Two-photon oxygen sensing with quantum dot-porphyrin conjugates

Article abstract of DOI:10.1021/ic4011168

Supramolecular assemblies of a quantum dot (QD) associated to palladium(II) porphyrins have been developed to detect oxygen (pO₂) in organic solvents. Palladium porphyrins are sensitive in the 0-160 Torr range, making them ideal phosphors for i

Full text of DOI:10.1021/ic4011168

Article
pubs.acs.org/IC
Two-Photon Oxygen Sensing with Quantum Dot-Porphyrin
Conjugates
Christopher M. Lemon,†,‡ Elizabeth Karnas,† Moungi G. Bawendi,*,† and Daniel G. Nocera*,‡
†Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139,
United States
‡Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United
States
S
* Supporting Information
ABSTRACT: Supramolecular assemblies of a quantum dot (QD)
associated to palladium(II) porphyrins have been developed to
detect oxygen (pO2) in organic solvents. Palladium porphyrins are
sensitive in the 0−160 Torr range, making them ideal phosphors
for in vivo biological oxygen quantification. Porphyrins with meso
pyridyl substituents bind to the surface of the QD to produce self-
assembled nanosensors. Appreciable overlap between QD emission
and porphyrin absorption features results in efficient Forster resonance energy transfer (FRET) for signal transduction in these
̈
sensors. The QD serves as a photon antenna, enhancing porphyrin emission under both one- and two-photon excitation,
demonstrating that QD-palladium porphyrin conjugates may be used for oxygen sensing over physiological oxygen ranges.
transduction.14−16 Additionally, QDs exhibit high two-photon
INTRODUCTION
■
absorption cross-sections (σ ∼ 104 Goppert−Mayer, 1 GM =
̈
2
Generating metabolic profiles of tumors provides a spatiotem-
poral map of the concentration of key species to assess and
quantify tumor growth, metabolism, and response to therapy.
Because the tumor microenvironment is characterized by
acidity and hypoxia,1 the concentration of protons and oxygen
are important indicators of tumor health.2 Understanding how
these two parameters change as a function of disease
progression is critical to develop novel targeted therapeutics
such as antiangiogenic agents, which can engender normal-
ization of the leaky, distended, and tortuous tumor vasculature.3
Upon normalization, blood flow, oxygen levels, and drug
distribution are increased; this represents an opportunity to
treat the tumor with a substantial dose of chemotherapeutics to
have a superior impact on tumor progression.4 However, the
normalization process must be efficiently monitored so that
doses of chemotherapeutics can be timed appropriately to have
a maximal effect on tumor viability. To address this challenge,
new non-invasive sensors must be developed that are small
enough to penetrate into the tumor and monitor dynamic
changes with high resolution.5 To this end, we have developed
self-referencing pH sensors6−8 and a high-pressure (160−760
Torr) oxygen sensor9 to probe biological microenvironments.
Herein, we report a low-pressure (0−160 Torr) oxygen sensor
that covers the oxygen concentration range of the hypoxic
tumor environment.
10−50 cm4·s/photon),17−19 making them attractive fluorophores
for multiphoton imaging. Near-IR excitation and detection
(600−1000 nm) in the so-called therapeutic window allows for
imaging with minimal background signal from cellular
autofluorescence.20 This spectral window is readily accessed
using two-photon excitation. This imaging technique is
nondestructive to tissue and provides high-resolution images
of live tissue at depths of several hundred micrometers with
submicrometer spatial resolution.21,22 Thus, QDs offer a
versatile platform on which to build supramolecular oxygen
sensing assemblies.
Many oxygen sensitive phosphors have been reported in the
literature using osmium,9 iridium,23 and ruthenium24 poly-
pyridine complexes as well as pyrene.25 Platinum and palladium
porphyrins are especially well-suited for O2 sensing applications
because of their strong room temperature phosphorescence in
the 650−800 nm range and long (∼102 μs) phosphorescence
lifetime.26 Most oxygen chemosensors heretofore have relied
on the immobilization of these porphyrins in polymer
matrixes,26−28 on solid surfaces,26,29 or in mesoporous
silica.26,30 Commercial palladium porphyrins (Oxyphor R2)
and benzoporphyrins (Oxyphor G2), which are available as
water-soluble glutamate dendrimers, have also been used for
oxygen sensing in solution.31
The exceptionally long phosphorescence lifetimes of
palladium porphyrins make them ideal for oxygen sensing in
the biologically relevant 0−160 Torr range. The heavy metal Pd
Fluorescent semiconductor quantum dots (QDs) have high
quantum yields, photostability, narrow emission bands, and
broad excitation profiles,10 rendering them ideal scaffolds for
constructing optical biosensors.11−13 By attaching an analyte-
sensitive fluorophore to the QD, Forster resonance energy
transfer (FRET) may be exploited as a means of signal
Received: May 4, 2013
̈
© XXXX American Chemical Society
A
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Figure 1. Schematic representation of the O2 sensing transduction mechanism of a self-assembled Pd porphyrin (2)-QD construct. The QD acts as
the two-photon antenna of NIR (700−1000 nm) excitation. The QD emission is quenched in the presence of a surface-bound Pd porphyrin (2) via
a FRET mechanism. The insensitivity of the QD emission to O2 affords an internal reference to establish a ratiometric O2 response.
promotes rapid intersystem crossing to produce the long-lived
triplet state. Molecular oxygen (a ground state triplet)
deactivates this excited state efficiently through collisional
quenching, as dictated by Stern−Volmer kinetics. By
monitoring the intensity or lifetime of the triplet excited state
of the Pd porphyrin, the amount of oxygen may be quantified.32
However, porphyrins alone have prohibitively low two-photon
absorption cross-sections (σ2 = 1−25 GM for free-base
tetraphenylporphyrin),33 and thus an ideal oxygen sensor
using Pd porphyrins combines the oxygen-sensitive properties
of porphyrins with an efficient two-photon antenna. In this
regard, coumarin dyes have served as two-photon antennae for
covalently attached Pd or Pt porphyrins.34−37 Herein, we
expand this approach by using a QD as the two-photon
antenna.
porphyrins are associated to a single two-photon QD antenna,
giving a sizable porphyrin response on a per sensor basis. Fifth,
the insensitivity of QD emission to O2 establishes a ratiometric
signal response. Finally, the construct exhibits greatest response
over the biologically important 0−160 Torr O2 pressure range.
EXPERIMENTAL SECTION
■
Materials. The following chemicals were used as received: hexanes,
diethylether (Et2O), anhydrous inhibitor-free tetrahydrofuran (THF),
dichloromethane (CH2Cl2), toluene, acetonitrile (MeCN), methanol
(MeOH), anhydrous methanol (MeOH), ethanol (EtOH), ethyl
acetate (EtOAc), 2-mercaptopyridine, isonicotinoyl chloride hydro-
chloride, nicotinoyl chloride hydrochloride, pyrrole, 4-pyridinecarbox-
aldehyde, 3-pyridinecarboxaldehyde, benzaldehyde, methyl 4-formyl-
benzoate, indium(III) chloride (InCl3), sodium hydroxide (NaOH)
beads, ethyl magnesium bromide 1 M solution in THF (EtMgBr),
bromine (Br2), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), magnesi-
um bromide (MgBr2), tris(2,2′-bipyridyl)dichlororuthenium(II) hex-
ahydrate ([Ru(bpy)]Cl2), 2′,7′-dichlorofluorescein (fluorescein 27),
trifluoroacetic acid (TFA), triethylamine (NEt3), 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ), and sodium borohydride
(NaBH4) from Sigma-Aldrich; sodium bicarbonate (NaHCO3),
sodium sulfate (Na2SO4), ammonium chloride (NH4Cl), ammonium
sulfate ((NH4)2SO4), and sodium chloride (NaCl) from Mallinckrodt;
potassium carbonate (K2CO3) and Celite 512 from Fluka; palladium-
(II) acetylacetonate (Pd(acac)2) and ytterbium(III) triflate (Yb-
(OTf)3) from Strem; benzoyl chloride from J. T. Baker; pyridine
from EMD; silica gel 60 Å 230−400 mesh ASTM from Whatman; and
chloroform-d (CDCl3) from Cambridge Isotope Laboratories. Nitro-
gen and argon gases (Airgas) were passed over a Drierite column prior
to use. Cadmium selenide core/shell QDs (518 nm emission, QD
Vision) were twice precipitated from toluene using EtOH and
redissolved in toluene prior to use. The following compounds were
prepared according to literature procedures or slight modifications
thereof: 5-(3-pyridyl)dipyrromethane (S1),38 S-2-pyridyl nicotino-
thioate (S2),39,40 5-phenyldipyrromethane (S3),41,42 S-2-pyridyl
benzoylthioate (S4),39,43 5-(4-methoxycarbonylphenyl) dipyrro-
methane (4),41,42 S-2-pyridyl isonicotinothioate (5),39,40 1-isonicoti-
noyl-5-(4-pyridyl) dipyrromethane (7),39,40 1-nicotinoyl-5-(3-pyridyl)-
dipyrromethane (8),39,40 1-benzoyl-5-phenyldipyrromethane (9),39,40
1,9-dibenzoyl-5-phenyldipyrromethane (10),38 and 5-(4-pyridyl)-
dipyrromethane (12).38 Exact procedures for the preparation of
these compounds and 1H NMR are provided in the Supporting
Information.
The porphyrin-QD construct shown in Figure 1 is prepared
by self-assembly, promoted by binding of Pd porphyrins with
meso-pyridyl substituents (compounds 1−3, Chart 1) to the
Chart 1
surface of a QD. In this sensing scheme, the QD is irradiated
with two-photon excitation and energy is transferred via FRET
to excite the Pd porphyrin, which is quenched by molecular
oxygen; the extent of quenching is proportional to the amount
of oxygen. Since the QD is unaffected by oxygen, its emission
may serve as an internal standard to afford a ratiometric oxygen
sensor by monitoring the QD:porphyrin emission intensity.
The system shown in Figure 1 offers several new advantages
for O2 sensing. First, the self-assembling process allows for
facile construction of a nanosensing construct. Second, QDs are
among the most efficient two-photon chromophores, exhibiting
absorption cross-sections that are 1000-fold greater than
common organic dyes (σ2 ∼ 104 GM for QDs versus σ2 = 20
GM for Coumarin-343).35 Third, the photophysical properties,
namely, the emission wavelength, of QDs are tunable,10
enabling optimized spectral overlap between Pd porphyrin
and QD, thus maximizing FRET efficiency. Fourth, several
1-Isonicotinoyl-5-(4-methoxycarbonylphenyl)-
dipyrromethane (6). This compound was prepared using a
procedure similar to that used for other 1-acyl dipyrromethanes,39,40
with minor modifications including the addition of acylating reagent as
a solid in one portion rather than a homogeneous solution. In an oven-
dried flask, 1.89 g of 5-(4-methoxycarbonylphenyl)dipyrromethane
(4)41,42 (6.74 mmol) was dissolved in 15 mL of anhydrous
B
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tetrahydrofuran (THF) under an argon atmosphere to afford a tan
solution. Seventeen milliliters of EtMgBr (1 M solution in THF) was
then slowly added, and the resultant dark brown solution was stirred at
room temperature for 10 min. The solution was cooled to −78 °C in a
dry ice/acetone bath, and 1.50 g (6.94 mmol) of S-2-pyridyl
isonicotinothioate (5)39,40 was added in one portion; the resultant
mixture was stirred at −78 °C for 1 h. The solution was warmed to
room temperature and stirred for an additional 3 h. Saturated NH4Cl
was added, and the product was extracted with EtOAc. The combined
organics were washed with water and brine, then dried over Na2SO4.
Solvent was removed by rotary evaporation to afford a brown oil. The
crude reaction mixture was loaded onto a silica gel column packed
with ethyl acetate. The product (second band, which turns yellow-
orange upon Br2 staining on TLC) was eluted with 100% EtOAc. After
solvent removal, the oily brown residue was dissolved in a minimal
amount of EtOAc and subsequently precipitated with a large excess of
hexanes, and the resultant solid was collected on a frit and washed with
hexanes to afford 1.67 g (64.2% yield) of the title compound as a tan
10 mL of CH2Cl2 and adding 0.2 mL of TFA (0.3 g, 2.6 mmol); the
resulting green solution was stirred at room temperature for 30 min
after which 0.4 mL of NEt3 (0.3 g, 2.9 mmol) was added. The reaction
mixture was washed with water and brine, dried over Na2SO4, and
brought to dryness. The free-base porphyrin (27 mg, 24% yield) was
1
used without further purification. H NMR (500 MHz, CDCl3) δ =
−2.81 (bs, 2H), 7.75−7.81 (overlapping m, 6H), 7.79 (overlapping m,
2H), 8.22 (d, J = 6.1 Hz, 4H), 8.53 (d, J = 6.8 Hz, 2H), 8.80 (d, J = 4.7
Hz, 2H), 8.84 (s, 2H), 8.87 (s, 2H), 8.91 (d, J = 4.7 Hz, 2H), 9.06 (m,
2H), 9.46 (s, 2H).
5,10,15-Triphenyl-20-(4-pyridyl)porphyrin (3-H2). In an oven-
dried flask, 90 mg of 1,9-dibenzoyl-5-phenyldipyrromethane (10)38
(0.21 mmol) was dissolved in 12 mL of anhydrous THF and 4 mL of
anhydrous MeOH under a nitrogen atmosphere to give a brown
solution. To this solution, 0.42 g of NaBH4 (11 mmol) was added, and
the resultant tan-orange mixture was stirred at room temperature for 1
h. Water was added to quench excess NaBH4, and the product was
extracted with CH2Cl2 and dried over K2CO3 to afford a solution of
the corresponding dicarbinol (11). Since the dicarbinol is prone to
decomposition, the solution was concentrated to near-dryness without
heating. The residue was dissolved in 50 mL of MeCN, and 45 mg of
5-(4-pyridyl)dipyrromethane (12)38 (0.20 mmol) and 1.22 g of
Yb(OTf)3 (2.0 mmol) were added; the resulting red solution was
stirred at room temperature for 1 h all the while protected from light.
DDQ (183 mg, 0.806 mmol) was added, and the solution was stirred
at room temperature for an additional 1 h. NEt3 (2 mL, 1.6 g, 16
mmol) was added to the solution and stirred for an additional 30 min.
Ethyl acetate was added, and the reaction mixture was washed with
water and brine, dried over Na2SO4, and brought to dryness. The
product was purified on a silica gel column using EtOAc as the eluent
1
solid. H NMR (500 MHz, CDCl3) δ = 3.92 (s, 3H), 5.61 (s, 1H),
5.97 (m, 1H), 6.08 (m, 1H), 6.19 (m, 1H), 6.76 (m, 1H), 6.81 (m,
1H), 7.30 (m, 2H), 7.63 (m, 2H), 8.01 (m, 2H), 8.04 (bs, 1H), 8.77
(m, 2H), 9.56 (bs, 1H).
5-(4-Methoxycarbonylphenyl)-10,15,20-tris(4-pyridyl)-por-
phyrin (1-H2). This compound was prepared using a microwave-
mediated [2 + 2] condensation.40 In a 10 mL microwave tube, 69 mg
(0.21 mmol) of 1-isonicotinoyl-5-(4-pyridyl)dipyrromethane (7)39,40
and 76 mg (0.20 mmol) of 1-isonicotinoyl-5-(4-
methoxycarbonylphenyl)dipyrromethane (6) were dissolved in 4 mL
of toluene to afford a dark brown-black suspension. The suspension
turned red after the addition of 0.6 mL of DBU (0.6 g, 4 mmol), and
was stirred for 5 min at room temperature. MgBr2 (420 mg, 2.3 mmol)
was added, and an additional 1 mL of toluene was added to rinse the
walls of the tube. The resultant orange-brown suspension was placed
in a microwave reactor (CEM Discover) and irradiated at 115 °C for 2
h. The reaction mixture was transferred to a flask, dissolved in THF,
and the solvent was removed by rotary evaporation. The residue was
dissolved in EtOAc, washed with water, and brought to dryness under
rotary evaporation. The magnesium porphyrins of the crude reaction
mixture were demetalated by dissolving the residue in 10 mL of
CH2Cl2 and adding 0.2 mL of TFA (0.3 g, 2.6 mmol); the resulting
green solution was stirred at room temperature for 30 min. Then 0.4
mL of NEt3 (0.3 g, 2.9 mmol) was added, and the reaction mixture was
washed with water and brine, dried over Na2SO4, and brought to
dryness. The residue was loaded onto a silica gel column and eluted
with EtOAc. After removal of the trans-A2B2 product, the solvent was
changed to 4% MeOH in EtOAc to give 15 mg (11% yield) of the title
compound. 1H NMR (500 MHz, CDCl3) δ = −2.90 (bs, 2H), 4.12 (s,
3H), 8.16 (m, 6H), 8.30 (m, 2H), 8.47 (m, 2H), 8.82−8.89 (bm, 8H),
9.06 (m, 6H).
1
to obtain 30 mg (24% yield) of the title compound. H NMR (400
MHz, CDCl3) δ = −2.80 (bs, 2H), 7.75−7.80 (m, 9H), 8.17 (m, 2H),
8.22 (m, 6H), 8.80 (d, J = 4.5 Hz, 2H), 8.86 (s, 4H), 8.90 (d, J = 4.7
Hz, 2H), 9.03 (m, 2H).
General Procedure for Palladium(II) Metalation. In a 10 mL
microwave tube, free-base porphyrin was dissolved in 3 mL of
pyridine, and Pd(acac)2 was added. An additional 1 mL of pyridine was
added to rinse solids down the tube. The resultant solution was placed
in a microwave reactor (CEM Discover) and irradiated at 180 °C for
20 min. The crude reaction mixture was filtered through a plug of
Celite and rinsed with CH2Cl2; the collected orange filtrate was
brought to dryness. The palladium complex was purified on a silica gel
column to afford the product.
5-(4-Methoxycarbonylphenyl)-10,15,20-tris(4-pyridyl)-
porphyrinopalladium(II) (1). Following the aforementioned general
procedure, 65 mg (0.10 mmol) of the free-base porphyrin 1-H2 was
treated with 175 mg (0.574 mmol) of Pd(acac)2. The compound was
purified using a gradient of 0−10% MeOH in EtOAc as the eluent to
1
give 39 mg (50% yield) of the title product. H NMR (400 MHz,
5,10-Diphenyl-15,20-bis(3-pyridyl)porphyrin (2-H2). This
compound was prepared using a microwave-mediated [2 + 2]
condensation.40 In a 10 mL microwave tube, 60 mg (0.18 mmol) of
1-nicotinoyl-5-(3-pyridyl)dipyrromethane (8)39,40 and 60 mg (0.18
mmol) of 1-benzoyl-5-phenyldipyrromethane (9)39,40 were dissolved
in 4 mL of toluene to afford a dark brown-black suspension. After the
addition of 0.6 mL of DBU (0.6 g, 4.0 mmol), the suspension became
red and was subsequently stirred for 5 min. MgBr2 (453 mg, 2.46
mmol) was added, and 1 mL of toluene was added to rinse the solids
down the tube. The resultant orange-brown suspension was placed in a
microwave reactor (CEM Discover) and irradiated at 115 °C for 2 h.
The reaction mixture was transferred to a flask, dissolved in THF, and
the solvent was removed by rotary evaporation. The residue was
dissolved in ethyl acetate, washed with water and brine, dried over
Na2SO4, and brought to dryness. The crude reaction mixture was
purified on a silica gel column using ethyl acetate as the eluent to
remove the first major band, Mg(TPP). The solvent was then switched
to 5% MeOH in EtOAc to elute the desired product, which moved as
the second fluorescent band on the column. The A4 complex with four
3-pyridyl groups remained at the top of the column and was not
isolated. The Mg complex was demetalated by dissolving the solid in
CDCl3) δ 4.12 (s, 3H), 8.12 (m, 6H), 8.25 (m, 2H), 8.45 (m, 2H),
8.80 (d, J = 5.1 Hz, 2H), 8.82 (s, 4H), 8.83 (d, J = 5.0 Hz, 2H), 9.05
(m, 6H). Anal. Calcd. for (M+H)+, M = C43H27N7O2Pd: 780.1339.
Found: 780.1507. Elem. Anal. Calcd.: 66.20% C, 3.49% H, 12.57% N.
Found: 66.14% C, 3.47% H, 12.53% N.
5,10-Diphenyl-15,20-bis(3-pyridyl)porphyrinatopalladium-
(II) (2). Following the aforementioned general procedure, 16 mg of the
free-base porphyrin 2-H2 (0.03 mmol) was treated with 55 mg (0.18
mmol) of Pd(acac)2 then purified using EtOAc as the eluent to give 7
1
mg (32% yield) of the title product. H NMR (500 MHz, CDCl3) δ
7.72−7.80 (m, 8H), 8.16 (m, 4H), 8.48 (m, 2H), 8.76 (d, J = 5.1 Hz,
2H), 8.80 (d, J = 2.2 Hz, 2H), 8.84 (s, 2H), 8.87 (d, J = 5.1 Hz, 2H),
9.04 (m, 2H), 9.42 (m, 2H). Anal. Calcd. for (M+H)+, M =
C42H26N6Pd: 721.1332. Found: 721.1335.
5,10,15-Triphenyl-20-(4-pyridyl)porphyrinatopalladium(II)
(3). Following the aforementioned general procedure, 30 mg (0.05
mmol) of the free-base porphyrin 3-H2 was treated with 136 mg
(0.446 mmol) of Pd(acac)2 then purified using CH2Cl2 as the eluent
1
to give 6 mg (17% yield) of the title product. H NMR (500 MHz,
CDCl3) δ 7.72−7.80 (m, 9H), 8.13 (m, 2H), 8.16 (m, 6H), 8.75 (d, J
= 5.1 Hz, 2H), 8.82 (s, 4H), 8.85 (d, J = 5.1 Hz, 2H), 9.02 (m, 2H).
C
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Anal. Calcd. for (M+H)+, M = C43H27N5Pd: 720.1380. Found:
720.1430.
using a BBO crystal; the pulse power was attenuated to 2.5 mW at the
sample. Emission lifetimes were measured on a Hamamatsu C4334
Streak Scope streak camera, which has been described elsewhere.50
The emission signal was collected over a 140 nm window centered at
475 nm using 100, 50, 20, 10, or 5 ns time windows. Delays in the 100
ns time window were generated using a Hamamatsu C1097-04 delay
unit, whereas a Stanford Research Systems DG535 delay generator was
used to generate delays for the other time windows.
Two-photon emission spectra were generated using the afore-
mentioned Libra-F-HE (Coherent) chirped-pulse amplified Ti:sap-
phire laser system. Excitation pulses of 800 nm were used directly from
the Libra output; the pulse power was attenuated to 6 mW using
neutral density filters, and the beam was focused onto the sample using
a 500 mm focal length lens. The emission spectrum was collected
using a Hamamatsu C4334 Streak Scope streak camera in 140 nm
windows centered at 500, 550, and 650 nm.
Two-photon lifetime measurements were performed using a
previously described custom-built multiphoton laser-scanning micro-
scope (MPLSM) in the Edwin L. Steele Laboratory at the Department
of Radiation Oncology at Massachusetts General Hospital.51 The
MPLSM system was modified to enable lifetime measurements to be
performed.52 Sub-100 fs laser pulses were generated at a repetition rate
of 80 MHz in a mode-locked Ti:sapphire oscillator (Spectra Physics
Mai Tai HP), which was pumped by a 14 W cw Spectra Physics
Millennia diode-pumped solid-state (DPSS) laser operating at 532 nm;
the output of the Mai Tai laser was tunable over the 690−1040 nm
range. The 850 nm laser output was adjusted using a 10RP52-2 zero-
order half-wave plate (Newport) and a 10GL08AR.16 Glan-Laser
polarizer (Newport) to attenuate the power to 700−800 mW for
aerated samples and 400 mW for FPT samples. The beam was passed
through a 350−50 KD*P Pockels cell (Conoptics) that amplified and
switched the triggering pulses from a DG535 digital delay generator
(Stanford Research Systems). The experimental square wave trigger
pulse from the delay generator defined the repetition rate while a
second delayed pulse defined the excitation pulse, which was 1.6 μs in
duration for aerated samples and 15.36, 30.72, or 51.20 μs in duration
for FPT samples. At the rejection site of the Pockels cell, a TDS-3052
oscilloscope (Tektronix) and photodiode were used to monitor the
applied voltage and the optical response; the Pockels cell attenuated
the beam to 10% of the incident power. The laser beam was directed
into a custom-modified multiphoton microscope based on the
Olympus Fluoview 300 laser scanner. The output beam from the
scanner was collimated through a scan lens into the back of an
Olympus BX61WI microscope. An Olympus LUMPlanFL 20×, 0.95
NA water immersion objective lens was used to focus the excitation
light and collect the emission light. NIR laser excitation light and
visible emission light were separated using a 750SP-2P AR-coated
dichroic mirror (Chroma Technology). A 690/90 bandpass filter
(Chroma Technology) and a focusing lens were used in front of the
GaAs H7421-50 photomultiplier tube (Hamamatsu) to collect
phosphorescent emission. Photon counting was performed using a
SR430 multichannel scaler (Stanford Research Systems) to histogram
the counts in 1024 or 2048 bins of 40 ns for aerated samples or 2.56 or
5.12 μs for FPT samples.
Preparation of QD/Porphyrin Conjugates. Toluene stock
solutions of each palladium porphyrin 1−3 (∼100 μM) and QD
(∼10 μM) were prepared. The concentration of the QD stock solution
was calculated using ε350 = 4.34 × 105 M−1 cm−1, as estimated using an
empirical formula based on the first absorbance feature at 501 nm.44
An aliquot of the QD stock (typically containing ∼1 nmol of QDs)
was dissolved in 4 mL of toluene; an appropriate volume of the
porphyrin stock was then added to give 10 mol equiv of porphyrin per
QD. The resultant mixture was stirred overnight at room temperature
to allow equilibration of the porphyrin to the QD surface, to furnish
conjugates QD1−QD3.
Physical Measurements. 1H NMR spectra were recorded at
room temperature on a Varian Inova-500 or Bruker Avance-400 NMR
spectrometer at the MIT Department of Chemistry Instrumentation
Facility (DCIF) and internally referenced to the residual solvent signal
(δ = 7.26 for CHCl3 in CDCl3).45 Mass spectra were recorded on a
Bruker micrO-TOF-QII LCMS ESI-TOF mass spectrometer in
positive ion mode. All spectra were externally calibrated with sodium
formate. Elemental analysis was performed by Complete Analysis
Laboratories, Inc. (Parsippany, NJ). UV−vis absorption spectra were
acquired using a Cary 5000 spectrometer (Agilent). Steady-state
emission and excitation spectra were recorded on a SPEX FluoroMax-
3 spectrofluorimeter (Jobin Yvon Horiba). Relative quantum yields of
porphyrins (Φsam) were calculated using [Ru(bpy)3]Cl2 in MeCN as
the reference according to the following equation,
2
⎛
⎝
⎞
⎠
⎛
⎜
⎝
⎞⎛
⎞
⎟
⎠
η
η
Aref Isam
sam
⎜
⎜
⎟
⎟
Φ
= Φ
⎟⎜
sam
ref
A
I
sam ⎠⎝
ref
(1)
ref
where A is the measured absorbance, η is the refractive index of the
solvent, I is the integrated emission intensity, and Φref is the emission
quantum yield of the reference. Φref was taken to be 0.094 for a
freeze−pump−thawed (FPT) sample of [Ru(bpy)3]Cl2 in MeCN.46
The quantum yield of QD was similarly determined using fluorescein
27 in 0.1 M NaOH (Φref = 0.87, η = 1.335)47 as the standard.
Porphyrin samples for quantum yield measurements, vacuum lifetime
(τo) measurements, and evacuated steady-state emission spectra were
prepared using three cycles of FPT to pressures below 10−5 Torr.
Nanosecond time-resolved emission measurements of porphyrin
lifetimes were acquired using a previously reported system.48,49 Pump
light was provided by the third harmonic (355 nm) of a Quanta-Ray
Nd:YAG laser (Spectra Physics) operating at 10 Hz. The pump light
was passed through a BBO crystal in an optical parametric oscillator
(OPO), yielding a visible frequency that was tuned to 525 nm to excite
1−3 or 450 nm to excite the assemblies QD1−QD3. Excitation light
was attenuated to 1−4 mJ per pulse for all experiments using neutral
density filters. Emitted light was first passed through a series of long
pass filters to remove excitation light and then entered a Triax 320
monochromator (Jobin Yvon Horiba) and was dispersed by a blazed
grating (500 nm, 300 grooves/mm) centered at 685 nm. The entrance
and exit slits of the monochromator were set to 0.36 mm in all
experiments herein, corresponding to a spectral resolution of 4.5 nm.
The signal was amplified by a photomultiplier tube (R928,
Hamamatsu) and collected on a 1 GHz digital oscilloscope
(9384CM, LeCroy); acquisition was triggered using a photodiode to
collect scattered laser excitation light.
Femtosecond emission lifetime measurements were acquired using
a Libra-F-HE (Coherent) chirped-pulse amplified Ti:sapphire laser
system. Sub-100 fs laser pulses were generated in a mode-locked
Ti:sapphire oscillator (Coherent Vitesse) which was pumped by a 5 W
cw Coherent Verdi solid-state, frequency-doubled Nd:YVO4 laser. The
80-MHz output was amplified in a regenerative amplifier cavity,
pumped by a diode-pumped, frequency-doubled Nd:YLF laser
(Coherent Evolution-30) to generate a 1 kHz pulse train with a
wavelength of 800 nm. This was then used to pump an OperA Solo
(Coherent) optical parametric amplifier (OPA), which is able to
generate frequencies between 285 and 2600 nm. Excitation pulses of
450 nm were produced via fourth harmonic generation of the idler
Energy Transfer Analysis. The efficiency of energy transfer (E)
from the QD to the porphyrin was evaluated using Forster
̈
analysis,32,53
mkD−A
mR06
mR06 + r6
E =
=
mkD−A + τD−1
(2)
where kD−A is the rate of energy transfer, r is the distance between the
donor and acceptor, R is the Forster distance or the distance at which
̈
0
the energy transfer efficiency is 50%, and m is the number of acceptor
molecules per donor. This quantity can be measured experimentally:
τD−A
τD
E = 1 −
(3)
where τD is the lifetime of the QD alone and τD−A is the lifetime of the
QD with surface-bound porphyrin (QD1−QD3). Although the
D
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efficiency can be experimentally determined from the excited-state
lifetime quenching, additional information is needed to quantify the
graphic separations. It also allows for the synthesis of porphyrin
isomers that are difficult to isolate under statistical conditions
(e.g., cis-A2B2 versus trans-A2B2). Additionally, up to four
different meso substituents can be incorporated, thus enabling
the formation of precise porphyrin isomers that are inaccessible
with statistical methods (e.g., cis-A2BC and ABCD). However,
the rational synthesis (Supporting Information, Scheme S2) of
1-H2 entails a series of nine synthetic steps, with a yield of 15%
(based on bilane precursor) for the combined bilane synthesis
and subsequent porphyrin-forming steps.
parameters R , r, and m. The Forster distance is calculated using the
̈
0
following equation
9000(ln 10)κ2ΦD
R06
=
∞ FD(λ) εA(λ)λ4 dλ
∫
128π5Nn4
(4)
0
where κ2 is the relative orientation factor of the dipoles, taken to be
0.476 for static donor-acceptor orientations,32,54 ΦD is the quantum
efficiency of the donor (determined to be 0.72 for QD in toluene), N
is Avogadro’s number, and n is the index of refraction of the medium,
which is taken to be 1.4961 for toluene.55 The latter half of the
equation represents the spectral overlap integral, often represented as
J, where FD(λ) is the normalized intensity of the donor and εA(λ) is
the extinction coefficient of the acceptor at wavelength λ. Thus, R0
may be calculated from the experimentally determined emission
spectrum of the donor and the absorption spectrum of the acceptor.
X-ray Crystallographic Details. Diffraction quality crystals of 3
were obtained via slow vapor diffusion of heptane into a toluene
solution of the compound, affording the crystals as orange blocks.
Low-temperature (100 K) X-ray diffraction data was collected on a
Siemens Platform three-circle diffractometer coupled to a Bruker-AXS
Smart Apex CCD detector with graphite-monochromated Mo Kα
radiation (λ = 0.71073 Å), performing φ- and ω-scans. The data were
processed and refined using the program SAINT supplied by Siemens
Industrial Automation. The structure was solved by direct methods
using SHELXS and refined by standard difference Fourier techniques
in the SHELXL program suite.56 All hydrogen atoms were included in
the model at geometrically calculated positions using a riding model
and refined isotropically; all non-hydrogen atoms were refined
anisotropically. Unit cell parameters, morphology, and solution
statistics for the structure are summarized in Supporting Information,
Table S1. Thermal ellipsoid plots are drawn at the 50% probability
level with hydrogen atoms omitted for clarity.
A more facile synthesis of 1−3 is outlined in Figure 2. The
method relies on the use of bilane precursors (1-acyl
RESULTS AND DISCUSSION
■
Porphyrin Synthesis. The methodology to synthesize the
free-base pyridyl porphyrins 1-H2−3-H2 was optimized by
comparing the merits of various synthetic protocols for 1-H2.
Initially, a mixed aldehyde condensation under standard
Lindsey conditions57 was attempted, as this synthesis is well-
known and works for a wide variety of meso-substituted
porphyrins. However, only an insoluble black material was
obtained in the attempted synthesis of 1-H2, likely because of
the formation of polypyrrolic species. To circumvent these side
reactions, a mixed aldehyde/dipyrromethane condensation58 of
5-(4-pyridyl)dipyrromethane (12), 4-pyridinecarboxaldehyde,
and methyl 4-formylbenzoate was performed under Lindsey
conditions; no macrocyclic products were identified from this
reaction. The failure of these reactions is presumably because of
coordination of BF3·OEt2 to a pyridyl nitrogen, thereby
sequestering the Lewis acid catalyst. While compound 1-H2
has been prepared using Adler−Longo59,60 porphyrin synthesis
(Supporting Information, Scheme S1),61−64 this protocol
suffers from difficult and repetitive chromatographic separations
as all six possible porphyrin isomers (A4, A3B, cis-A2B2, trans-
A2B2, AB3, and B4) are formed in addition to other tetrapyrrolic
products, thus resulting in a low yield (4.3−5.9%)61−64 of the
desired A3B product. Indeed, we could only obtain 1-H2 under
Adler−Longo conditions at 3.9% yield (see Supporting
Information). An alternative to statistical methods is rational
porphyrin synthetic methods, which involve the construction of
a linear tetrapyrrole (i.e., bilane) that is cyclized and
subsequently oxidized to afford the corresponding porphyr-
in.65,66 This method has the advantage of forming a single
porphyrin product, thereby circumventing difficult chromato-
Figure 2. Synthesis of 1−3 from metal-catalyzed [2 + 2] cyclo-
additions of appropriate bilane precursors.
dipyrromethanes) in a MgBr2-catalyzed [2 + 2] cycloaddition
under microwave irradiation.40 While this is a statistical method
of synthesis for 1, the formation of only three porphyrin
isomers (trans-A2B2, A3B, and A4) is possible. Using this
method, porphyrin 1-H2 was isolated in 11% yield. This
nominal decrease in yield relative to the aforementioned bilane
route is more than compensated by the elimination of three
synthetic steps. This route is particularly amenable to the
synthesis of cis-A2B2 porphyrins such as 2-H2 for which the
separation of the three porphyrins is facile. Similarly, 3-H2 was
synthesized in a [2 + 2] cycloaddition of a 1,9-diacyldipyrro-
methane and a dipyrromethane. By replacing the typical TFA
acid catalyst38 with Yb(OTf)3, the yield of 3-H2 was increased
dramatically from 3.9% to 24%. In this case, the formation of
only one porphyrin isomer is possible. Given the simplicity of
this route and its advantages over both completely statistical
and rational methods, the [2 + 2] route was preferred in the
synthesis of 1-H2−3-H2.
E
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Palladium was introduced into the porphyrin macrocycle by
using Pd(acac)2 in pyridine under microwave irradiation.67 This
dramatically reduces the reaction time from as long as 4 d
under conventional heating68 to 20 min with microwave
irradiation. Although the metalation reaction using this method
is typically quantitative,67 the observed yields here are low (17−
50%). This is attributed to the formation of insoluble
coordination oligomers in which Pd2+ coordinates to pyridyl
groups of two different porphyrins, as has previously been
observed.69−72
Diffraction quality crystals of 3 were obtained as orange
blocks by slow vapor diffusion of heptane into a toluene
solution of the compound. The thermal ellipsoid plot of the
refined structure is shown in Figure 3, and a summary of the
Figure 3. Solid-state crystal structure of compound 3. Thermal
ellipsoids are drawn at the 50% probability level and hydrogen atoms
have been removed for clarity.
crystallographic data is presented in Supporting Information,
Table S1. To identify the unique 4-pyridyl group of the
structure, the 4 position of each meso substituent was refined as
a carbon atom, and the resultant electronic density difference
map was examined. The 10 largest residual density peaks (Q
peaks) of this refinement cycle are shown in Supporting
Information, Figure S1. Three of the four meso substituents
show a Q peak adjacent to the 4 position of the ring, indicating
the presence of a hydrogen atom, while the fourth does not,
thereby differentiating the 4-pyridyl ring from the phenyl rings.
Qualitatively, the structure is very similar to other Pd porphyrin
complexes, such as 5,10,15,20-tetrakis(4-carboxyphenyl)-
porphyrinatopalladium(II).73 The palladium atom resides in
the center of the 24 atom macrocycle plane with an average
Pd−N distance of 2.011 Å (as compared to 2.009 Å for
5,10,15,20-tetraphenylporphyrinatopalladium(II), PdTPP).74
The porphyrin plane exhibits an S4 ruffle with an average
deviation of 0.196 Å from the mean 24 atom plane. Such
nonplanarity is also observed in the solid state structure of
PdTPP. Each of the pyrrole nitrogen atoms deviates from the
mean N4 plane by 0.030 Å, a displacement that is identical to
that observed for PdTPP.74
Electronic Properties of Porphyrins. Porphyrins 1−3
display absorption spectra (Figure 4) that are typical of
hypsoporphyrins.75,76 Table 1 summarizes the spectroscopic
data for these porphyrins. The B band (Soret), centered at 415
nm, is intense and flanked by the weaker Q(1,0) and Q(0,0)
bands at 523 and 554 nm, respectively. This absorption profile
is similar to other meso-substituted Pd porphyrins. For
comparison, PdTPP exhibits analogous absorbance features at
416, 522, and 551 nm.77 The porphyrin emission spectra
(Figure 4) (λexc = 525 nm) exhibits two broad emission bands
Figure 4. Comparison of the steady state absorption (red lines) and
emission spectra (λexc = 525 nm) of (a) 1, (b) 2, and (c) 3 in the
presence of air (∼160 Torr O2) (gray lines) and under vacuum (blue
lines) in toluene.
with maxima observed at 690 nm for T(0,0) and 760 nm for
T(0,1); these spectral features are similar to those observed for
PdTPP (688 and ∼760 nm).77 Each porphyrin also exhibits a
small emission feature at about 606 nm that is attributed to the
Q(0,1) fluorescence transition. This assignment is consistent
with the insensitivity of the intensity of this band to oxygen;
such a transition has been observed at 606 nm for PdTPP.78
The additional feature observed in the emission spectrum of
2 centered at 650 nm is assigned to the Q(0,0) transition of 2-
H2. Based on the size of the emission band and the ten-fold
higher emission quantum yield for H2TPP (ϕf = 0.13)79 as
compared to that of the Pd complex (ϕp = 0.011 for 2), it is
estimated that 2-H2 is present at most as a 1% impurity. In
accordance with this contention, no trace of 2-H2 was visible by
NMR or TLC. Since the 2-H2 fluorescence band is eclipsed by
the phosphorescence bands of 2 in the absence of oxygen
(Figure 4b) and the fluorescence lifetime of 2-H2 (τair ∼ 9 ns
for free-base pyridyl porphyrins)80 is much shorter than 2 (τair
∼ 300 ns), the trace presence of this species has little impact on
the results presented herein.
The phosphorescence quantum yields of 1−3 are between
1−2% and are comparable to other Pd porphyrins (ϕp = 0.02
for PdTPP).81 As shown in Figure 4, the phosphorescence is
quenched significantly by air, establishing that these com-
pounds are responsive to oxygen in the 0−160 Torr range.
F
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Table 1. Summary of Linear Spectroscopic Data for Pd Porphyrins and QD Conjugates
a
b
b
b
c
c
d
compound
B(0,0), ε
Q(1,0), ε
Q(0,0), ε
QDEm
T(0,0)
T(0,1)
ϕp
1
414, 240
416, 290
415, 250
416
522, 22
523, 24
523, 22
523
554, 3.6
554, 2.6
554, 2.8
554
684
691
691
684
692
691
753
760
760
754
763
761
0.014
0.011
0.014
0.040
0.027
0.018
2
3
QD1
QD2
QD3
517
518
516
418
524
555
417
523
553
a
b
c
Toluene solution, transition wavelengths are in units of nm. ε in 103 M−1 cm−1, as determined in CH2Cl2. Triplet transitions for evacuated
d
samples (<10−5 Torr). Phosphorescence quantum yield of freeze−pump−thawed samples.
Indeed, the phosphorescence emission intensity from a freeze−
pumped−thawed (FPT) sample of 1 is 500-fold that observed
for the sample in air; a 100-fold to 200-fold enhancement was
observed for 2 and 3.
The triplet lifetime (λexc = 525 nm) data of 1−3 in toluene at
room temperature in the presence and absence of air is
summarized in Table 2. Decay traces were fit at t > 100 ns to a
450 nm) centered at 519 nm (Supporting Information, Figure
S2).
The quenching of QD luminescence via energy transfer was
examined as a function of the disposition of pyridyl rings at the
meso positions: two cis 4-pyridyl rings (1), two cis 3-pyridyl
rings (2), and a single 4-pyridyl ring (3). Titrations were
performed in which the same amount of QD (∼400 picomoles
or ∼100 nM) was treated with 1, 2, 5, or 10 equiv of 1−3. Each
sample point in the titration was prepared independently and
incubated overnight at room temperature to ensure porphyrin
binding on the QD surface. Figure 5 shows the absorption,
steady-state emission and time-resolved emission profiles for
the titration of QD with 1; similar data for compounds 2 and 3
are presented in Supporting Information, Figures S3 and S4,
respectively. The excitation wavelength of 450 nm was chosen
for these experiments because the absorbance of the porphyrin
is minimal whereas that of the QD is relatively high.
Table 2. Summary of Lifetime Data for Pd Porphyrins
b
b
τair (ns)
1-photon
τo (μs)
τair (ns)
2-photon
τo (μs)
d
a
c
c
d
compound
1-photon
2-photon
e
1
2
3
332
289
305
3
7
6
154 12
556 40
354 14
365 12
157
77
3
5
3
133
144
5
6
80
a
b
Toluene solution. Freeze−pump−thawed samples (<10−5 Torr).
c
d
e
λexc = 525 nm. λexc = 850 nm. 95% confidence interval.
The absorption profile (Figure 5a) is effectively the sum of
the QD and compound profiles. Figures 5b and 5c establish
significant quenching of the QD emission with increasing
concentration of the porphyrin. The quenching lifetimes
obtained from the decay profiles in Figure 5c are provided in
Supporting Information, Table S2. We note that the emission
2
monoexponential decay curve (Radj > 0.96). The significant
difference in the lifetimes of 1−3 in the presence (∼300 ns)
and absence of air (∼150 μs) suggests that these phosphors
have a sufficient dynamic range to accurately quantify oxygen
over a 0−160 Torr range. Although porphyrins have low two-
photon cross sections, sufficient signal was acquired to measure
the lifetimes of 1−3 under two-photon (λexc = 850 nm)
excitation (Table 2). To the best of our knowledge, this is the
first study that compares porphyrin lifetimes under one- and
two-photon excitation. Because of differences in selection rules
for one- and two-photon transitions, different states are
accessed with different vibronic coupling.82 It has been shown
for linear polyenes that these differences lead to different one-
and two-photon lifetimes.83 Accordingly, the differences in
lifetimes of 1−3 under one- and two-excitation are not
unexpected.
2
decay of QD in the absence of porphyrin is biexponential (Radj
> 0.99) with components that reflect surface trapped states (17
ns) and exciton emission (5 ns).84,85 While QD lifetimes have
been fit to triexponential functions86,87 to include Auger
recombination,88 the inclusion of a third term is an over-
parameterization of our data. Biexponential behavior is
preserved upon titration with porphyrin. Of the three
compounds, 2, with its cis disposition of pyridyl rings, quenches
QD emission most effectively whereas 3, with its single pyridyl
ring is the least effective quencher. These results clearly suggest
that two cis pyridyl rings facilitate porphyrin binding to the QD
surface.
Since the formation of the QD/porphyrin conjugate occurs
under dynamic self-assembly, there exists a discrete distribution
of the number of porphyrins attached to the QD; hence there is
a distribution of QD:porphyrin ratios at any point in the
titration. Such a distribution is best described with Poisson
statistics.80 The Poisson probability distribution provides a
model for the probability, p(Y), that the number of events Y
(i.e., a given QD:porphyrin ratio) occur, where λ is the average
value of Y89
QD Quenching Titrations with 1−3. Titration experi-
ments were performed to assess surface binding of pyridyl
porphyrins 1−3 to the surface of QD. In these titrations, there
exists the following equilibrium,
KA
QD + Por ⇄ QD−Por
KD
(5)
with an equilibrium constant KA. Upon increasing the amount
of porphyrin, the equilibrium is driven to conjugate formation.
The QD emission provides a convenient probe of the
equilibrium. For these experiments, the QD was judiciously
chosen such that its emission overlapped with the absorbance
of Q(1,0) of 1−3, thereby maximizing the spectral overlap
integral J (eq 4) and accordingly enabling quenching of QD
emission by energy transfer. Specifically, we selected a QD with
λY
Y!
p(Y) =
e−λ
(6)
where Y = 0, 1, 2, ..., and λ > 0. For example, after the addition
of 1 equiv of porphyrin per equiv of QD (λ = 1), the probability
p(Y) that there are 0, 1, 2, or ≥3 porphyrins per QD is 0.368,
0.368, 0.184, and 0.080, respectively.
a first absorbance feature at 501 nm and emission band (λexc
=
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For 1 equiv of 1, the long component (surface trapped
states) is reduced to 12% of the overall fit (compared to 53%
for free QD), suggesting that ∼20% of the QDs in solution lack
a surface bound porphyrin. If the distribution of species in
solution were purely statistical (i.e., non-specific interactions or
encounter complexes), one would expect that ∼37% of QDs
would lack a surface bound porphyrin based on the Poission
distribution. Since we observe fewer free QDs in solution, it is
clear that 1 is binding to the QD surface in a directed manner.
This is more clearly illustrated with 2, which shows virtually no
free QDs in solution after the addition of 1 equiv of porphyrin.
The fact that these two compounds do not follow the expected
Poission distribution indicates that the QD-porphyrin inter-
action is specific (i.e., directed bidentate surface binding) and
not just a statistical non-specific interaction. These observations
are in stark contrast to compound 3. After the addition of 1
equiv of 3, the amplitude of the long component is reduced to
25%, suggestive of 47% free QDs in solution. This is consistent
with the expected value of 37% base distribution. The binding
of 3 is so inefficient that ∼34% of QDs are free in solution after
the addition of 2 equiv (compare to an expected 13% based on
the Poission distribution). Since the titration with 3 matches
the expected Poisson distribution, the interaction between this
molecule and the QD is nonspecific, suggesting that the
interaction is statistical rather than directed. These results are
consistent with the proposal that 1 and 2 are immobilized via a
two point attachment, while porphyrin 3, with a single 4-pyridyl
group, results in less efficient luminescence quenching.
Whereas the distribution of species at low porphyrin
concentration can be probed using QD lifetime data as a
measure of free QD in solution, the lack of a suitable
spectroscopic signature at high porphyrin concentrations
renders the experimental determination of QD-porphyrin ratios
intractable. At low porphyrin concentrations, the distribution of
species in solution is dictated by the porphyrin’s affinity for the
surface (i.e., equilibrium constants as described below). At high
porphyrin concentrations, geometric and steric factors control
the distribution of QD-porphyrin species. Therefore, it is
expected that this phenomenon is stochastic and follows a
Poisson distribution.80
Determination of Equilibrium Constants. Using both
the QD emission intensity and the lifetime titration data, the
equilibrium constant (KA) for the formation of the QD-
porphyrin conjugate (eq 5) was determined. The association
constant can be determined using the Stern−Volmer equation:
Figure 5. Spectral changes associated with titration of a toluene
solution of QD (gray lines) with 1 (red lines), 2 (yellow lines), 5
(green lines), and 10 (blue lines) equiv of 1. Similar data for 2 and 3
are shown in Supporting Information, Figures S3 and S4. (a) The
intensity of the Soret and Q-band absorption profiles increases. (b)
The QD emission (λexc = 450 nm) intensity is quenched, and (c) the
decay profiles of the QD emission (λexc = 450 nm) decrease with
increasing equiv of 1.
As with the QD alone, the presence of porphyrin gives rise to
a biexponential decay. Both the exciton emission (short lifetime
component) and surface trapped state (long lifetime
component) are affected by the presence of porphyrin. Because
the surface state is most perturbed by porphyrin binding, the
amplitude of the long lifetime component was used as a metric
for determining the amount of free QD in solution. To this
end, the long component of each decay was fixed at 16.93 ns,
which is the calculated lifetime of the surface-trapped states for
the QD alone. The relative reduction in the amplitude of this
component serves as a measure of free QD at various points in
the titration. The lifetime data is summarized in Supporting
Information, Table S2. Over the course of the titration, the
surface trapped state is quenched, resulting in the formation of
a non-fluorescent QD-porphyrin complex (i.e., static quench-
ing). Based on the data, 10 equiv of 1 and 2 reduce the QD
lifetime to ∼300 ps with a similar emission intensity (vide
supra), indicating that the cis pyridyl groups enhance QD
surface binding and result in efficient luminescence quenching.
Io
I
τ
τ
o
=
= 1 + KA[Por]
(7)
where Io and τo are the emission intensity and lifetime,
respectively, in the absence of added porphyrin, I and τ are the
emission intensity and lifetime, respectively, in the presence of
a given concentration of porphyrin, [Por]. For all three
porphyrins, both the lifetime (short component of Supporting
Information, Table S2) and intensity data are consistent, giving
qualitatively similar plots (Supporting Information, Figure S5).
Downward curvature in the plots of the stronger ligators 1 and
2 at high porphyrin concentrations suggests a binding
saturation event while 3 exhibits a linear plot. The linear
regions of the data (Supporting Information, Table S3) were fit
to eq 7 to give average values of KA = 2.50 × 107 M−1 (1), 2.83
× 107 M−1 (2), and 1.36 × 106 M−1 (3). These results are
consistent with the data of Figure 5, Supporting Information,
Figures S3 and S4, indicating that 1 and 2 bind to the QD
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surface with similar efficacy whereas 3 binds more weakly. At
high porphyrin concentrations, the curvature of 1 and 2 may be
modeled with the Hill equation, which accounts for binding
cooperativity:
[Por]n
y = ymax
KDn + [Por]n
(8)
where ymax is the theoretical maximum value of y (I0/I or τ0/τ)
at infinite substrate concentration and is a measure of the
maximum extent of quenching of lifetime or emission intensity.
KD is the dissociation constant (KD = 1/KA), and n is the Hill
coefficient, which is a measure of the degree of binding
cooperativity. Positive cooperativity, that is, the binding of one
molecule facilitates the subsequent binding event, is indicated
for n > 1. For n < 1, there is negative cooperativity such that the
binding of one molecule impedes the binding of additional
molecules. If n = 1, binding is not cooperative, and each binding
event is independent. The fits of the data for 1 and 2 to eq 8 are
presented in Supporting Information, Table S4. For both
intensity and lifetime data, the calculated ymax is very close to
the observed y values at 10 equiv, suggesting that binding
saturation to a QD is reached at a limiting value of 10 equiv of
1 and 2. The different values of n = 1.8 for 1 and n = 0.8 for 2,
may reflect the different coordination modes of the porphyrins
on the QD surface. The π-stacking of 1 between porphyrin
rings could lead to a positive cooperativity through π−π
stacking interactions that facilitate the binding of subsequent
porphyrin molecules. Conversely, an angled conformation in
which the porphyrin covers the surface of the QD, would
inhibit the binding of neighboring porphyrin molecules. This
binding model is supported by titration experiments in which 1
equiv of 2 dramatically quenches QD (Supporting Information,
Figure S3 and Table S2); we suspect this is because of the
proximity of the porphyrin macrocycle to the QD surface.
Characterization of QD−Porphyrin Conjugates. Given
the results of the titration experiments, 10 equiv of porphyrin
drives the equilibrium in eq 5 far to the right and ensures that
each QD is associated to porphyrin. From eq 6, the probability
of having a free QD at 10 equiv of porphyrin in solution is
essentially 0 (0.0045%). Conjugates prepared from 10 equiv of
porphyrin are designated QD1−QD3.
Figure 6. Comparison of the absorption spectra of 1 (gray lines), QD
(red lines) and QD1 (blue lines) in toluene. The spectrum of QD1
represents a composite of the two constituent spectra.
Table 1 lists selected steady-state absorption and emission
spectroscopic data for the self-assembled QD1−QD3 con-
jugates. The absorption spectrum of the conjugates is a
composite sum of the porphyrin and QD spectra, as illustrated
in Figure 6 for QD1. The emission spectra (λexc = 450 nm) also
exhibit features characteristic of both the porphyrin and the
QD. As shown in Figure 7, a slight 1−2 nm (∼ 100 cm−1 for B
bands and ∼40 cm−1 for Q bands) red shift was observed in the
porphyrin absorption and emission bands of QD1−QD3 as
compared to 1−3. This phenomenon, which has been observed
for free-base porphyrins90 and pyrene25 bound to a QD surface,
has been attributed to differences between the average dielectric
constant of the lumophore in solution as compared to that near
the surface of the QD. The QD emission from the conjugate is
slightly blue-shifted (1−3 nm, 40−100 cm−1) relative to free
QD, which has previously been ascribed to modulation of the
QD surface states91,92 upon ligand binding.93 More pronounced
than these slight wavelength shifts is the increase in porphyrin
phosphorescence quantum yield when bound to the QD (Table
1); QD1 and QD2 show the greatest enhancement (nearly a
three-fold increase) in quantum yield upon surface binding as
Figure 7. Comparison of the steady state absorption (red lines) and
emission spectra (λexc = 525 nm) of (a) QD1, (b) QD2, and (c) QD3
in the presence of air (∼160 Torr O2) (gray lines) and under vacuum
(blue lines) in toluene.
compared to QD3. This phenomenon has been attributed to a
decrease in knr when the porphyrin is intercalating within the
capping ligand environment.9 These observations are con-
cordant with stronger association of 1 and 2 to the QD surface
than as compared to 3 owing to the cis-disposition of pyridyl
ligands at the meso sites of the porphyrin framework of the
former. Because the intensity of the T(0,0) porphyrin and QD
emissions of QD1 and QD2 are comparable, a self-referencing
scheme is easily established with these conjugates. This is not
I
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Inorganic Chemistry
Article
the case of QD3, which displays weak porphyrin emission as
compared to the QD emission. The O2 response is reversible,
as the phosphorescence intensity is recovered upon subjecting
an aerated sample to a freeze−pump−thaw cycle. This process
may be repeated with no change in intensity.
The enhancement of porphyrin emission upon association of
the QD is a result of FRET. In these assemblies, the QD serves
as the antenna for photon absorption. Energy is passed from
the QD donor to the porphyrin acceptor by the FRET
mechanism. Under linear excitation (λexc = 450 nm), a nearly
four-fold enhancement in emission is observed when QD is
present (Supporting Information, Figure S6) owing to the
greater absorbance of the QD at 450 nm relative to 1.
would look like the absorption profiles of 1−3. Because of the
similar extinction coefficients of 1−3, the excitation spectra of
these compounds should be the same in the absence of other
effects. However, this is not the case because the FRET is
modulated by the association of porphyrin to the QD.
Accordingly, concentration-matched solutions of QD1−QD3
show that QD3 produces the least emission signal (since 3 is
the weakest binder), having an intensity that is approximately
50% of QD1.
The FRET enhancement of porphyrin emission via the QD
donor is more dramatic under two-photon excitation (λexc
=
800 nm), as the two-photon absorption cross-section for QD is
∼104 times greater than for the porphyrin itself. Figure 9 shows
The FRET parameters for QD1−QD3 are summarized in
Supporting Information, Table S5, and the spectral overlap of
QD donor and porphyrin acceptor is illustrated in Supporting
Information, Figure S7. J is similar for all three assemblies and
thus Forster distances (R ) for the QD-porphyrin conjugates
̈
0
are also similar. Nevertheless, the FRET efficiencies, as
calculated from the short component of the QD lifetimes at
10 equiv (Supporting Information, Table S2) and eq 3, vary
significantly owing to differences in the interaction of the
porphyrin with QD. The efficiency is 94% for 1 and 2, but only
67% for 3. The disparate conjugate interactions are unveiled in
the donor−acceptor distances calculated from eq 2. r is
significantly (∼1.5 nm) longer in QD3 as compared to QD1
and QD2. These data again indicate that the cis disposition of
the meso pyridyl substituents results in a tighter and more
compact QD:porphyrin conjugate. The similarity of r for QD1
and QD2 indicate that the substitution motif (4-pyridyl versus
3-pyridyl) is not crucial to the FRET efficiency.
Figure 9. Two-photon emission spectra (λexc = 800 nm) of
concentration-matched toluene solutions of QD (black lines) and
freeze−pump−thawed solutions of 1 (blue lines) and QD1 (red lines).
1 does not emit under two-photon excitation, and emission is only
observable in the presence of QD.
The consequences of the FRET efficiency calculations are
revealed in the excitation spectra of QD1−QD3 in Figure 8,
the two-photon emission spectrum for concentration-matched
solutions of QD, 1, and QD1. The porphyrin emission in QD1
relative to 1 is enhanced by 10-fold and at the same time, the
QD emission is reduced by ∼70% in QD1 relative to QD.
These results indicate that the QD serves as a photon antenna
and FRET donor, resulting in enhanced porphyrin emission
under both one- and two-photon excitation.
Although QD1−QD3 show a qualitatively similar response
to oxygen as do 1−3, evidenced by the difference in the
emission intensity (Figure 7) in the presence and absence of
air, the relative extent of quenching is not as great as the free
porphyrin in solution. This observation is attributed to the
overall lower amount of signal due to the FRET excitation of
the porphyrin in QD1−QD3 relative to direct excitation of
Q(1,0) in 1−3. Air quenches the T(0,0)emission of QD1 by
100- and by 50-fold for T(0,1). QD2 is somewhat less sensitive
with approximately a 50-fold effect for both transitions whereas
only a 10-fold effect is observed for QD3, presumably because
of the lower FRET efficiency and thus lower amount of total
signal.
Figure 8. Excitation spectra (λem = 685 nm) of concentration-matched
toluene solutions of QD1 (black lines), QD2 (red lines), and QD3
(blue lines).
which were recorded by monitoring the porphyrin emission at
685 nm with a 5 nm bandwidth. All of the collected emission is
attributed to the porphyrin, as QD emission is spectrally
separated by over 150 nm. At wavelengths where the
porphyrins do not readily absorb (<375 nm), a substantial
emission signal is observed. This cannot be attributed to
porphyrin absorption and subsequent emission. Instead, the
QD absorbs the incident photons and transfers energy to the
porphyrin which then emits. These excitation spectra
correspond to the absorption profile of the conjugates (Figure
6), reflecting both porphyrin and QD absorption profiles. If the
QD were not acting as a FRET donor, the excitation spectra
Triplet lifetimes (λexc = 450 nm) of QD1−QD3 were
determined in toluene at room temperature under air and for
FPT solutions (Table 3). Decay traces at t > 100 ns were fit to a
2
biexponential decay curve (Radj > 0.96). With an increase in
quantum yield of the porphyrin within the QD construct, a
similar increase in lifetime is expected based on
kr
Φ =
= krτo
kr + knr
(9)
J
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Article
Table 3. Summary of Porphyrin Triplet State Lifetime Data for Assemblies QD1−QD3
a
b
b
c
compound
sample
λexc/nm
τ1
A1 (%)
τ2
A2 (%)
τavg
d
QD1
QD2
QD3
air
air
air
450
450
450
638 64 ns
575 5 ns
550 6 ns
17
1
69 11 ns
29 1 ns
41 1 ns
83
99
94
174 92 ns
37 1 ns
72 7 ns
6
e
QD1
QD2
QD3
fpt
450
450
450
590 79 μs
482 41 μs
75
65
69
149 51 μs
143 34 μs
166 51 μs
25
35
31
479 26 μs
363 15 μs
386 106 μs
fpt
fpt
578 111 μs
QD1
QD2
QD3
air
air
air
850
850
850
537 14 ns
401 4 ns
430 5 ns
100
100
100
f
f
f
537 14 ns
401 4 ns
430 5 ns
QD1
QD2
QD3
fpt
fpt
fpt
850
850
850
270 11 μs
203 8 μs
101 5 μs
62
60
60 10 μs
57 15 μs
g
38
40
190 16 μs
145 11 μs
100
101 5 μs
a
b
c
d
Toluene solution. Relative contribution to the biexponential fit. Weighted average lifetime, calculated as τavg = (A1τ1 + A2τ2)/100. 95%
e
f
Confidence interval. Freeze−pump−thawed samples (<10−5 Torr). Because of instrumental limitations, the fast component could not be resolved
and the data was fit to a monoexponential decay. A biexponential fit was found to be an overparameterization of the data and was thus fit to a
g
monoexponential decay.
As expected, the long component of the decay is significantly
longer than free porphyrin by over 300 ns for air measurements
and 450 μs for FPT measurements (see Table 2). Such
observations have been previously reported for other
fluorophore systems.9,25,85 The significantly longer component
is attributed to the intercalation of the porphyrin into the
passivating ligand that coats the QD surface; this protects the
porphyrin from molecular collisions and other nonradiative
decay processes, thereby extending the radiative lifetime. The
shorter component is likely to be free porphyrin as this lifetime
is comparable to free porphyrin in solution (∼150 μs, Table 2).
In air, the average lifetimes for the QD−porphyrin assemblies
are significantly shortened, reflecting the quenching process
observed by steady-state emission.
In addition to the one-photon lifetimes, the lifetimes of FPT
and aerated samples of QD1−QD3 were measured under two-
photon irradiation (λexc = 850 nm) (Table 3). The observed
average lifetimes for QD1 and QD2 are consistent with the
results of one-photon excitation of 1 and 2. The FPT samples
exhibit long (∼200 μs) and short lifetime components (∼50 μs,
compared to ∼150 μs for free porphyrin). The lifetimes of the
conjugates in air under two-photon excitation reflect
quenching, but the short lifetime, akin to what was observed
in the one-photon case, is not resolved from the instrument
response function.
porphyrin assemblies have been previously studied as FRET
systems,80,86,90 they have not been examined under two-photon
excitation nor have they been explored as sensors. Both
ruthenium bipyridine complexes94 and Pt porphyrins95,96 have
been used as O2 sensors in conjunction with QDs, but in these
systems the QD has served only as an internal intensity
standard. Indeed the few examples of authentic FRET-based O2
sensing include pyrene,25 platinum octaethylporphyrin ke-
tone,97,98 or osmium bipyridine complexes9 as the FRET
acceptor. Of these, two-photon excitation of the FRET acceptor
by QD has been achieved only with the osmium bipyridine
complex. This system, however, suffers from low dynamic range
at biologically relevant oxygen pressures (0−160 Torr).9 We
now establish a viable two-photon O2 sensing mechanism over
this pressure range with QD1−QD3. Having fully characterized
this system in organic solvents, we are currently developing
methods to translate this system to water so that it can be used
for in vivo two-photon oxygen sensing applications.
ASSOCIATED CONTENT
* Supporting Information
Detailed synthetic procedures for porphyrin precursors and
■
S
1
alternative methods, in addition to H NMR spectra of all
compounds, are presented in the Supporting Information.
Summaries of crystallographic data, QD luminescence lifetime
titration data, equilibrium constants, FRET parameters, and
other relevant spectral data are included. This material is
available free of charge via the Internet at http://pubs.acs.org.
CONCLUSIONS
■
Self-assembled QD−porphyrin nanosensor conjugates detect
O2 in the 0−160 Torr range under both one- and two-photon
excitation. Two cis pyridyl groups in the meso position of the Pd
porphyrin macrocycle promote the most effective binding of
the palladium porphyrin to the surface of a QD. The QD serves
as one- or two-photon light harvesting antennae of the Pd
porphyrin. Owing to the insensitivity of the QD to O2, a
ratiometric signal transduction mechanism may be established.
Because of superior spectral overlap and efficient surface
binding, the FRET efficiency in these systems is 67−94%.
The QD is essential in implementing sensing under two-
photon conditions owing to the QD’s high two-photon
absorption cross-section (σ2). Although free-base QD−
AUTHOR INFORMATION
Corresponding Author
*E-mail: dnocera@fas.harvard.edu (D.G.N.), mgb@mit.edu
(M.G.B.).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the U.S. National Cancer
Institute grants R01-CA126642. C.M.L. acknowledges the
National Science Foundation’s Graduate Research Fellowship
K
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Article
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using the Libra−F−HE laser system, Andrew Ullman for
assistance in collecting X-ray diffraction data, Dr. Xiaoxing Han
for assistance in acquiring two-photon lifetimes, and Dr.
Thomas Kempa for acquiring the TEM images used in the
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