Microwave-assisted synthesis of thiophene fluorophores, labeling and multilabeling of monoclonal antibodies, and long lasting staining of fixed cells

Article abstract of DOI:10.1021/ja902416s

We report the expedient microwave-assisted synthesis of thiophene based 4-sulfo-2,3,5,6,-tetrafluorophenyl esters whose molecular structure was engineered to achieve blue to red bright fluorescence. The reactivity toward monoclonal antibodies of the newly

Full text of DOI:10.1021/ja902416s

Published on Web 07/20/2009
Microwave-Assisted Synthesis of Thiophene Fluorophores,
Labeling and Multilabeling of Monoclonal Antibodies, and
Long Lasting Staining of Fixed Cells
Massimo Zambianchi,† Francesca Di Maria,† Antonella Cazzato,‡ Giuseppe Gigli,§
Manuel Piacenza,§ Fabio Della Sala,§ and Giovanna Barbarella*,†
Consiglio Nazionale Ricerche CNR-ISOF and Mediteknology srl, Via Gobetti 101,
40129 Bologna, Italy, and National Nanotechnology Laboratory (NNL) of INFM-CNR and Dip.
Ingegneria InnoVazione, UniVersita` del Salento, Via Arnesano, 73100 Lecce, Italy
Received March 26, 2009; E-mail: barbarella@isof.cnr.it
Abstract: We report the expedient microwave-assisted synthesis of thiophene based 4-sulfo-2,3,5,6,-
tetrafluorophenyl esters whose molecular structure was engineered to achieve blue to red bright
fluorescence. The reactivity toward monoclonal antibodies of the newly synthesized fluorophores was
analyzed in comparison with that of the corresponding N-succinimidyl esters. Single-fluorophore and multiple-
fluorophore labeled antibodies were easily prepared with both types of esters. Multiple-fluorophore labeling
with blue and orange emitting fluorophores resulted in white fluorescent antibodies. Thiophene based
fluorophores displayed unprecedented fluorescence stability in immunostaining experiments. First-principles
TD-DFT theoretical calculations helped us to interpret the behavior of fluorescence emission in different
environments.
1. Introduction
become one the most exploited techniques owing to its
sensitivity and noninvasiveness.2 Unfortunately, a universal
class of fluorophores applicable to all the fields of interest
has not yet been described. There are many classes of
fluorophoressmolecular, macromolecular, dendrimeric, nano-
structured, some of which already commercialsthat are
employed more or less efficiently in specific applications.1
One of the best classes of fluorophores described so far is
that of Green Fluorescent Proteins (GFPs), worthy of the
Chemistry Nobel Prize 2008.3 GFPs are genetically encoded
fluorescent tags; joining GFPs to a protein of interest allows
the fluorescent imaging of the protein within the cell.4 Variants
of GFP are able to emit light in many different colors.5
Fluorescent proteins are a great achievement, yet still other
biomolecules not directly encoded by the genome such as
nucleic acids, glycans, lipids, or secondary metabolites cannot
be imaged by this method.
Expanding the tool box of currently available fluorescent
probes for the monitoring of biological systems and processes
is a challenge crucial to several research areas spanning from
medical diagnostics to genomics and live cell metabolism.1
In the past few years understanding the molecular basis of
diseases and developing new methods for genetic testing as
well as environmental monitoring, security problems, and
forensic analyses have become topics of central importance.
In all these fields, new and more efficient analytical tools to
characterize nucleic acids, proteins, and cells and the
production of low cost, easy to manipulate systems for fast
sample analysis and large-scale screening are highly required.
In this rapidly developing context, fluorescent detection has
† Consiglio Nazionale Ricerche CNR-ISOF.
‡ Mediteknology srl.
Another class of fluorescent probes that has enjoyed great
attention in the past few years is that of quantum dots (QDs),
nanocolloids made of CdSe or other inorganic nanocrystals.6,1c
Different strategies of QD surface modification and coating
§ Universita` del Salento.
(1) (a) Sameiro, M.; Gonc¸alves, T. Chem. ReV. 2009, 109, 190–212. (b)
Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2008, 3, 142–155. (c)
Mann, S. Angew. Chem., Int. Ed. 2008, 47, 2–17. (d) Resch-Ganger,
U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Nat.
Methods 2008, 5, 763–775. (e) Chen, A. K.; Cheng, Z.; Behlke, M. A.;
Tsourkas, A. Anal. Chem. 2008, 80, 7437–7444. (f) Simeonov, A.;
Jadhav, A.; Thomas, C. J.; Wang, Y.; Huang, R.; Southall, N. T.;
Shinn, P.; Smith, J.; Austin, C. P.; Auld, D. S.; Inglese, J. J. Med.
Chem. 2008, 51, 2363–2371. (g) Longmire, M. R.; Ogawa, M.; Hama,
Y.; Kosaka, N.; Regino, C. A. S.; Choyke, P. L.; Kobayashi, H.
Bioconjugate Chem. 2008, 19, 1735–1742. (h) Johnsson, N.; Johnsson,
K. ACS Chem. Biol. 2007, 2, 31–38. (i) Mart´ı, A. A.; Jockusch, S.;
Stevens, N.; Ju, J.; Turro, N. J. Acc. Chem. Res. 2007, 40, 402–409.
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Y.; Koyama, Y.; Bernardo, M.; Choyke, P. L.; Kobayashi, H.
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2005, 2, 902–904. (p) Lichtman, J. W.; Fraser, S. E. Nat. Neuroscience
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(2) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.;
Springer: New York, 2006.
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Ai, H.; Hazelwood, K. L.; Davidson, M. W.; Campbell, R. E. Nat.
Methods 2008, 5, 401–403. (c) Shaner, N. C.; Lin, M. Z.; McKeown,
M. R.; Steinbach, P. A.; Hazelwood, K. L.; Davidson, M. W.; Tsien,
R. Y. Nat. Methods 2008, 5, 545–551. (d) Shaner, N. C.; Campbell,
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9
10892 J. AM. CHEM. SOC. 2009, 131, 10892–10900
10.1021/ja902416s CCC: $40.75  2009 American Chemical Society

Microwave-Assisted Synthesis of Thiophene Fluorophores
A R T I C L E S
procedures have been described to make them biocompatible.
The development of a sophisticated QD technology has allowed
their application to many biological problems, including in ViVo
experiments with NIR fluorescent QDs.6 QDs have demonstrated
superior performance in applications requiring single particle
imaging.6,7 However, there are serious problems with the
reproducibility in the quality of biocoated QDs, and reliable
tests for the quality of surface coating have not yet been
established.1c Moreover, the cytotoxicity of QDs in the intra-
cellular metabolism is still a debated question.8
Another class of fluorescent dyes that have been extensively
studied in the last years is that of conjugated polymer electrolytes
for the detection of molecules of biological interest or biologi-
cally important ions.9 Thanks to positively charged pendant
groups these polymers can coordinate oppositely charged
biological molecules via electrostatic interactions, in particular
DNA, while the fluorescence emission of the conjugated
backbone is sensitive even to very minor environmental changes.
In the presence of polyelectrolytes of biological interest,
conjugated polyelectrolytes act as optical transducers of bio-
chemical events by means of changes in fluorescence. Several
investigations have concerned the interactions of polythiophene
polyelectrolytes with nucleic acids and proteins for the realiza-
tion of homogeneous biosensors for real-time sequence-specific
detection.9a Polythiophene electrolytes have also been used for
the hystological staining of amyloid deposits in ex vivo tissue
samples associated with several pathogenic states.10 However,
the methods based on conjugated polyelectrolytes suffer from
the fact that the nature of the complexes they form with
biopolymers is poorly known11 and that in biological fluids other
charged macromolecules are generally present besides those of
interest, which might contribute to the observed changes in
fluorescence.
in particularshave demonstrated high performance levels in
diagnostic applications such as flow cytometry and immun-
ostaining, in the imaging of biological compounds, in DNA
labeling, and in FRET experiments.12 However, molecular
fluorescent dyes present limitations in photostability and
sensitivity preventing, for example, the long-term imaging of
stained and living cells. Having generally narrow bandwidths
and small Stokes shifts,1c another inconvenience is the limited
possibility for multiplexing analysis, which requires irradiation
at a single wavelength and the simultaneous observation of the
emission of different fluorophores. Nevertheless the appeal of
molecular fluorophores lies in the possibility to enhance
performance by design and to introduce functional groups that
can react with target biocompounds even within living cells.
Being small molecules of a few nanometer size, they are ideal
candidates to penetrate live cells and illuminate them without
altering their metabolism. Owing to the resources of synthetic
chemistry, they could in principle be functionalized with almost
any functional group and shaped on demand for almost any
possible application. In other words, and paraphrasing the title
of a well know article from a pioneer in the field of semicon-
ductor oligo and polythiophenes,14 they are ideal fluorophores
a` la carte, i.e., custom fluorescence dyes to develop and
manufacture for the most diverse application areas. However,
new systematic studies are needed to renew the toolbox of
available molecular fluorophores and push farther their perfor-
mance. Unquestionably, the better the fluorophores, the easier
their manipulation and reproducibility, and the lower their cost,
then the better and more numerous the tests are that can be
performed.
Owing to the versatile chemistry of thiophene, its easy
functionalization, and the fluorescence properties of its
oligomers,15a we started a few years ago research aimed to
explore the possibility of functionalizing these compounds with
groups capable of covalently binding relevant biomolecules such
as DNA or monoclonal antibodies.15b-f These first attempts
having been successful, we have now started a more ambitious,
wide scope investigation aimed to transform the potentialities
into real advantages and original experiments. We wished, on
one hand, to control the reactivity of these dyes toward
compounds of biological importance by tuning the functional-
ization of the backbone in view of their use for selective site
binding to biocomponents within living cells and, on the other,
to enhance and orient their fluorescence properties toward
innovative technological applications.
Molecular fluorescent probesssmall organic molecules, the
prototype of which is fluorescein isothiocyanateshave been
employed for a long time as reporters of events of biological
interest.12 Significant improvements in their optical properties
have been achieved over the years. New molecular structures
are continuously being engineered.13 A few families of these
compoundssfluorescein and its derivatives and cyanine dyes,
(6) (a) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose,
S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S.
Science 2005, 307, 538–544. (b) Gill, R.; Zayats, M.; Willner, I.
Angew. Chem., Int. Ed. 2008, 47, 7602–7625. (c) Medints, I. L.;
Tetsuo, H.; Goldman, U. E. R.; Mattoussi, H. Nat. Mater. 2005, 4,
436–4462. (d) Mahler, B.; Spinicelli, P.; Buil, S.; Quelin, X. Nat.
Mater. 2008, 7, 659–664. (e) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.;
Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat.
Biotechnol. 2002, 21, 41–46.
In this paper we report the rapid, efficient, cost-effective, and
clean synthesis of a new class of molecular fluorophores more
soluble under physiological conditions and more reactivite
toward monoclonal antibodies compared to previously described
thiophene fluorophores. An easy protocol for multiple labeling
of monoclonal antibodies was developed leading also to brightly
white emitting antibodies. The use of TFs in immunostaining
(7) Temirov, J. P.; Bradbury, A. R. M.; Werner, J. H. Anal. Chem. 2008,
80, 8642–8648.
(8) (a) Kim, B. Y. S.; Jiang, W.; Oreopoulos, J.; Yip, C. M.; Rutka, J. T.;
Chan, W. C. W. Nano Lett. 2008, 8, 3887–3892. (b) Chang, E.;
Thekkek, N.; Yu, W. W.; Colvin, V. L.; Drezek, R. Small 2006, 2,
1412–1417. (c) Jamieson, T.; Bakhshi, R.; Petrova, D.; Pocock, R.;
Imani, M.; Seifalian, A. M. Biomaterials 2007, 28, 4717–4732.
(9) (a) Ho, H. A.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168–
178. (b) Herland, A.; Thomsson, D.; Mirzov, O.; Scheblykin, I. G.;
Ingana¨s, O. J. Mater. Chem. 2008, 18, 126–132. (c) Liu, B.; Bazan,
G. C. Chem. Mater. 2004, 16, 4467–4476. (d) Thomas, S. W., III;
Joly, J. D.; Swager, T. M. Chem. ReV. 2007, 107, 1339–1386.
(10) Nilsson, K. P. R.; Hammarstro¨m, P.; Ahlgren, F.; Herland, A.; Schnell,
E. A.; Lindgren, M.; Westermark, G. T.; Ingana¨s, O. ChemBioChem
2006, 7, 1096–1104.
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(13) (a) Ozhalici-Unal, H.; Pow, C. L.; Marks, S. A.; Jesper, L. D.; Silva,
G. L.; Shank, N. I.; Jones, E. W.; Burnette, J. M.; Berget, P. B.;
Armitage, B. A. J. Am. Chem. Soc. 2008, 130, 12620–12621. (b)
Richard, J. A.; Massonneau, M.; Renard, P. Y.; Romieu, A. Org. Lett.
2008, 10, 4175–4178. (c) Mottram, L. F.; Boonyarattanakalin, S.;
Kovel, R. E.; Peterson, B. R. Org. Lett. 2006, 8, 581–584. (d) Peneva,
K.; Mihov, G.; Herrmann, A.; Zarrabi, N.; Bo¨rsch, M.; Duncan, T. M.;
Mu¨llen, K. J. Am. Chem. Soc. 2008, 130, 5398–5399. (e) Kim, D. S.;
Ahn, K. H. J. Org. Chem. 2008, 73, 6831–6834. (f) Umezawa, K.;
Nakamura, Y.; Makino, H.; Citterio, D.; Suzuki, K. J. Am. Chem.
Soc. 2008, 130, 1550–1551. (g) Li, L.; Han, J.; Nguyen, B.; Burgess,
K. J. Org. Chem. 2008, 73, 1963–1970.
(11) Chi, C.; Chworos, A.; Zhang, J.; Mikhailovsky, A.; Bazan, G. C. AdV.
Funct. Mater. 2008, 18, 3606–3612.
(12) Haugland, R. P. A Guide to Fluorescent Probes and Labeling
Technologies; Molecular Probes Inc.: Eugene, OR, 2005.
(14) Garnier, F. Acc. Chem. Res. 1999, 32, 209–215.
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A R T I C L E S
Zambianchi et al.
Chart 1. Molecular Structure of Thiophene Fluorophores
tion (pages S19-S22). The fluorophore to protein molar ratio was
estimated spectrophotometrically according to standard proce-
dures.21
and unprecedented fluorescence stability for this type of
experiments are demonstrated. Finally, theoretical calculations
furnishing guidelines for further molecular engineering of
thiophene based fluorophores are also reported.
2.5. Immunostaining Experiments. Lymphocytes from an
MOLT-4 cell line were employed. The cell staining protocol is
described in detail in the Supporting Information (pages S24-S26).
A fluorescence microscope (Olympus, Model BX52) equipped
with a halogen lamp (100 W) for transmitted light, 100 W HBO
(mercury) lamp for fluorescence, and mirror units for ultraviolet,
violet, blue violet, and blue excitation was employed to observe
fluorescence emissions of MOLT-4 cells stained with different
MoAbs labeled with TFs.
2. Experimental Details
2.1. Synthesis. The synthesis of compounds 2-8, their charac-
terization, and the materials used for their preparation are described
in detail in the Supporting Information (pages S1-S15).
2.2. Optical Characterization. Absorption and photolumines-
cence spectra were obtained using a Perkin-Elmer Lamda 20
spectrophotometer and a Perkin-Elmer LS50B spectrofluorometer,
respectively. For all dyes photoluminescence was recorded exciting
at the maximum wavelength absorption. Solvents were spectral
grade. Details of quantum yield measurements and other additional
information are reported as Supporting Information (pages S20-S22).
2.3. Computational Details. All theoretical calculations were
performed using the TURBOMOLE 5.9 program package.16 Ground
state and excited state geometries were fully optimized employing
the B3-LYP17,18 hybrid density functional and triple-ꢀ basis set
with polarization functions on all atoms (TZVP).19 Dipole moments
were obtained from the Kohn-Sham densities for the ground state
and from the excited-state relaxed density for the excited state.20
More details are given in the Supporting Information (page S18).
2.4. Labeling and Multiple Labeling of Monoclonal Antibod-
ies (MoAbs). MoAb anti-CD38 was furnished by Professor Fabio
Malavasi, Laboratory of Immunogenetics, Department of Genetics,
Biology and Biochemistry, University of Torino Medical School,
Torino, Italy. MoAb anti-CD4 was furnished by Beckman Coulter,
Miami, USA. The detailed procedure for conjugation of the
fluorophores to the antibodies is described in Supporting Informa-
3. Results and Discussion
Two classes of thiophene based fluorophores (TFs) have
already been described: isothiocyanates (TF-NCS) and N-
hydroxysuccinimidyl esters (TF-NHS). To improve solubility
in aqueous media and employ more effective synthetic protocols,
we have now developed a third class of TFs, namely thiophene
based 4-sulfo-2,3,5,6-tetrafluorophenyl esters (TF-STP). The
molecular structures of TFs-STP and TFs-NHS analyzed in this
study are shown in Chart 1.
3.1. Synthesis of Thiophene Based 4-Sulfo-2,3,5,6-tetrafluo-
rophenyl Esters (TFs-STP). Tetrafluoro-phenyl esters of bi-, ter-,
and quaterthiophene (compounds 3a-8a) are described for the
1
first time. The detailed synthesis and H, 13C, and 19F NMR
spectra are reported as Supporting Information (pages S9-S16).
Compared to N-hydroxysuccinimidyl esters (3b-8b) syn-
thesized by conventional methodologies,15b,c,e TFs-STP 3a-8a
were obtained much more rapidly and in higher yields by taking
advantage of microwave irradiation.22 The synthetic strategy is
illustrated in Scheme 1. It consists of building up the aromatic
skeleton by sequential Pd(II) catalyzed Suzuki cross couplings
of newly synthesized or commercial borylated and halogenated
building blocks with the final addition of thienyl tetrafluoro-
sulphonate (2) prepared starting from commercial 5-bromo-2-
thiophenecarboxylic acid (1). The coupling of 2 to boronic esters
to obtain the desired TF-STP was complete after only 2 min of
microwave irradiation. Due to the hydrophilic character of TFs-
STP, aqueous workup was avoided.
(15) (a) Barbarella, G.; Melucci, M.; Sotgiu, G. AdV. Mater. 2005, 17, 1581–
1593. (b) Barbarella, G.; Zambianchi, M.; Pudova, O.; Paladini, V.;
Ventola, A.; Cipriani, F.; Gigli, G.; Cingolani, R.; Citro, G. J. Am.
Chem. Soc. 2001, 123, 11600–11607. (c) Barbarella, G.; Zambianchi,
M.; Ventola, A.; Fabiano, E.; Della Sala, F.; Gigli, G.; Anni, M.;
Bolognesi, A.; Polito, L.; Naldi, M.; Capobianco, M. Bioconjugate
Chem. 2006, 17, 58–67. (d) Zambianchi, M.; Barbieri, A.; Ventola,
A.; Favaretto, L.; Bettini, C.; Galeotti, M.; Barbarella, G. Bioconjugate
Chem. 2007, 18, 1004–1009. (e) Capobianco, M. L.; Cazzato, A.;
Alesi, S.; Barbarella, G. Bioconjugate Chem. 2008, 19, 171–177. (f)
Quarta, A.; Di Corato, R.; Manna, L.; Argentiere, S.; Cingolani, R.;
Barbarella, G.; Pellegrino, T. J. Am. Chem. Soc. 2008, 130, 10545–
10555. (g) Piacenza, M.; Zambianchi, M.; Barbarella, G.; Gigli, G.;
Della Sala, F. Phys. Chem. Chem. Phys. 2008, 10, 5363–5373.
(16) Ahlrichs, R, et al. Universita¨t Karlsruhe2007. See also: http://
www.turbomole.com.
A rapid and high-yield purification by chromatography on
florisil removed the excess of boronic ester and the base used.
(21) Brinkley, M. Bioconjugate Chem. 1992, 3, 2–13.
(17) Becke, A. D. J. Chem. Phys. 1993, 98, 5648–5652.
(22) (a) Polshettiwar, V.; Varma, R. S. Chem. Soc. ReV. 2008, 37, 1546–
1557. (b) Alesi, A.; Di Maria, F.; Melucci, M.; Macquarrie, D. J.;
Luqueb, R.; Barbarella, G. Green Chem. 2008, 10, 517–523. (c)
Melucci, M.; Barbarella, G.; Zambianchi, M.; Di Pietro, P.; Bongini,
A. J. Org. Chem. 2004, 69, 4821–4828.
(18) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B 1988, 37, 785–789.
(19) Scha¨fer, A.; Huber, C.; Ahlrichs, R. J. Chem. Phys. 1994, 100, 5829–
5835.
(20) Furche, F.; Ahlrichs, R. J. Chem. Phys. 2002, 117, 7433–7447.
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Microwave-Assisted Synthesis of Thiophene Fluorophores
A R T I C L E S
Scheme 1. Synthetic Pattern for the Preparation of TFs-STPa
a Reagents and conditions: (i) 4-sulfotetrafluorophenol, sodium salt, DCC, acetone/DMF 15:1, rt, 16 h; (ii) NaHCO3, PdCl2dppp, DMF/H2O 9:1, µV 2
min, 80 °C. Yields in isolated product in parentheses.
This simple protocol leads to very rapid reaction times, is
environmentally friendly and cost-effective, and allows easy
tuning of the molecular structure, hence of the optical properties,
by changing the size and the substituents of the aromatic
backbone prior to the addition of thienyl tetrafluorosulphonate.
The use of microwaves prevents the formation of undesired
products arising from the occurrence of boron-halogen ex-
change, a slower reaction compared to cross coupling, allowing
a dramatic simplification of the purification procedures of the
crude reaction products. The synthesis could be scaled up to
300 mg at a time. The synthetic details, in particular the
synthesis of methylthio-thienyl, -bithienyl, and -terthienyl
boronic acid pinacol esters, are reported as Supporting Informa-
tion (pages S2-S8).
3.2. Optical Properties and Theoretical Calculations. The
absorption and emission spectra of oligothiophene 4-sulfo-
2,3,5,6,-tetrafluorophenyl esters 3a-8a measured in DMSO are
shown in Figure 1, whereas those of compounds 3b-10b have
already been reported.15c,e Table 1 gives the absorption and
emission wavelengths and the molar absorption coefficients of
3a,b-8a,b in DMSO.
The spectral characteristics of TFs-STP are similar to those
of the corresponding TFs-NHS, and band shapes and bandwidths
(60-120 nm) are essentially the same for both classes of
fluorophores, which is not surprising since the fluorophores have
the same emitting aromatic component and only differ in the
Figure 1. Normalized absorption and fluorescence emission spectra of
compounds 3a-8a in DMSO and palette of the fluorescence colors. The
PL spectra were obtained by irradiating each compound at the maximum
nature of the amine-reactive substituent. All compounds are
characterized by large Stokes shifts from absorption to emission,
much greater than those of common molecular fluorophores.1c
wavelength absorption. The photograph shows the fluorescence emission
While the absorption wavelengths vary in the range 350-450
nm, the emission wavelengths are spread from 450 to 650 nm,
spanning almost over the entire visible range.
of all compounds under irradiation at λexc ) 364 nm with a 15 W UV lamp.
that of fluorescein under irradiation with a 100 W mercury lamp
is reported as Supporting Information (page S21).
Table 1 reports the fluorescence quantum yield of compounds
3a-7a and 3b-7b measured in dimethylsulfoxide. The quantum
yields of compounds 9b and 10b (Chart 1) in the same solvent
are 0.42 and 0.34, respectively.
The most important property of TFs is their photostability.
No matter what the functional group is, -NCS, -NHS or -STP,
the fluorophores can be excited continuously for hours without
loosing their brightness. As powders, in air, they are stable for
years. In solution, the esters may slightly be hydrolyzed while
only the fluorescence frequency, but not the intensity, is modified
from that of an ester to that of the corresponding acid. An
experiment comparing the photostability of 6a in solution with
Table 1 shows that the quantum yields are very similar for
NHS and STP esters, not unexpectedly considering that the two
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A R T I C L E S
Zambianchi et al.
Table 1. Absorption (λmax, nm) and Emission (λPL, nm)
Wavelengths, Molar Absorption Coefficients (ꢀ, M-1 cm-1), and
Fluorescence Quantum Yields (φ)a of 3a,b-8a,b 10-5 M in DMSO
item λmax
ꢀ
λPL
φ
item λmax
ꢀ
λPL
φ
3a 353 23.200 430 0.78
3b 350 22.600 430 0.71
4a 376 19.900 503
4b 380 22.700 500
0.70
0.74b
5a 400 24.100 508 0.27
5b 400 34.400 507 0.42
6a 410 21.100 580
6b 412 28.600 580
0.35
0.37b
7a 429 31.900 555 0.26
7b 430 25.600 565 0.31
8a 434 34.300 515, 625c 0.26
8b 438 39.000 625 0.31
a Estimated error: (0.05. b 1.00 ( 0.05 and 0.76 ( 0.05 for 4b and
6b, respectively, in CH2Cl2. From ref 15d. c DMSO/H2O 90:10.
types of fluorophores have the same emitting components.
Interestingly, for NHS as well as for STP esters, φ decreases
on increasing the size of the fluorophore, from the >0.7 value
of the bithiophene esters to the ∼0.3 value of the quater-
thiophene esters. Both the quantum yield values and the trend
of variation with size are remarkably different from those of
the corresponding unsubstituted bi-, ter-, and quaterthiophene
(quantum yield ranging from ∼0.02 for bithiophene to ∼0.06
for terthiophene and ∼ 0.2 for quaterthiophene).23 The reasons
for such behavior have not yet been elucidated and require much
deeper photophysical study.
We noticed that for all esters the fluorescence efficiency was
drastically reduced on passing from DMSO to a H2O buffered
solution, probably due to aggregation phenomena since we also
observed that the decrease was progressively attenuated on
decreasing the concentration of the solution. Judging from the
intensity of the corresponding PL spectra, the fluorescence
efficiency increased again when the fluorophore in a buffered
solution was reacted with the antibody, probably because in the
bioconjugate the fluorophores are well separated from each
other. Using commercial molecular dyes, we obtained similar
results, but we were unable to find accurate investigations on
this point. Thus, we are currently carrying out a comparative
study on this important point.
As a result of increasing the number of thiophene rings, or
introducing a SCH3 substituent, the photoluminescence shifts
toward longer wavelengths. The SCH3 substituent is an electron
donor, while the carbonyl group at the opposite end of the
molecule is an electron acceptor; hence the fluorophore is a
“push-pull” compound with an increased π-electron delocal-
ization. In compound 9b, where the SCH3 group is in the
ꢁ-position, the electron donor effect of the substituent is partially
counteracted by increased molecular distortion due its steric
effects.24 As a consequence, the shift from absorption to
emission for 9b is smaller than that for 6b. The photolumines-
cence of these “push-pull” fluorophores is very sensitive to
environment changes, as shown in Figures 23S, 24S and Table
1S reporting the changes in λPL observed on varying the solvent.
Figure 2 compares the changes in photoluminescence wave-
length observed on going from toluene (with a dielectric constant
of 2.4) to DMSO (with a dielectric constant of 48.9) for a dimer-
NHS (4b, λPL from 458 to 502 nm), a linear trimer-NHS (6b,
λPL from 509 to 582 nm), a linear trimer-STP (6a, λPL from
519 to 580 nm), and a branched trimer-NHS (9b, λPL from 489
Figure 2. Normalized fluorescence emission spectra of compounds 4b,
9b, 6b, and 6a in toluene (blue line) and in DMSO (red line), obtained by
exciting each compound at the maximum wavelength absorption.
Table 2. TD-DFT Calculated Absorption (EABS) and Emission (EPL
Energiesa, Molecular Dipole Moments in the Ground (µg) and
Excited State (µe)b and Experimental and Theoretical Emission
Peak Shiftsc from Toluene to DMSO
)
EABS
EPL
µg
µe
∆Eexp
∆Etheor
4b
6b
9b
3.41 (363)
2.94 (421)
2.94 (421)
2.82 (439)
2.49 (497)
2.44 (507)
1.7992
2.7927
1.5725
7.7835
10.4534
10.9622
0.24
0.24
0.31
0.26
0.24
0.30
a In eV (nm in brackets). b In debye. c In eV.
to 539 nm). On increasing the dielectric constant value, a
progressive displacement toward longer wavelengths is observed.
The observed effect only concerns the excited states since
the corresponding absorption spectra (Figure 23S) are insensitive
to solvent changes. Note that the fluorescence emission wave-
lengths of unsubstituted thiophene oligomers do not vary with
the solvent.23
To shed light on the solvent effect observed on the photo-
luminescence of “push-pull” TFs, we have carried out first-
principles TD-DFT theoretical calculations in the homogeneous
series of N-succinimidyl esters 4b, 9b, and 6b, where the
electron donor substituent is either R or ꢁ to sulfur. The
corresponding computed excitation and emission energies are
reported in Table 2, while Figure 3 shows the optimized
molecular structure and molecular electrostatic potential of the
fluorophores in the ground state (left) and excited state (right),
together with the direction and size of the molecular dipole
moments.
All transitions can be traced back to strongly dipole allowed
excitations from the highest occupied (HOMO) to the lowest
unoccupied (LUMO) molecular orbital. The calculated values
of absorption and photoluminescence energies agree very well
(i.e., within the accuracy of the method) with the experimental
results.
The absorption energy of dimer 4b is calculated at 3.41 eV
(363 nm) and the emission energy at 2.82 eV (439 nm), which
corresponds to a large Stokes shift of 0.59 eV. For trimers 9b
and 6b TD-DFT computed energies are equivalent in absorption,
whereas the emission energy of 9b is slightly red-shifted with
respect to that of 6b, as found experimentally. The resulting
(23) Becker, R. S.; Seixas de Melo, J.; Macu¨anita, A. L.; Elisei, F. J. Phys.
Chem. 1996, 100, 18683–18695.
(24) Barbarella, G.; Zambianchi, M.; Antolini, L.; Folli, U.; Goldoni, F.;
Iarossi, D.; Schenetti, L.; Bongini, A. J. Chem. Soc. Perkin Trans. 2
1995, 1869–1873.
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Microwave-Assisted Synthesis of Thiophene Fluorophores
A R T I C L E S
more importantly, it clearly reflects an increased solvent effect
for dye 6b due to the much larger dipole changes in the excited
state. Thus, 6b should present red-shifted emission in high-
dielectric constant environments.
The calculation results, in agreement with experimental data,
indicate that the insertion of electron-donor substituents of
different “push” strength in different positions of the aromatic
backbone is a way to achieve fine-tuning of the fluorescence
emission and to realize fluorescent probes that are fine reporters
of environment changes.
3.3. Labeling and Multiple Labeling of Monoclonal
Antibodies. The labeling of antibodies with fluorescent dyes,
namely immunofluorescence, is widely used in different fields
such as medical diagnostics, molecular biology, or environ-
mental monitoring, owing to the high specificity and versatility
of immunoassays.12
Dyes 3-10 are amine reactive compounds and can be
covalenty bound to monoclonal antibodies (MoAbs) through
reaction of the X ) NHS or STP functional groups with the
NH2 groups of ꢀ-lysine residues of MoAb under basic
conditions.12,15c
Figure 3. Optimized molecular structure and molecular electrostatic
potential in the ground state (left) and excited state (right) for compounds
4b, 9b, and 6b. The direction and size of the molecular dipole moments
are reported as red arrows.
Stokes shifts are 0.45 and 0.50 eV for 9b and 6b, respectively.
Comparison of the values for 4b and 6b shows that the increase
in conjugation length leads to a red shift in absorption of 0.47
eV and in emission of 0.27 eV, whereas the different positions
of the SCH3 substituent in 9b and 6b have a much smaller
impact.
Biopolymer-NH2 + TF-C(O)-OX f
Biopolymer-NH-C(O)-TF
3.3.1. Labeling Experiments with TFs. Both classes of
fluorophores, TFs-NHS and TFs-STP, were reacted with the
anti-CD38 monoclonal antibody in the same experimental
conditions to allow the comparison of the fluororophore to
protein (F/P) molar ratios achieved for the same aromatic moiety
but different functional group. Anti-CD38 is a human mono-
clonal antibody directed against the cell surface glycoprotein
CD-38 present on various immune cells and with potential
antineoplastic activity.25,26 In the Experimental Details we report
the protocol used to bind 3a,b-8a,b with anti-CD38. The
reaction time was arbitarily fixed at 30 min.
Figure 4 shows the absorption and emission spectra of the
anti-CD38 MoAb labeled with TFs-STP and TFs-NHS.
Table 3 reports the absorption and emission wavelengths of
anti-CD38 MoAbs measured in PBS buffer pH ) 7.4, together
with the fluorophore to protein molar ratios, F/P, estimated from
the relative intensities of protein and dye absorptions.21,15d In
the table the absorption signal arising from the protein, ∼280
nm in all cases, is omitted.
The F/P values achieved in 30 min with TFs-STP are
generally higher than those achieved with the corresponding
TFs-NHS, indicating a greater reactivity of the -STP with respect
to the -NHS functional group, the lowest F/P ratios having been
obtained with quaterthiophene derivatives.
The insertion of an SCH3 group causes a marked red shift in
the antibody emission wavelength for dimers and trimers but
not for tetramers, as already observed for the free fluorophores.
For MoAbs labeled with dimers and trimers, the emission
Table 2 also reports the dipole moments in the ground state
(µg) and in the first optically allowed excited state (µe), which
are also shown in Figure 3 together with the molecular
electrostatic potential. Dipole moments are much larger than
those previously reported for unsubstituted N-succinimidyl
bithiophenes,15g due to the presence of the SCH3 group.
The ground state dipole moments of 4b, 9b, and 6b have
different intensities and directions due to a complex interplay
of the charges localized on SCH3 and on the succinimidyl
groups. In the excited state, on the other hand, the calculations
predict a strong increase of the molecular dipole moment, which
arises from an accumulation of negative charge on the succin-
imidyl group and of positive charge on the methyl group of the
methylsulfinyl substituent. In contrast to 4b and 6b, in 9b the
dipole moment in the ground and excited state are almost parallel
owing to the reduced molecular size caused by the different
position of the SCH3 substituent. In addition, the changes in
the molecular geometries induced by electronic excitation
contribute to a change in the dipole moment, as shown in Figure
3 where the optimized molecular geometries in the ground and
excited states are reported. In the ground state geometry, the
thiophene rings are in the anti orientation. The corresponding
dihedral angles are found in the range 158°-164°, and the
methylsulfanyl group adopts an approximately perpendicular
orientation with respect to the neighboring thiophene ring. In
the first excited state, the bithiophene and terthiophene cores
planarize and the SCH3 substituent rotates until the previously
perpendicular dihedral angle is reduced to 45° for TF 4b, 36°
for 9b, and 29° for 6b.
(25) (a) Deaglio, S.; Mallone, R.; Baj, G.; Donati, D.; Giraudo, G.; Corno,
F.; Bruzzone, S.; Geuna, M.; Ausiello, C.; Malavasi, F. FASEB J.
2001, 15, 580–582. (b) Alessio, M.; Roggero, S.; Funaro, A.; De
Monte, L.B.,; Peruzzi, L.; Geuna, M.; Malavasi, F. J. Immunol. 1990,
145, 878–3884. Patten, P. E. M.; Buggins, A. G. S.; Richards, J.;
Wotherspoon, A.; Salisbury, J.; Mufti, G. J.; Hamblin, T. J.; Devereux,
S. Blood 2008, 111, 5173–5181.
The dipole moments in the ground and excited states were
PL
used to compute the photoluminescence energy (E ) as a
solv.
function of the dielectric constant of the solvent according to
the Amos-Burrows equation (see Supporting Information, p
S18). In the last two columns of Table 2 the experimental and
calculated photoluminescence energy differences from toluene
(26) (a) Zhu, S.; Zhang, Q.; Guo, L. H. Anal. Chim. Acta 2008, 624, 141–
146. (b) Zhang, Q.; Guo, L. H. Bioconj. Chem 2007, 18, 1668–1672.
(c) Luchowski, R.; Matveeva, E. G.; Gryczynski, I.; Terpetschnig,
E. A.; Patsenker, L.; Laczko, G.; Borejdo, J.; Gryczynski, Z. Curr.
Pharm. Biotechnol. 2008, 9, 411–420.
PL
PL
to dimethylsulfoxide (∆E ) E - E
) are compared. The
tol
DMSO
agreement between theory and experiment is very good, and
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A R T I C L E S
Zambianchi et al.
Figure 4. Normalized absorption and fluorescence emission spectra of anti-CD38 monoclonal antibody (MoAb) labeled with TFs-STP (3a-8a) and TFs-
NHS (3b-8b) fluorophores (incubation time: 30 min).
in the detection of molecules of biological interest25a,b or in
Table 3. Absorption (λmax, nm) and Emission (λPL, nm)
Wavelengths of Anti-CD38 Monoclonal Antibodies Labeled with
single molecule studies for the understanding of the behavior
Fluorophores 3a,b-8a,b in PBS Buffer pH ) 7.4 and Fluorophore
of complex biomolecules in real time.25
to Protein Molar Ratios (F/P)
The easy manipulation of thiophene fluorophores and their
item
λmax
λPL
F/P
item
λmax
λPL
F/P
photostability allowed us to perform facile multiple labeling of
monoclonal antibodies with TFs. Here we report experiments
carried out using the same antibody (anti-CD4 or anti-CD38
MoAbs) simultaneously labeled with different TFs, excited using
one single light source and resulting in intense white light
emission.
MoAb-3a 346 427
MoAb-3b 336 415
11
8
MoAb-4a 370 487
MoAb-4b 365 488
8
2
MoAb-5a 395 492
MoAb-5b 365 480
5
2
MoAb-6a 405 550
MoAb-6b 405 550
12
11
MoAb-7a 420 520
MoAb-7b 420 465, 490
1
1
MoAb-8a 420 500, 580
MoAb-8b 420 510
3
1
At the beginning, our intention was, on one hand, to verify
the possibility to create new color shades by mixing TFs of
different colors as we had already realized with oligothiophene
thin films27 and, on the other, to explore the possibility of FRET
(Fluorescence Resonance Energy Transfer)2 to enhance the
intensity of the red emitters through irradiation of appropriate
blue emitters with much higher fluorescence efficiencies. For
fluorescence resonance energy transfer to be observed, the
excited fluorophore must emit at a wavelength matching the
absorption wavelength of the second fluorophore and the two
fluorophores must be located in close proximity (generally 5-6
nm).2,28
bandwidth is remarkably sharper for TFs-STP compared to the
corresponding TFs-NHS. We temptatively assume that this is
the result of the greater reactivity of TFs-STP allowing the
binding of all fluorophores to the same site of the antibody, an
interpretation which also explains the differences in absorption
and emission between the two classes of compounds reported
in Table 3.
In all cases a blue shift is observed for both the absorption
and emission signals compared to those of the free fluorophores
(Table 1), but the Stokes shifts from absorption to emission
remain quite large.
The labeled antibodies are highly stable, chemically as well
optically. We have now been keeping labeled antibodies (anti-
CD4, anti-CD3 from different sources and anti-CD38) for years
at 4 °C and verified their unaltered biological activity and
fluorescence properties. More detailed information on this point
and the comparison with the same antibodies labeled with
commercial molecular fluorophores will be reported in a separate
paper.
Since we had control of the choice of the fluorophores but
not of the position at which they would be attached in the
antibody, we reasoned that with the lack of FRET we could
verify the occurrence of multiple labeling by choosing comple-
mentary emission colors, blue and orange, in particular, and
observe whether there was white light emission from the
antibody. The protocol used for the multilabeling experiments
is reported in detail in the Supporting Information.
(27) Anni, M.; Gigli, G.; Paladini, V.; Cingolani, R.; Barbarella, G.;
Favaretto, L.; Sotgiu, G.; Zambianchi, M. Appl. Phys. Lett. 2000, 77,
2458–2460.
3.3.2. Multiple-Labeling Experiments with TFs and White
Emitting Antibodies. Multiple labeling of antibodies is starting
to be investigated in fluorescence immunoassays to implement
new labeling strategies to meet the demand for high sensitivity
(28) Kozlov, V. G.; Bulovic, V.; Burrows, P. E.; Baldo, M.; Khalfin, V. B.;
Parthasarathy, G.,; Forrest, S. R. J. Appl. Phys. 1998, 84, 4096–4108.
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Microwave-Assisted Synthesis of Thiophene Fluorophores
A R T I C L E S
Figure 5. Normalized absorption and emission spectra of white fluorescent anti-CD38 and anti-CD4 monoclonal antibodies, labeled with compounds 3a/6a
and 4b/10b, respectively. The fluorescence spectra were obtained by exciting the samples with λexc ) 347 and 420 nm, respectively. The photograph is that
of the anti-CD38 antibody within a Sephadex column under irradiation with a 15 W UV lamp at λexc ) 364 nm.
Figure 6. Fluorescence microscopy images (× 20) of human T lymphocytes (MOLT-4 cell line) stained using the anti-CD4 monoclonal antibody labeled
with fluorophore 6b. (A) Light transmission image. (B) Fluorescence microscopy image after staining with 6b and counterstaining with DAPI. The inset
shows the magnified image of one of the cells.
Despite the large choice of fluorophores allowed by the TFs
available to us, and the use of two different antibodies (anti-
CD4 and anti-CD38), we did not observe any fluorescence
transfer energy from blue to red. We did instead observe the
occurrence of white fluorescence upon excitation with one single
light source. Thus, the relative amounts of blue and orange fluo-
rophores were accurately weighed to obtain intense and neat
white emission from the antibody (see the Supporting Informa-
tion).
Figure 5A shows the absorption spectrum of the anti-CD38
MoAb labeled with both 3a and 6a and the corresponding
emission spectrum obtained by irradiating at λexc ) 347 nm. At
this wavelength both dyes are simultaneously excited. By
irradiating with a 15 W UV lamp at λexc ) 364 nm, it was
possible to observe with the naked eye the intense white
fluorescence of the antibody within a Sephadex column.
red emitting fluorophore was obtained by exciting at λexc ) 420
nm. A further example is reported in the Supporting Information
(p S21).
From these results we conclude that the fluorophore binding
sites in the MoAb are too distant to allow the energy transfer
to occur from the blue to the orange emitter. Nevertheless, to
our surprise, we obtained experimental evidence that energy
transfer does occur when the multiple-labeled MoAb is fixed
on the cell membrane in immunostaining experiments. This is
difficult to assess quantitatively, and we are currently trying to
put our initial observations on a more solid basis. Also difficult
to quantify is the gain in fluorescence intensity associated with
multiple labeling compared to the monolabeling of a given
antibody. In principle, the more photons are absorbed, the more
intense the fluorescence signal is, and indeed qualitatively the
signal arising from antibodies labeled with multiple TFs is very
intense and bright. However, again, a methodology to ensure
signal quantification and reproducibility has still to be developed.
What at the moment is clear is that the procedure described
above to immobilize multiple fluorophores on a single antibody
is very simple to implement and is worthy of further study.
3.4. Immunostaining Experiments with TFs. The labeling of
antibodies with fluorescent dyes may be used to visualize the
subcellular distribution of biomolecules of interest. For example,
a primary monoclonal antibody such as anti-CD4 may be used
The frequency of the single light excitation source could be
finely tuned toward longer wavelengths by choosing the
appropriate fluorophores. Figure 5B shows the absorption and
emission spectra of the anti-CD4 monoclonal antibody labeled
with fluorophores 4b and 10b. The optical characteristics of
10b (λmax ) 470 nm, λPL ) 590 nm in CH2Cl2, and λPL ) 615
nm in DMSO) are described in the Supporting Information and
in ref 15f. In this case simultaneous excitation of the blue and
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A R T I C L E S
Zambianchi et al.
4. Conclusions
to specifically recognize and bind the corresponding antigen
protein located on the cell membrane (immunostaining). If the
antibody is labeled with a fluorescent dye, analysis with a
fluorescence microscope indicates the location of the antigen.
Immunostaining experiments require a meticulous setup, from
cells preparation and fixation to fluorescence microscopy
analysis, to optimize the signal arising from the antibody bound
to the corresponding antigen on the cell membrane.
Molecular fluorophores stand out from the many classes of
available fluorescent dyes because they can easily be built up
Via organic synthesis to meet the growing demand of tailor-
made tools for biomolecular detection, sensing, and multiplexing
analysis.
The results reported here indicate that thiophene based
molecular fluorophores, TFs, can be tailored to satisfy the
conditions for a wide range of applications requiring highly
stable and long lasting fluorescence emission, facile emission
tunability through appropriate substitution, and multilabeling
experiments employing a single light excitation source. A
desired functional group can be added to a preselected emitting
core employing standard modalities to increase hydrophilicity
and reactivity, to optimize photophysical properties and/or the
specific linkage to target biomolecules. Belonging to the same
chemical family, TFs can easily be combined for multicolor
applications in bioresearch. For all these aspects, thiophene
fluorophores are true “fluorophores a` la carte”.
One of the most interesting achievements realized with TFs
is their outstanding optical stability allowing a significant
extension of fluorescence observation times with respect to
currently employed dyes. Monoclonal antibodies labeled and
multilabeled with TFs are brightly fluorescent and can undergo
repeated excitation cycles without any fluorescence loss. In
immunostaining experiments using TFs, unaltered fluorescence
emission was observed from the cells for at least 3 weeks and
repeated exposition to laser excitation. These results show that
TFs are highly suitable for analyses by fluorescence microscopy
in clinical diagnostics, and laboratory research and pave the way
to applications in cytofluorimetry, in which cells are immun-
ostained in solution.
We have developed a protocol for the staining of fixed cells
with TFs which is reported in detail in the Supporting Informa-
tion (pp S24-S26). In Figure 6 we show the immunostaining
of the MOLT 4 cell line (T lymphocytes) with the anti-CD4
MoAb labeled with fluorophore 6b.
Cells are a very complex environment which is difficult to
mimic in vitro. The cellular environment of our samples under
the experimental conditions employed for staining was such that
6b was intensely orange-red fluorescent, as predicted by
theoretical calculations (see above) in environments with a high
dielectric constant.
Figure 6B shows the orange-red fluorescence emission of the
cell membrane of the lymphocytes stained with the anti-CD4/
6b conjugate and counterstained with DAPI, namely 4′,6-
diamidino-2-phenylindole, a blue emitting dye which colors
selectively the nucleus of fixed cells.2 Normally, the antigen
on the cell surface is present only on a fraction of the cells.
Anti-CD4 is a Mouse IgG2 isotype monoclonal antibody
reacting with CD4, a 55 kDa molecular weight glycoprotein
expressed on the cell surface of nearly 60% of peripheral blood
T lymphocytes. In agreement with this, in Figure 6B only a
portion of the cells display orange-red staining of the cell
membrane while all cells display the blue staining of the nucleus
due to DAPI. Figure 6B shows great morphological detail and
the fine intense orange-red fluorescence of the cell membrane
well separated from the fluorescence of the nucleus.
We are currently working to the realization of a set of
thiophene fluorophores for spontaneous uptake by live cells
which are also functionalized to target specific cell components.
Acknowledgment. F.D.S. and M.P. acknowledge support from
the ERC-Starting Grant FP7-Project DEDOM (no. 207441). This
work was partially supported by the project SYNERGY (FIRB
RBNE03S7XZ_005). We are grateful to Prof. Fabio Malavasi,
University of Torino and to Beckman Coulter, Miami, USA, for
the generous gifts of anti-CD38 and anti-CD4 monoclonal antibod-
ies. Thanks are also due to Laura Favaretto and Cristian Bettini
(CNR-ISOF Bologna, Italy) and to Dr. Alfredo Ventola (INFM-
CNR, Lecce) for expert technical assistance. We are also grateful
to Dr. Andrea Barbieri (CNR-ISOF Bologna, Italy) for help with
fluorescence quantum yield measurements.
Loss of fluorescence caused by photobleaching (conversion
of a fluorophore into a nonfluorescent state by the absorbed
photons) is the source of many problems in immunostaining
experiments, mainly due to the need for reduced times for light
excitation exposure. Time course examination of our samples
showed that after 3 h from the first observation with repeating
light exposure the emission signal had not changed and was
still intensely bright. During the 3 h time course, with the slides
being exposed to microscope light continuously for 5 min each
10 min for a total of 12 times, we did not observe any change
in the fluorescence image. The same slides of Figure 6B, kept
in air into a clean room at 18-22 °C without any light
protection, were observed 1 and 3 months after immunostaining
(see Figure 23SA and 23SB of the Supporting Information).
The quenching of the blue fluorescence of DAPI was observed,
while there was only a slight loss of the orange-red fluorescence
due to 6b. Further examples of fixed cell staining will be
reported in a separate paper.
Supporting Information Available: Detailed synthesis, NMR
spectra and mass spectra of 3a-8a; details of optical properties
and theoretical calculations; detailed procedures for labeling and
multiple labeling of monoclonal antibodies; detailed procedures
for immunostaining. This material is available free of charge
via the Internet at http://pubs.acs.org.
JA902416S
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