, Mn(NO3)2, Cd(NO3)2, Ni
(NO3)2, Mg(NO3)2, Zn(NO3)2, KNO3, Fe(NO3)3, Cu(NO3)2 and Ca
(NO3)2 was dissolved in 1 × 103 μL of buffer. 1.65 μL of each cation
and 1.65 μL of Fe2+ (or 1.5 μL for Fe3+) (20 mM) were diluted into
buffer (3 mL). 6 μL of HPHN was added to the mixed solution to
make 2 × 10−5 M. The solutions were stirred for 7 s before getting
UV–visible spectra.
2.2. Synthesis of HPHN
6-Hydroxypicolinohydrazide was synthesized with the literature
method [65]. Yield: 0.20 g (65.3%). 1H NMR (deuterated DMSO): δ =
11.18 (s, 1H), 10.57 (s, 1H), 7.77 (t, J = 7.2 Hz, 1H), 7.37 (s, 1H), 6.81
(d,
J = 9.7 Hz, 1H). HPHN was given by the reaction of
6-hydroxypicolinohydrazide (0.15 g, 1 × 10−3 mol) and 2-hydroxy-1-
naphthaldehyde (0.17 g, 1 × 10−3 mol) in ethanol. The starting mate-
rials were dissolved and stirred in ethanol for 3 h at 23 °C. The yellow
precipitate was filtered and rinsed with ether (0.13 g, 42.3%). 1H NMR
(deuterated DMSO): δ = 12.76 (s, 1H), 12.19 (s, 1H), 9.68 (s, 1H),
8.26 (d, J = 12.8 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 4.3 Hz,
2H), 7.83 (t, J = 6.4 Hz, 1H), 7.62 (t, J = 6.4 Hz, 1H), 7.48 (s, 1H), 7.42
(t, J = 6.4 Hz, 1H), 7.24 (d, J = 8.5 Hz, 1H), 6.90 (s, 1H); 13C NMR (deu-
terated DMSO): δ = 162.61, 158.10, 148.22, 140.51, 132.92, 131.76,
128.96, 127.81, 123.60, 120.80, 118.89, 109.55, 180.59. ESI-MS (m/z):
calcd for [C21H25N4O5 + H+ + DMF + MeOH]+, 413.18; found,
413.50. Anal. Calc. for C21H25N4O5 .2H2O: C, 59.47; H, 4.99; N, 12.24.
Found: C, 59.57; H, 4.76; N, 12.94.
For ClO−, HPHN (0.31 mg, 1 × 10−6 mol) was dissolved in MeOH
(1 × 103 μL). 1 × 10−4 mol of tetraethylammonium salts (X = Br−
,
,
CN−, Cl−, and F−) or tetrabutylammonium salts (X = BzO−, OAc−
and N−3 ) or Na2S, NaH2PO4, NaNO2 and NaOH was dissolved in PBS
buffer (1 × 103 μL). 0.5 mmol of H2O2, tBuOOH, AcOOH and NaClO
was diluted with distilled water to make 5 mL of 0.1 M stock. 3.6 μL of
each anion or oxidizing agent and 3.6 μL of ClO− (100 mM) were diluted
into 3 mL buffer. 60 μL of HPHN was added to the mixed solution to
make 2 × 10−5 M. The solutions were stirred for 7 s before getting
UV–visible spectra.
2.7. pH effect
2.3. UV–visible titrations
For iron, bis-tris buffers (1 × 10−2 M) with pH values (2−12) were
made by mixing KOH and HCl. HPHN (3.19 mg, 1 × 10−5 mol) was dis-
solved in DMSO (1 × 103 μL). 6 μL of the HPHN (1 × 10−2 M) was diluted
to 2.992 mL of buffer to make 2 × 10−5 M. 1 mL of buffer solution was
used to dissolve Fe(NO3)3 (or Fe(ClO4)2) (2 × 10−5 mol). Then,
1.65 μL of the Fe2+ (or 1.5 μL of Fe3+) stock was added to each HPHN.
The solutions were well blended for 7 s before getting UV–visible
spectra.
For iron, HPHN (3.1 mg, 1 × 10−5 mol) was dissolved in DMSO
(1 × 103 μL) and 6 μL of it was diluted to 2.994 mL bis-tris buffer
(pH 7, 1 × 10−2 M) to produce 2 × 10−5 M. 1 mL of buffer was used
to dissolve Fe(NO3)3 (or Fe(ClO4)2) (2 × 10−5 mol). Then, 0.15–1.8 μL
(2 × 10−2 M) of the Fe2+ stock (or 0.15–1.65 μL of the Fe3+) were
added to the HPHN (2 × 10−5 M). The solutions were well blended
for 7 s before getting UV–visible spectra.
For ClO−, HPHN (0.31 mg, 1 × 10−5 mol) was dissolved in 1 × 103 μL
of MeOH and 60 μL of it was diluted to 2.940 mL buffer (PBS, pH 7.4,
1 × 10−2 M) to produce 2 × 10−5 M. 256 μL of NaClO solution
(1.95 M) was diluted with 4.744 mL of distilled water to make 0.1 M.
0.3–3.9 μL of the ClO− stock was added to the HPHN (2 × 10−5 M).
The solutions were well blended for 7 s before getting UV–visible
spectra.
For ClO−, a series of PBS buffer (1 × 10−2 M) with pH values
(2–12) were made by mixing KOH and HCl. HPHN (0.32 mg,
1 × 10−6 mol) was dissolved in MeOH (1 × 103 μL). 60 μL of HPHN
(1 × 10−3 M) was diluted to 2.940 mL of buffer to make 2 × 10−5
M. 256 μL of NaClO (1.95 M) was diluted with 4.744 mL of distilled
water to make 0.1 M. Then, 3.6 μL of the ClO− stock was added to
each HPHN. The solutions were well blended for 7 s before getting
fluorescence spectra.
2.4. Fluorescence titration
HPHN (0.31 mg, 1 × 10−6 mol) was dissolved in 1 × 103 μL of MeOH
and 60 μL of it was diluted to 2.940 mL PBS buffer to afford 2 × 10−5 M.
256 μL of NaClO (1.95 M) was diluted with 4.744 mL of distilled water to
make 0.1 M. 0.3–4.2 μL of the ClO− stock was added to the HPHN
(2 × 10−5 M). The solutions were well blended for 7 s before getting
fluorescence spectra.
2.8. EPR
Liquid N2 was employed to freeze the reaction solution 5 min after
Fe2+ (1 × 10−3 M) reacted with HPHN (2 × 10−3 M) in a EPR tube.
The EPR spectrum was recorded under microwave power conditions
(1.0 mW and 9.153 GHz).
2.5. Job plots
2.9. Imaging of live cells
For iron, HPHN (3.1 mg, 1 × 10−5 mol) was dissolved in 1 × 103 μL of
DMSO. 5.4–0.6 μL of the HPHN were put to vials, which were diluted
with buffer to give 2.994 mL. 1 mL of buffer was used to dissolve Fe
HeLa cells were kept under the previous conditions [66]. For the
fluorescence quenching, they were treated with HPHN (5 × 10−5
M) for 3 h prior to additional incubation with NaClO (2 × 10−4 M) for
15 min. Imaging was executed with a fluorescence microscope using a
GFP light cube (λex = 470 nm and λem = 510 nm).
(NO3)3 (or Fe(ClO4)2) (1 × 10−5 mol). 0.6–5.4 μL of the Fe2+ (or Fe3+
)
were added to each diluted HPHN. Each vial was filled with buffer to