Photophysical properties of a new water soluble tetra thiamine substituted zinc phthalocyanine conjugated to gold nanorods of different aspect ratios

Article abstract of DOI:10.1039/c4dt00197d

A water soluble zinc phthalocyanine substituted with thiamine is reported in this work. The aggregation of this compound in aqueous solutions causes quenching of the fluorescence quantum yields. Gold nanospheres and nanorods were linked to the phthalocyanine. X-ray photoelectron spectroscopy showed that both the amine and the sulphur groups on the thiamine substituent of the zinc phthalocyanine were involved in the linking to gold nanoparticles. The Pc showed an increase in the fluorescence quantum yields in the presence of the nanoparticles. The singlet oxygen quantum yield increased when the Pc was conjugated to the nanoparticles and even higher for larger aspect ratio gold nanorods. the Partner Organisations 2014.

Full text of DOI:10.1039/c4dt00197d

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Photophysical properties of a new water soluble
tetra thiamine substituted zinc phthalocyanine
conjugated to gold nanorods of different
aspect ratios
Cite this: Dalton Trans., 2014, 43,
8230
Thandekile Mthethwa, Edith Antunes and Tebello Nyokong*
A water soluble zinc phthalocyanine substituted with thiamine is reported in this work. The aggregation of
this compound in aqueous solutions causes quenching of the fluorescence quantum yields. Gold nano-
spheres and nanorods were linked to the phthalocyanine. X-ray photoelectron spectroscopy showed that
both the amine and the sulphur groups on the thiamine substituent of the zinc phthalocyanine were
involved in the linking to gold nanoparticles. The Pc showed an increase in the fluorescence quantum
yields in the presence of the nanoparticles. The singlet oxygen quantum yield increased when the Pc was
conjugated to the nanoparticles and even higher for larger aspect ratio gold nanorods.
Received 20th January 2014,
Accepted 28th February 2014
DOI: 10.1039/c4dt00197d
www.rsc.org/dalton
Gold nanoparticles have been vastly studied for biological
applications because they are non-toxic in the biological
Introduction
Metallophthalocyanines (MPcs) have found diverse appli- systems. Gold nanoparticles (AuNPs) are also known to be
cations such as in non-linear optics,1 electrocatalysis2 and effective for photothermal therapy (PTT).10 Conjugation of
photodynamic therapy (PDT).3 MPcs absorb strongly in the red AuNPs to Pcs should result in the combined PDT and PTT. In
region of the UV-visible spectrum and produce high singlet addition, AuNPs can facilitate photodynamic therapy by
oxygen quantum yields which make them very interesting for increasing singlet oxygen quantum yields through increased
photodynamic therapy. Solubility of phthalocyanines is a intersystem crossing.11 Our group has reported on the conju-
major limitation. Substituents can be introduced at the peri- gation of AuNPs to Pcs with improved photophysical and
pheral and non-peripheral positions of the phthalocyanine photochemical properties.12–15 The improvement in singlet
ring to improve solubility.4 Water solubility is important for oxygen quantum yield12 in particular is of significance for PDT
applications of Pcs in PDT. There have been a number applications. Conjugates of AuNPs with Pcs may also be used
of studies on functionalization of phthalocyanines with bio- for drug delivery.16,17
molecules including polylysine,5 carbohydrates6 and folic
Of the AuNPs, gold nanorods (AuNRs) have become more
acid.7 These molecules provide biocompatibility and water interesting because their absorption wavelength can be tuned
solubility for phthalocyanines. In this work thiamine (vitamin to near infrared (NIR) regions where tissue penetration is more
B1) is employed as a ring substituent in order to form a water efficient.18 The combined PDT and PTT is expected to be more
soluble zinc phthalocyanine. Thiamine is employed as a sub- prominent for AuNRs which absorb in the NIR regions where
stituent for phthalocyanines for the first time.
phthalocyanines absorb, allowing for the use of one wave-
Thiamine is a water soluble vitamin8 which is deficient in length for excitation of both. In this study, we report on the
patients with advanced stage cancer. It has been reported that conjugation of a water soluble zinc phthalocyanine containing
if thiamine is administered in high doses, it can reduce tumor thiamine to AuNRs for potential coupled PDT and PTT. This
proliferation.9 Thus the conjugation of thiamine to phthalo- work reports on the effects of the aspect ratio of gold nanorods
cyanine is expected to enhance water solubility and improve on the photophysical and photochemical properties of the
cancer treatment through the synergistic effect.
thiamine substituted phthalocyanine. It has been reported
that AuNRs possess superior long blood circulation time due
to the anisotropic geometry.19 It is with this in mind that we
study the effects of different sizes (aspect ratios) of AuNRs on
the photophysical behavior of zinc tetra (4-(thiamine)phthalo-
cyanine) (ZnTTAPc) (4).
Department of Chemistry, Rhodes University, Grahamstown 6140, South Africa.
E-mail: t.nyokong@ru.ac.za; Fax: +27 46 6225109; Tel: +27 46 6038260
8230 | Dalton Trans., 2014, 43, 8230–8240
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were taken for each sample of gold nanoparticles. Gold was
analysed at wavelengths of 208, 242 and 267 nm.
Experimental
Materials
Ground state electronic absorption spectra were performed
Zinc chloride, gold(III) chloride trihydrate, sodium boro- using a Shimadzu UV-2550 spectrophotometer. Fluorescence
hydride, cetyltrimethylammonium bromide, (CTAB), silver excitation and emission spectra were recorded on a Varian
nitrate and ascorbic acid, thiamine hydrochloride, potassium Eclipse spectrofluorimeter. Fluorescence lifetimes were
carbonate and zinc phthalocyanine (ZnPc) were purchased measured using a time correlated single photon counting
from Sigma Aldrich. Dimethyl formamide (DMF), dimethyl (TCSPC) setup (FluoTime 200, Picoquant GmbH). The
amino ethanol and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) excitation source was a diode laser (LDH-P-670 driven by PDL
were obtained from Fluka. Deionized water was used for 800-B, 670 nm, 20 MHz repetition rate, 44 ps pulse width,
all aqueous solution preparations. AlPcSMix (containing a Picoquant GmbH), as described before.13
mixture of sulfonated derivatives and used as a standard in
Transmission electron microscope (TEM) images were
the determination of singlet oxygen quantum yields in obtained using a Zeiss Libra TEM 120 model operated at 90 kV
water) was synthesized according to literature methods.20 accelerating voltage. TEM samples were prepared by placing a
4-Nitrophthalonitrile (1) was synthesized and purified accord- drop of the nanoparticle solution on the sample grid and
ing to literature methods.21
allowing it to dry before measurements.
Laser flash photolysis experiments were performed to deter-
mine the triplet decay kinetics. The excitation pulses were pro-
duced by a tunable laser system consisting of an Nd:YAG
laser (355 nm, 135 mJ/4–6 ns) pumping an optical parametric
oscillator (OPO, 30 mJ/3–5 ns) with a wavelength range of
420–2300 nm (NT-342B, Ekspla) as described in detail
before.22,23
The time resolved phosphorescence of singlet oxygen at
1270 nm was used to determine the singlet oxygen quantum
yield of the Pc and Pc–gold nanoparticle conjugates in DMF
and aqueous solution, details have been described before.23
The singlet oxygen phosphorescence signal was compared to
AlPcSmix as the standard for aqueous solutions and ZnPc as a
standard in DMF.
Instrumentation
Infrared spectra were recorded on a Perkin Elmer Spectrum
100 FT-IR spectrometer. X-ray photoelectron spectroscopy
(XPS) data were collected using a Kratos Axis Ultra DLD, using
an Al (monochromatic) anode, equipped with a charge neutral-
izer. The operating pressure was maintained below 5 × 10−9
torr. The resolution was 10 eV pass energy in the slot mode.
Mass spectral data were collected with a Bruker AutoFLEX III
Smartbeam TOF/TOF Mass spectrometer using dithranol as
the MALDI matrix. Details have been provided before.13
The concentration of gold atoms (Au0) in AuNPs was deter-
mined using a ThermoElectron ICAP 6000 inductively coupled
plasma (ICP) spectrometer with an optical emission
spectroscopy (OES) detector. Gold nanoparticles were digested
by addition of 3 mL of aqua regia for ICP measurements.
Standard calibration was achieved at concentrations ranging
Synthesis (Scheme 1)
Synthesis of 4-(thiamine)phthalonitrile (3). Thiamine
from 0.5 to 5 ppm. A minimum of three measurements hydrochloride 2 (6.34 g, 18.8 mmol) was added to compound 1
Scheme 1 Synthesis of thiamine substituted zinc phthalocyanine (ZnTTAPc).
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(1.5 g, 8.67 mmol) in DMF under argon. After stirring for for coordination to ZnTTAPc, AuNR solution was centrifuged
15 min at room temperature K2CO3 was added (5 g, 36 mmol) for the third time for 5 min to remove excess CTAB. The clear
in portions. The reaction mixture was stirred at room tempera- liquid was discarded and the resulting gold pellet was dis-
ture for 2 days. The mixture was then poured into ice and the solved in 1 mL distilled water. The AuNRs of different aspect
precipitate was filtered and dried. Yield: 2.4 g (65%) IR (νmax
/
ratios are represented as AuNRs (2.0), AuNRs (4.7) and AuNRs
cm−1): 2228 (C–N), 3032–3044 (Ar–CH), 2954 (Alph-CH), 3361 (7.1).
1
(N–H), 1594 (CvC). H NMR (acetone-D6): 8.18 (s, 1H, Ar-H),
7.97 (d, 1H, Ar–H), 7.90 (d, 1H, Ar-H), 7.79 (s, 1H, Ar–H), 7.76
Conjugation of ZnTTAPc to gold nanoparticles
(s, 1H, Ar-H), 4.33 (s, 2H, NH2), 3.11 (t, 2H, O–CH2), 2.89 (t,
2H, –CH2), 2.75 (s, 2H, –CH2), 2.36 (s, 3H, CH3), 2.23 (s, 3H,
CH3). Anal. Calc. for C20H19N6OSCl: C 61.45, H 4.86, N 21.51,
S 8.19; Found: C 61.26, H 4.76, N 21.35, S 7.98.
The solution of gold nanoparticles (1 mL) from above was
mixed with 1 mL aqueous solution (pH 9) of ZnTTAPc (2 mg
mL−1) and this mixture was stirred for about 5 min, then
further diluted with 3 mL distilled water and stirred overnight.
The conjugate was purified in a size exclusion column of Bio-
Beads using pH 9 buffer as an eluting solvent.
Synthesis of zinc tetra (4-(thiamine)phthalocyanine)
(ZnTTAPc) (4). A mixture of 3 (0.3 g, 0.703 mmol), anhydrous
zinc chloride (0.024 g, 0.18 mmol) and DBU (1 mL, 6.3 mmol)
was refluxed in dimethyl amino ethanol for 8 h. The product
was cooled to room temperature and precipitated with
ethanol. After filtration the product was washed repeatedly
with ethanol. The product was purified in a reverse phase
column using a mixture of methanol and acetonitrile (1 : 2) as
an eluent. Yield: 0.19 g (63%). IR: 3492 (N–H), 3051, 3079 (Ar–
CH), 2958, 2921 (Alph-CH), 1610 (N–H bending), 1226 (C–O–
Data analysis
The total number of Au atoms per AuNP, the surface area of
AuNPs and the number of surface Au atoms per AuNP were cal-
culated using established methods.25–27 The extinction coeffi-
cients of AuNPs were determined using Beer’s law and ICP as
reported before.28,29
The number of Pc molecules on AuNPs was also obtained
using the Beer’s law and the Q band absorption maximum of
the phthalocyanines, assuming the extinction coefficient does
not change upon conjugation.
1
C), 1576 (CvC). H NMR (DMF-D7): 9.53–9.89 (m, 12H, Ar–H),
8.41 (s, 4H, S–CH), 7.27 (s, 4H, CHvN), 5.76 (s, 8H, CH2–N),
3.66 (s, 8H, NH2), 3.29 (t, 8H, O–CH2), 2.90 (t, 8H, –CH2), 2.47
(t, 12H, CH3), 2.21 (s, 12H, CH3), MALDI-TOFMS Anal. Calc.
m/z 1631.27, Found: [M]+: 1630.51. UV-Vis (DMF) λmax/nm
(log ε) 358 (4.6), 617 (4.2), 686 (4.97). Anal. Calc. for
C80H76N24O4S4Zn: C 58.89, H 4.69, N 20.61, S 7.86; Found: C
58.49, H 4.66, N 21.09, S 7.87.
Results and discussion
Characterization of Zn thiamine phthalocyanine (ZnTTAPc (4))
Synthesis of gold nanoparticles
Thiamine was employed as a substituent for complex 4 in
order to provide the solubility in aqueous media. However, the
complex was not soluble in water unless at high pH. Hence pH
9 buffer was employed. Furthermore the thiamine substituent
will allow for the binding with gold nanoparticles. Characteriz-
ation of the compound was achieved using infra-red (IR), UV-
visible, proton nuclear magnetic resonance (NMR) and matrix-
assisted laser desorption/time of flight (MALDI-TOF) spectro-
scopies and elemental analysis. 1H NMR showed all the
protons in their correct positions. The mass of the molecule
was confirmed by MALDI-TOF spectrometry. Elemental analy-
sis confirmed the absence of chloride and hence the non-ionic
nature of the molecule following an extensive purification
procedure.
Seed solution (used as nanospheres, AuNS). The seed AuNS
solution was prepared following literature methods,24 with
slight modifications: aqueous solution of 2.5 × 10−4 M (5 mL)
HAuCl4 was mixed with 10 mL of 0.1 M CTAB solution, and
the mixture was stirred for 2 min. Then, 0.6 mL of an ice cold
aqueous solution of 0.01 M NaBH4 was added, and the
mixture was shaken by hand at 2 min intervals. The solution
was allowed to stand for 2 h and then used for the subsequent
synthesis of shaped AuNPs.
Synthesis of gold nanorods (AuNRs (2.0), AuNRs (4.7) and
AuNRs (7.1)). The growth solution was prepared according to
the literature24 by mixing 10 mL of 0.2 M CTAB, 5 mL of 1 ×
10−4 M HAuCl4 and 0.004 M AgNO3. The aspect ratio of the
nanorods was adjusted by adding different amounts of 0.004
M AgNO3 from 0.19, 0.22 and 0.25 mL for AuNRs with aspect
ratios of 7.1, 4.7 and 2.0, respectively. To this solution,
UV-Vis spectra
0.07 mL of 0.1 M ascorbic acid was added and the reaction The UV-Vis spectrum of the ZnTTAPc shows two absorption
turned colourless. Then the seed solution (15 μL) prepared peaks at 638 and 696 nm due to aggregation in aqueous solu-
above was added and the mixture was shaken by hand and left tions, Fig. 1 and Table 1. The blue shifted band at 638 nm is
undisturbed between 30 and 37 °C for 24 h. Gold nanorods due to the aggregates and the band at 696 nm is the monomer
were purified by centrifuging for 20 min at 2000 rpm to peak. After the addition of Triton X-100 the aggregate peak
remove the unreacted gold and spherical gold nanoparticles. decreased and the monomer peak intensity increased, but
The supernatant was removed and the resulting pellet of there is still some aggregation. In DMF only one peak due to
AuNRs was dissolved in 3 mL of distilled water. Before using the monomer is observed showing no aggregation.
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Fig. 1 UV-Vis spectra of ZnTTAPc in (i) pH 9 buffer, (ii) pH 9 buffer +
Triton X-100 and (iii) DMF.
Table 1 Spectral properties and size of AuNPs–ZnTTAPc conjugates of
ZnTTAPc and conjugatesa
Sample
Solvent
λabs/nm
λexc/nm λem/nm
ZnTTAPc
ZnTTAPc
ZnTTAPc
Pc–AuNS (1.0) pH 9 buffer
Pc–AuNR (2.0) pH 9 buffer
Pc–AuNR (4.7) pH 9 buffer
Pc–AuNR (7.1) pH 9 buffer
DMF
pH 9 buffer
pH 9 buffer + Triton X-100 633, 696 696
688
688
699
704
704
704
706
703
712
638, 696 696
692
694
692
692
693
694
692
692
a λabs = absorption wavelength, λexc = excitation wavelength, λem = emission
wavelength.
Fig. 2 (i) Absorption, (ii) excitation and (iii) emission spectra of ZnTTAPc
in (A) pH 9 buffer + Triton X-100 and (B) in DMF.
Fluorescence behaviour
The emission spectrum of the ZnTTAPc in aqueous solution
was very weak due to aggregation, but improved on addition of
Triton X-100, Fig. 2A. The absorption spectrum is not in agree-
ment with the excitation spectrum for ZnTTAPc in Fig. 2A due
to effects of aggregation. The emission spectrum was however
the mirror image of the excitation spectrum. The excitation
spectrum was in agreement with the corresponding absorption
spectrum for ZnTTAPc in DMF, Fig. 2B, and the emission spec-
trum was the mirror image of the excitation spectrum.
It was not possible to calculate the fluorescence quantum
yield (ΦF) in aqueous media without Triton X-100 due to very
weak fluorescence as a result of the highly aggregated nature
of this molecule. The fluorescence quantum yield in pH 9
buffer + Triton X-100 was ΦF = 0.01, a value lower than in DMF
at ΦF = 0.09 (Table 2). The value in DMF is still lower than is
typical of unaggregated ZnPc derivatives in organic solvents,12
due to the known30 quenching of excited states by amino
groups which form part of the thiamine substituent on the Pc
ring.
(Table 2). Two fluorescence lifetimes were observed for
ZnTTAPc in pH 9 + Triton X-100, which is attributed to the
presence of aggregates which tend to quench the fluorescence
and hence the longer unquenched and shorter quenched life-
times were obtained.31
Triplet quantum yields and lifetimes
The triplet decay curve for ZnTTAPc is shown in Fig. 4. Triplet
quantum yields and lifetimes were obtained for the Pc alone
in DMF and pH buffer + Triton X-100 (Table 2). Triplet
quantum yields can be used to explain the ability of the mole-
cules to undergo intersystem crossing to the triplet state.
Triplet quantum yields were calculated using comparative
methods described before32 and ZnPc in DMF (ΦTStd = 0.5833)
and AlPcSmix in aqueous media ΦSTtd = 0.4434 as standards.
The triplet quantum yields of the Pc were 0.28 and 0.21 in
DMF and pH 9 buffer + Triton X-100, respectively. Triplet life-
times of the Pc in DMF are 35 µs and 25 µs in pH 9 buffer +
Triton X-100. Both the triplet quantum yield and lifetime
values are lower in aqueous media due to aggregation.
Fluorescence lifetimes of ZnTTAPc were measured using
the TCSPC method and the fluorescence decay curves are
shown in Fig. 3. ZnTTAPc gave one lifetime in DMF
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Table 2 Spectral properties and photophysical parameters of ZnTTAPc and conjugates
Sample
ΦF ( 0.01)
τF a (ns)
τ0 (ns)
ΦT
τT (μs)
ΦΔ
ZnTTAPc (DMF)
ZnTTAPc (pH 9 buffer + Triton X-100)
0.09
0.01
2.43
27.0
293.4
0.28
0.21
35
25
0.44
0.19
2.71 (0.94)
0.69 (0.56)
2.39 (0.86)
0.49 (0.13)
2.64 (0.85)
0.88 (0.15)
2.71 (0.88)
0.91 (0.11)
2.73 (0.91)
0.83 (0.09)
Pc–AuNS (1.0)
pH 9 buffer
Pc–AuNR (2.0)
pH 9 buffer
Pc–AuNR (4.7)
pH 9 buffer
Pc–AuNR (7.1)
pH 9 buffer
0.02
0.03
0.05
0.06
106.0
79.2
49.7
42.7
—
—
—
—
—
—
—
—
0.23
0.23
0.25
0.29
a Abundances in brackets.
UV-Vis spectra
Spherical gold nanoparticles have a characteristic surface
plasmon resonance (SPR) absorption in the visible region of
the electromagnetic spectrum. Gold nanorods have two charac-
teristic SPR peaks (transverse and longitudinal).36 Fig. 5 shows
the UV-Vis spectra of the gold nanospheres and nanorods with
varying aspect ratios. Gold nanospheres showed one absorp-
tion peak at 518 nm, Table 3. However, for nanorods there
were two absorption peaks observed and the longitudinal peak
was tuned to the near infrared by increasing the aspect ratio,
Table 3. The aspect ratio of the gold nanorods was controlled
by the amount of silver nitrate added and was found to
decrease with an increase in AgNO3.
Transmission electron microscopy (TEM)
Fig. 3 Fluorescence decay curves of ZnTTAPc in (a) DMF and (b) pH 9
buffer plus Triton X-100.
Fig. 6 shows TEM images of the gold nanospheres and nano-
rods (the latter at different aspect ratios). TEM images of gold
nanospheres show the distribution of small particles with an
estimated particle size of 2.1 nm (average). Gold nanorods
shown in this figure have the aspect ratio of 2.0, 4.7 and
7.1 nm (Tables 1–3), represented as AuNRs (2.0), AuNRs (4.7)
and AuNRs (7.1), respectively.
Characterization of ZnTTAPc–AuNPs conjugates
Gold is known to form strong bonds with amines and
sulphur. ZnTTAPc contains sulphur on the thiazole group
and the amine from its pyrimidine group of the thiamine.
The phthalocyanine is expected to form the amine–gold or
sulphur–gold bonds with the nanoparticles.
As stated above, the surface area of the nanorods was found
to increase as the aspect ratio increases. The larger surface
area results in an increase in the number of surface atoms
which are important for attachment with the phthalocyanine
molecules on the surface. The ratio of the number of phthalo-
cyanine molecules to the number of gold nanoparticles was
found to range from 27 to 243, increasing with increase in the
surface area of the NPs (Table 3).
Fig. 4 Triplet decay curves of ZnTTAPc in DMF.
Characterization of gold nanoparticles
The aspect ratio of the Au nanospheres will be taken as one.35
The surface area of the gold nanoparticles increased as the
aspect ratio increased, Table 3. This has been observed
before.35 The total number of Au atoms as well as surface Au
atoms also increased as the aspect ratio increased. The extinc-
tion coefficients of the gold nanoparticles reported in Table 1
are comparable to those found in the literature.28,29
TEM images
TEM images of Pc–gold conjugates are shown in Fig. 7. The
morphology of the gold nanoparticles did not change upon
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Table 3 Properties of gold nanoparticles and loading
Sample
λ SPR band (nm)
Ε (M−1 cm−1
)
Surface area (NPs) (nm2)
Total Au atoms
Surface Au atoms
Pcs/NPs
AuNS (1.0)
AuNR (2.0)
AuNR (4.7)
AuNR (7.1)
518
1.3 × 108
2.4 × 108
5.6 × 108
6.9 × 108
14
477
733
450
168 214
354 660
743 236
235
8103
124 396
215 671
27
49
111
243
528, 643
518, 745
518, 825
1271
possibly due to aggregation. The aspect ratios of the conju-
gated nanorods were 2.9, 4.9 and 7.8 nm for AuNRs (2.0),
AuNRs (4.7) and AuNRs (7.1), respectively, showing a slight
increase.
XPS studies
In order to determine if the amine or sulphur groups on the
thiamine substituent on the Pc ring are coordinated to AuNPs,
high resolution XPS was employed.
The high resolution scans of the N (1s) and S (2p) for
the ZnTTAPc alone and ZnTTAPc–gold conjugates are
shown in Fig. 8. Fig. 8A(i) shows C–NH2, CvN and
metal–N bonds present in the phthalocyanine molecule.
Conjugation of the Pc to the nanoparticles resulted in the
disappearance of the C–NH2 peak, Fig. 8A(ii), which proves
the participation of the amine in conjugation with gold
nanorods.
Fig. 5 UV-Vis spectra of gold nanoparticles (i) nanospheres, (ii) Pc–
AuNRs (2.0), (iii) Pc–AuNRs (4.7) and Pc–AuNRs (7.1).
The sulphur group of the thiazole ring was also analyzed
with XPS to confirm the interaction with gold nanoparticles.
The deconvoluted spectra for S (2p) show a doublet of
(2p3/2) and (2p1/2) (Fig. 8B(i)) which is common for S (2p).37
conjugation except for the slight changes in particle sizes. The
average particle size of the conjugated spherical nanoparticles
is 4.6 nm, an increase from 2.1 nm of the nanospheres alone,
Fig. 6 TEM images of gold (i) nanospheres, (ii) Pc–AuNRs (2.0), (iii) Pc–AuNRs (4.7) and Pc–AuNRs (7.1).
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Fig. 7 TEM images of Pc–gold conjugates (i) nanospheres, (ii) Pc–AuNRs (2.0), (iii) Pc–AuNRs (4.7) and Pc–AuNRs (7.1).
Fig. 8 XPS deconvoluted spectra of (A) N 1s and (B) S 2p of (i) ZnTTAPc and (ii) ZnTTAPc–Au.
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Fig. 9 UV-Vis spectra of (A) AuNSs, (B) AuNRs (2.0), (C) AuNRs (4.7) and (C) AuNRs (7.1), and their conjugates. (i) Gold nanoparticles and (ii) Pc and
(iii) gold nanoparticles–Pc conjugates in pH 9 buffer (without Triton X-100).
Upon binding to gold nanoparticles the first doublet dis- induced by the heavy atom effect. This behavior has been
appeared and the second doublet was observed at higher observed before for other Pc–AuNP conjugates.38
binding energies. This shows that sulphur also played a role in
the formation of the phthalocyanine–gold NP conjugate.
Fluorescence behaviour. The excitation spectra of the conju-
gates were not similar to the corresponding absorption
spectra, Fig. 10 (showing AuNRs (4.7) and AuNRs (7.1) as
examples), due to remaining aggregation and the presence of
SPR bands of the AuNPs. The emission spectra were all mirror
images of the excitation spectra though emission was weak for
gold nanospheres (figure not shown).
The fluorescence behavior of the fluorophore in the pres-
ence of nanoparticles is affected by factors such as the size of
the nanoparticle and the distance separating them.39 Hence,
the effect of the aspect ratio of the nanorods is investigated.
The fluorescence quantum yields measured in the presence of
gold nanoparticles are all slightly higher than the phthalo-
cyanine alone in pH 9 buffer + Triton X-100 due to reduced
aggregation. The presence of metallic particles can modify the
local field around the fluorescing molecule by causing an
increase of the rate of excitation at larger distances which
would result in an increase in the quantum yield.40 Fluo-
rescence quantum yields were found to marginally increase
with increasing aspect ratio. It has been reported that the
UV-Vis absorption spectra
Fig. 9 shows the UV-Vis spectra of the conjugates in pH 9
buffer. The conjugation of gold nanoparticles to ZnTTAPc
did not significantly shift the Q band (Table 1) maxima of the
monomer peak of ZnTTAPc but the aggregation was reduced
as judged by the decrease in the aggregate peak near 630 nm.
Thus linking of AuNPs to ZnTTAPc reduces aggregation. Dis-
aggregation of Pc molecules has been observed before for star
shaped AuNPs but not for spherical ones.15 However, in this
work the spherical nanoparticles also break aggregates,
Fig. 9A. The spectra of the conjugates show the characteristic
SPR peaks from gold nanoparticles.
Photophysical studies
No triplet decay curves were observed for the Pc in the pres-
ence of AuNPs, probably due to the short triplet lifetime
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the fluorescence and decreases the decay time. Hence, the
radiative lifetime (τ0), which is the lifetime of the molecule in
the absence of the non-radiative processes, can be used to
explain the quenching of lifetimes. The radiative lifetime can
be calculated from the measurement of the fluorescence
quantum yield (ΦF) and lifetime (τF) using eqn (1).43
τ0 ¼ τF=ΦF
ð1Þ
The radiative lifetimes in Table 2 were estimated for nano-
spheres and different sizes of the gold nanorods. It was
observed that when the aspect ratio increases, τ0 decreases.
The τ0 values can be affected by the distance and orientation
between the molecule and metal particles, and can either
increase or decrease depending on the molecular dipole and
the distance between the fluorophore and the metal. Both the
size and shape of nanoparticles can also alter the radiative
decay.44,45 Higher photonic mode density increases in the
presence of nanoparticles and causes a decrease in radiative
decay.46 The decrease in radiative lifetime at a larger aspect
ratio is caused by the increasing photonic mode density. As
the aspect ratio increased, the absorption of the nanoparticles
also shifts towards higher wavelengths towards the absorption
of the Pc, hence increasing the interference between their
excited dipoles. The absorption and scattering coefficients of
nanorods are also larger than that of spherical nanoparticles,39
therefore nanorods are expected to increase the effects felt by
the Pc molecules.
Singlet oxygen quantum yields. Singlet oxygen causes cell
death in photodynamic therapy hence monitoring it is very
important. Phthalocyanines are well known to produce singlet
oxygen where they undergo intersystem crossing which makes
them good photosensitizers for photodynamic therapy. Singlet
oxygen was measured for the Pc alone and Pc with gold nano-
spheres and rods (Table 2). In DMF the singlet oxygen
quantum yield of the Pc was 0.44 while in pH 9 buffer (when
Triton X-100 was added) it was 0.19. There was an increase in
the singlet oxygen quantum yields when the Pc is conjugated
to gold NPs (Table 2). This could be due to the effect of heavy
atoms which promote intersystem crossing.
The moderate increase in singlet oxygen quantum yield
could have been affected by the so-called screening effect,
where the AuNPs prevent the oxygen molecule from interacting
with the excited triplet phthalocyanine molecule47 and/or
by the fact that the AuNPs and the Pc absorb at the same
wavelength hence absorption by the former could affect the
excitation of the latter.
AuNS and AuNR (2.1) produced the equal amounts of
singlet oxygen (ΦΔ = 0.23, Table 2). The number of Pc mole-
cules per AuNP was slightly different for the two at 27 and 49
(Table 3), respectively. The amount of singlet oxygen produced
from AuNR (4.7) at ΦΔ = 0.25 was slightly larger than for AuNR
(2.0) corresponding to the larger Pc molecules per AuNP at 111
and 243, respectively. The largest ΦΔ value at 0.29 was
observed for AuNR (7.1) with the largest number of Pcs per
AuNP.
Fig. 10 (i) Absorption, (ii) excitation and (iii) emission spectra of (A)
ZnTTAPc–AuNRs (4.7) and (B) ZnTTAPc–AuNRs (7.1) in pH 9.
fluorescence emission is more polarized along the length of
the nanorods and the degree of polarizability by nanorods
increases with their aspect ratio.41,42 Hence, the increase in
fluorescence quantum yields could also be due to the increas-
ing excitation rate as the aspect ratio increases. The fluo-
rescence quantum yields in the presence of Au nanoparticles
will also be affected by intersystem crossing to the triplet state
due to the heavy atom effect of gold. Thus decreased aggrega-
tion will improve the values, but the heavy atom effect will
decrease them.
Two fluorescence lifetimes were observed for ZnTTAPc in
the conjugates, Table 2, as was the case for ZnTTAPc alone
in Triton X-100. Fluorescence lifetimes (τF) decreased (consid-
ering the long lifetime) for the nanospheres and smaller
aspect ratio nanorods (AuNRs (2.0)) when compared with the
phthalocyanine alone in pH 9 + Triton X-100. The τF value for
AuNRs (4.7) was the same as for ZnTTAPc in pH 9 + Triton
X-100, with a slight increase for AuNRs (7.1).
Radiative and non-radiative processes compete in the de-
excitation of the molecules. The presence of metal nanoparti-
cles can influence decay of the fluorophore by inducing non-
radiative processes. Non-radiative processes usually quench
8238 | Dalton Trans., 2014, 43, 8230–8240
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Dalton Transactions
Paper
13 N. Masilela and T. Nyokong, J. Photochem. Photobiol., A,
2011, 223, 124–131.
Conclusions
Water soluble zinc thiamine substituted phthalocyanine was 14 N. Nombona, K. Maduray, E. Antunes, A. Karsten and
successfully synthesized. The compound shows aggregation T. Nyokong, J. Photochem. Photobiol., B, 2012, 107, 35–44.
in aqueous solution, which led to the quenching of the fluo- 15 S. D’ Souza, S. Moeno, E. Antunes and T. Nyokong, New
rescence quantum yields. The photophysical properties of the J. Chem., 2013, 37, 1950–1958.
Pc improved by the addition of Triton X which is a surfactant. 16 D. C. Hone, P. I. Walker, R. Evans-Gowing, S. FitzGerald,
The Pc was also conjugated to gold nanospheres and nanorods
A. Beeby, I. Charmbrier, M. J. Cook and D. A. Russell,
of different aspect ratios. An increase in fluorescence quantum
Langmuir, 2002, 18, 2985–2987.
yields in the presence of the nanorods shows that the aspect 17 M. E. Wieder, D. C. Hone, M. J. Cook, M. M. Handsley,
ratio has an effect on the photophysical behavior of this mole-
cule. Singlet oxygen quantum yields were larger for complex 4
linked to AuNPs of higher aspect ratios.
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Acknowledgements
This work was supported by the Department of Science 20 M. Ambroz, A. Beeby, A. J. McRobert, M. S. C. Simpson,
and Technology (DST)/Nanotechnology (NIC) and the National
Research Foundation (NRF) of South Africa, through DST/NRF
R. K. Svensen and D. Phillips, J. Photochem. Photobiol., B,
1991, 9, 87–95.
South African Research Chairs Initiative for Professor of Medi- 21 D. Wöhrle, M. Eskes, K. Shigehara and A. Yamada, Syn-
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