A selective optode membrane for histidine based on fluorescence enhancement of meso - Meso-linked porphyrin dimer

Article abstract of DOI:10.1021/ac010386b

A plasticized polymer film, poly(vinyl chloride) (PVC) incorporated with a specific porphyrin dimer, is shown to exhibit significant and analytical usefulness for optical response toward histidine. The porphyrin dimer containing a free-base porphyrin and a covalently linked metalloporphyrin is shown to be weakly fluorescent as a result of the photoinduced intramolecular electron transfer from the inner free-base porphyrin in singlet excited state to a low-spin cobalt(II). The fluorescence enhancement of the membrane by histidine is based on favorable extraction of histidine into the bulk organic membrane and complexation with the inner metallopophyrin moiety and inhibiting the electron transfer process. With the optode membrane described, histidine in a sample solution from 0.0045 to 1.53 mM can be determined. The calibration curve of the optode membrane for histidine shows a good correlation with the mathematically derived formalism and, thus, confirms the theoretically expected behavior. The sensor presented exhibits high selectivity toward histidine over several amino acids and common inorganic anions. The optical selectivity coefficients obtained for histidine over other biologically relevant amino acids and anions are shown to meet the selectivity requirements for the monitoring concentration levels of histidine in biological samples. The selective characteristic of the sensor has been discussed in the view of the coordination chemistry of metalloporphyrin.

Full text of DOI:10.1021/ac010386b

Anal. Chem. 2002, 74, 1088-1096
A S e le c t ive Op t o d e Me m b ra n e fo r His t id in e Ba s e d
o n Flu o re s c e n c e En h a n c e m e n t o f
Me s o -Me s o -Lin k e d P o rp h yrin Dim e r
Ronghua Ya ng, Ke m in Wa ng,* Liping Long, Da n Xia o, Xia oha i Ya ng, a nd We ihong Ta n
College of Chemistry and Chemical Engineering, State Key Lab of Chemo/Biosensing and Chemometrics, Hunan University,
Changsha, 410082, China
developed so far. The most widely used techniques for quantifica-
tion of amino acids are liquid chromatographic methods.5,6
Unfortunately, they are not suitable for monitoring the analytes
on-line. Recently, an electrode for histidine that is based on PVC
membrane of Mn(TPP)Cl7 was proposed; however, it is easily
affected by the fluctuation in electric potential or electric field.
There is still a significant need for the genesis of a new optical
chemical sensor (optode) for amino acids, because the optode
can be miniaturized, can be manufactured at low cost, and are
quite safe. Analytical detection using an optical chemical sensor
can be performed by connecting a chemically sensitive layer with
a transducer device. The sensing material determines the main
features of the device, such as sensitivity, selectivity, and dynamic
parameters. So far, a few optical chemical sensors for direct
determination of amino acids have been reported by Fabrizzi et
al.,8 Preuschoff et al.,9,10 and Wolfbeis et al.11
The major challenge for the application of optical chemical
sensors for the detection of amino acids is the lack of photo-
chemically sensing materials that fulfill the requirements of
recognition of the analytes and expression of the relation to the
recognition by optical signals. One of the most important recogni-
tion elements in optical sensors involves the specific metal-ligand
interactions.12 Such interactions have been used in the develop-
ment of anion-selective optical sensors based on different iono-
phores and chromophores.13-15 Among them, metalloporphyrins
are attractive condidates in light of their roles in biological
systems. The specific interaction between metalloporphyrins and
biological compounds makes the biological functions of them
important. A naturally occurring porphyrin is vitamin B12, which
contains the cobalt(II) cation.16 Synthetic metalloporphyrins have
A plasticized polymer film, poly(vinyl chloride) (P VC)
incorporated with a specific porphyrin dimer, is shown
to exhibit significant and analytical usefulness for optical
response toward histidine. The porphyrin dimer contain-
ing a free-base porphyrin and a covalently linked metal-
loporphyrin is shown to be weakly fluorescent as a result
of the photoinduced intramolecular electron transfer from
the inner free-base porphyrin in singlet excited state to a
low-spin cobalt(II). The fluorescence enhancement of the
membrane by histidine is based on favorable extraction
of histidine into the bulk organic membrane and com-
plexation with the inner metallopophyrin moiety and
inhibiting the electron transfer process. With the optode
membrane described, histidine in a sample solution from
0 .0 0 4 5 to 1 .5 3 mM can be determined. The calibration
curve of the optode membrane for histidine shows a good
correlation with the mathematically derived formalism
and, thus, confirms the theoretically expected behavior.
The sensor presented exhibits high selectivity toward
histidine over several amino acids and common inorganic
anions. The optical selectivity coefficients obtained for
histidine over other biologically relevant amino acids and
anions are shown to meet the selectivity requirements for
the monitoring concentration levels of histidine in biologi-
cal samples. The selective characteristic of the sensor has
been discussed in the view of the coordination chemistry
of metalloporphyrin.
The direct and selective analysis of minute quantities of
biologically important compounds is particularly demanding, since
it requires the specific recognition of particular element in the
presence of closely related species. The recent rapid advance in
genetic engineering and biotechnology and the need for nutritional
assessment necessitate the development of a method for deter-
mination of trace concentrations of amino acids. Detection of
amino acids in physiological fluids in the diagnosis of certain
metabolic disorders is one of the important applications of such
analysis.1-4 Many analytical methods for amino acids have been
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* Corresponding author. Tel: +86-731-8822701. Fax: +86-731-8824525.
E-mail: kmwang@mail.hunu.edu.cn.
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1088 Analytical Chemistry, Vol. 74, No. 5, March 1, 2002
10.1021/ac010386b CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/29/2002

attracted attention in relation to chemical or biological molecular
recognition;17 however, they were mainly confined to those of
electrochemical18-20 or absorption measurements.21,22 To the best
of our knowledge, the only transition metal coordination com-
pound of this kind that has been employed as a fluorescent sensor
is a porphyrin zinc derivative.23 On the other hand, molecular
fluorescence spectroscopy is one of the most important spectro-
scopic techniques, since it can can have an enormously lower limit
of detection and more intense response signals than absorbance
spectroscopy. Furthermore, the fluorescent sensors provide a
means to significantly reduce the required membrane volume
while retaining the same level of the signal-to-noise ratio.
There are numerous mechanisms by which fluorescence signal
transduction may be affected. One of the most frequently used
systems to vary fluorescence intensity is the photoinduced electron
transfer (PET) mechanism.24-27 Typical PET sensors are designed
by following a two-component approach, tha is, by covalently
linking a receptor subunit displaying selective affinity toward the
envisaged substrate to a fluorescent fragment. The concept of PET
sensor has been exploited by de Silva, Shinkai, and others for
spectroscopic sensing of H+,25,28 metal cations,29,30 anions,26,31 and
neutral analytes.27,32
The success of the above outlined modular approach prompted
us to design a two-component fluorescent sensor for amino acids.
In a previous report, it was demonstrated that porphyrin cobalt-
(II) displays a selective affinity toward histidine via a strong metal
ion-nitrogen ligation reaction.33 In the present work, we make
the first attempt to design and use a porphyrin-dimer-based
fluorescent sensor for selective histidine detection that does not
require any chemical pretreatment of the analyte. The design of
the approach consists of meso-tetraphenylporphyrin cobalt(II)
(CoTPP) as a histidine-bonding site and a covalent attachment of
meso-tetraphenylporphyrin(TPP) as a fluorescent site, where the
cobalt(II)-nitrogen ligation reaction is responsible for the en-
hancement of the fluorescence emission of the fluorescent site.
The sensor was constructed by immobilizing the sensing material
in a plasticized PVC membrane, and the resulting optode mem-
brane was characterized in terms of sensitivity, selectivity, and
the principle of operation.
EXPERIMENTAL SECTION
Reagents. Bis(2-ethylhexyl) sebacate (BOS), high relative
molecular mass poly(vinyl chloride) (PVC), and tetrahydrofuran
(THF) were purchased from Fluka (Switzerland) and used for
membrane preparation. Other chemicals were of analytical reagent
grade and were used without further purification. Buffer solution
(pH 8.0) was prepared by dissolving 0.1 mol of Tris-(hydroxy-
methyl)amino methane (Tris) in 1000 mL of water and then
adjusting the pH with 0.1 M HCl. All solutions were prepared with
twice-distilled water.
A stock solution of 0.2 M histidine [2-amino-3-(4-imidazolyl)-
propionic acid] was prepared by dissolving L-histidine in water.
Solutions of 0.2 M interferences were prepared by dissolving the
appropriate amount of each compound in water. Working solutions
were prepared by successive dilution of the stock solutions with
water. All of the working solutions were buffered at pH 8.0 using
Tris/ HCl buffer solution.
Synthesis of the P orphyrin Dimer (4 ) (Figure 1 ). 5-(4-
Hydroxyphenyl)-10,15,20-triphenylporphyrin (1).34 Benzaldehyde
(708 mg, 66.7 mmol) and 4-hydroxybenzaldehyde (482 mg, 33.4
mmol) were dissolved in 250 mL of propionic acid. The mixture
was brought to reflux, and 6.92 mL of pyrrole (100 mmol) was
added within 15 min. The resulting black solution was refluxed
for 30 min, then cooled to room temperature. After the addition
of 100 mL of methanol, the resulting solution was allowed to stand
for 24 h. Filtration through a coarse sintered glass funnel, followed
by methanol washes and drying at 120 °C, gave 960 mg of blue
crystals. This mixture of porphyrins was dissolved in 5 mL of
chloroform and loaded onto a 4 × 40 cm column of ∼100-200-
mesh silica gel then eluted with 10:1 chloroform/ ethanol(v/ v).
The first band was meso-tetraphenylporphyrin, and the second was
the desired monohydroxyphenylporphyrin 1 . 1H NMR(400 MHz,
CDCl3, relative to TMS): δ ppm 8.84-9.01 (d, 8H, â pyrrole),
8.212 (d, 6H, ortho-H of triphenyl), 7.768 (m, 9H, meta/ para-H of
triphenyl), 8.08 (d, 2H, ortho-H of hydroxyphenyl), 7.217 (m, 2H,
meta-H of hydroxyphenyl), -2.784 (s, 2H, pyrrole NH). IR (KBr,
cm-1): 3423 (νOH), 3313 (νNH), 1595 (νCdC), 1258 (νC-O).
(16) Geno, M. K.; Halpern, J. J. Am. Chem. Soc. 1 9 8 7 , 109, 1238-1240.
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930.
5-(4-Bromohexyloxyphenyl)-10,15,20-triphenylporphyrin (2).35 To
a solution of 700 mg of 5-(4-hydroxyphenyl)-10,15,20-triphenylpor-
phyrin (1 ) in 80 mL of dry dimethylformamide (DMF), 1.2 g of
1,6-dibromohexane and 1.0 g of K2CO3 were added. The mixture
was refluxed 6 h, and the tarry solution was allowed to cool and
stand for 24 h. A dark solid was collected by filtration, washed
successively with methanol and water, and dried. The resulting
product was purified by flash column chromatography (silica gel,
∼100-200 mesh, elute, chloroform). After the removal of the
solvent on a rotary evaporator, a red solid was obtained in 47%
yield. 1H NMR(400 MHz, CDCl3, relative to TMS): δ ppm 8.860-
9.014 (d, 8H, â pyrrole), 8.202 (d, 6H, ortho-H of triphenyl), 8.101
(d, 2H, ortho-H of bromohexyloxyphenyl), 7.774 (m, 9H, meta/
para-H of triphenyl), 7.26 (m, 2H, meta-H of bromohexyloxyphen-
yl), 4.259 (m, 2H, OCH2), 1.253-3.743 (m, 10H, CH2), -2.805 (s,
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7519.
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1712.
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L.; Foekema, J.; Schenning, A. P. H. J.; Meijer, E. W.; de Schyver, F. C.;
Nolte, R. J. M. J. Am. Chem. Soc. 1 9 9 8 , 120, 11054-11060.
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Analytical Chemistry, Vol. 74, No. 5, March 1, 2002 1089

Fig u re 1 . Synthesis of the porphyrin dimer.
2H, pyrrole NH). IR (KBr, cm-1): 3383 (νN-H), 2858 (νC-H), 1595
(νCdC), 1243 (νC-O-C).
impurity of a molecular weight (784 g/ mol) higher than CoTPP
(735 g/ mol), and the mass spectrum showed no evidence of the
presence of free porphyrin. The UV-vis spectra bands of the
product had reached constant values at 530 nm (1.660 × 104),
416 nm (2.58 × 105), ∼331 nm (1.03 × 104) and 264 nm (2.70 ×
104). IR (KBr, cm-1): 3432 (νO-H), 3020 (νC-H), 1597 (νCdC), 1259
(νC-O-C).
Porphyrin Dimer (4). A 60-mL amount of dry pyridine was
added to 370 mg of crude 3 and 20 mg of potassium metal. The
resulting solution was refluxed with stirring for 2 h and then
cooled to room temperature. After removing the excess potassium,
430 mg of crude 2 was added, and the reflux was continued for
6 h. Then 30 mL of pyridine was removed under reduced pressure
on a rotary evaporator, and 50 mL of water was added to the
remaining liquid. The tarry solution was allowed to stand for 24
h. A dark solid was collected by filtration, washed with five 50-
mL portions of methanol and a large amount of water, and dried.
The residue was purified by chromatography on a column packed
with 40 g of ∼100-200 mesh silica gel in chloroform. Elution with
5-(4-Hydroxyphenyl)-10,15,20-triphenylporphyrin Cobalt(II) (3).
The Co(II) complex of porphyrin 1 was obtained according to a
procedure reported by Buchler.36 Under N2, dissolving 5-(4-
hydroxyphenyl)-10,15,20-triphenylporphyrin and Co(acetate)2 in
DMF, the solution was refluxed 2 h. After the addition of an
appropriate amount of water, the tarry solution was allowed to
cool and stand for 24 h. The product was purified by column
chromatography as described for complex 2 , but only the center
of the reddish purple band was collected, and no attempt was
made to exclude oxygen from the purification procedure. After
the chromatographed sample was crystallized from chloroform
and the crystals were heated to 120 °C for 30 min, a red product
containing no solvent was obtained in 68% yield. Calcd for
CoC48H28N4O4: C, 78.36; H, 3.81; N, 7.62. Found: C, 79.01; H,
3.74; N, 7.46. A mass spectrum of a pure product showed <1%
(36) Buchler, J. W.; In Porphyrins and Metalloporphyrins; Smith, K. M., Ed.;
Elsevier: Amsterdam, 1976; p 185.
1090 Analytical Chemistry, Vol. 74, No. 5, March 1, 2002

8% ethanol in chloroform (v/ v) removed a fast-moving minor band
of unreacted starting materials, then 3% ethanol in chloroform
(v/ v) was used to remove the product. Removal of solvent on a
rotary evaporator afforded 491 mg (60%) of porphyrin dimer 4 .
1H NMR (400 MHz, CDCl3, relative to TMS): δ ppm 15.839 (s,
8H â pyrrole of the inner metalloporphyrin moiety), 12.98 (s, 8H,
ortho-H of all phenyl groups of the inner metalloporphyrin moiety),
9.870 (s, 9H, meta/ para-H of triphenyl of the inner metallopor-
phyrin moiety), 9.641 (s, 2H, meta-H of hexyloxyphenyl of the
inner metalloporphyrin moiety), 8.834-9.042 (s, 8H, â pyrrole of
the inner free-base porphyrin), 8.208 (d, 6H, ortho-H of triphenyl
of the inner free-base porphyrin), 8.108 (d, 2H, ortho-H of hexyl-
oxyphenyl of the inner free-base porphyrin), 7.749 (m, 9H, meta/
para-H of triphenyl of the inner free-base porphyrin), 7.241 (d,
2H, meta-H of hexyloxyphenyl of the inner free-base porphyrin),
4.252 (m, 2H, C-H of hexyl group tail), 2.379 (m, 2H, C-H of
hexyl group tail), ∼1.248-1.984 (m, 8H, C-H of hexyl group),
-2.786 (s, 2H, NH of the inner free-base porphyrin moiety). IR
(KBr, cm-1): 3317 (νN-H), 2922 (νC-H), 2851 (νC-H), 1597 (νCdC),
1259 (νC-O-C).
Preparation of Sensing Membrane. The sensing membrane M1
was prepared by using a spin-coating technique. A membrane
cocktail was obtained by dissolving 2.5 mg of porphyrin dimer 4 ,
80 mg of PVC, and 160 mg of BOS in 2 mL of freshly distilled
THF. An aliquot of 0.2 mL of this solution was applied to the
surface of a circular 35-mm diameter quartz plate that was
mounted on a rotating (rotating frequency 600 rpm) aluminum
alloy rod under a THF-saturated atmosphere.37 After a spinning
time of only 5 s, a membrane of ∼4 µm thickness was obtained
on the quartz plate. In a similar manner, membrane M2 containing
1.5 mg of TPP as sensing material was prepared from the
described membrane cocktail.
Measurement Setup. The quartz plate with sensing membrane
was first fitted onto the front side of a measuring cell described
elsewhere37 that was suitable for fluorescence detection. A 35-
mm-diameter black PVC plate without a sensing membrane was
then fitted onto the other side to complete the cell. The cell was
introduced in a Hitachi spectrophotometer (model F-2500) at the
appropriate position38 to measure the fluorescence intensity. About
3.4 mL of sample solution was introduced using a syringe, and
the fluorescence emission spectra of the optode membrane were
recorded in the range between 560 and 760 nm with an excitation
wavelength of 423 nm. All fluorescence measurements were taken
under batch conditions. Before measurements, all sensing mem-
branes were conditioned in Tris/ HCl plain buffer solution until
the fluorescence intensity was stabilized. The limiting fluorescence
intensities F0 and F1 were determined with the sensing membrane
in contact with Tris/ HCl buffer solution and 3.0 mM histidine
solution, respectively.
Fig u re 2 . Fluorescence emission spectra of the porphyrin dimer
(1) and TPP (2) in CH2Cl2 solution (porphyrin concentration 0.002
mM; excitation wavelength, 423 nm).
level in human serum.40 The response to histidine was used as
the standard.
RESULTS AND DISCUSSION
Optical P roperties of the P orphyrin Dimer. The optical
properties of a porphyrin compound can be related to its molecular
skeleton. The nucleus of porphyrin consists of four pyrrole rings
linked by a methine bridge: the corresponding macrocycle is fully
conjugated, containing an 18-electron aromatic π-system and
planar. The extended aromatic system is responsible for the high
molar absorbance (4.33 × 105 cm-1 M-1 at 419 nm in dodecane41)
and, finally, for the intense fluorescence (fluorescence quantum
yield æ ) 0.13)42 of the porphyrin. From an optical point of view,
the porphyrin molecules are characterized by the presence of a
pronounced absorption band (the Soret band) in the blue region
of the visible spectra and the large Stockes shift (∆λ > 200 nm).43
Meanwhile, TPP is also a highly hydrophobic reagent and retains
almost the same optical properties after being immobilized in a
plasticized PVC membrane.38
When TPP is covalently linked with a metalloporphyrin moiety,
which is insulated by at least one methylene group, fluorescence
quenching by the intramolecular metal center is, of course, a well-
known phenomenon.44 Figure 2 shows the fluorescence emission
spectra of the porphyrin dimer 4 and TPP in CH2Cl2 (0.002 mM).
The excitation wavelength is 423 nm, near the Soret band. Since
the absorbances of these two compounds are similar to each other
at this wavelength, one can directly compare their fluorescence
intensities. It can be seen from Figure 2 that the fluorescence
intensity of the porphyrin dimer (at 657 nm) is only 16% of that of
TPP, indicating that the inner metalloporphyrin efficiently quenches
the fluorescence of the inner free-base porphyrin moiety. This
result has been ascribed to the photoinduced intramolecular
electron transfer from the inner free-base porphyrin in the singlet
excited state to a low-spin cobalt(II) (Figure 3a).45 For such a
derivative, electron transfer is the primary de-excitation path for
For the selectivity measurements, a separate solution method
(SSM)39 was used throughout by using solutions of corresponding
amino acids or anions in Tris/ HCl buffer solution. The selectivity
coefficients were evaluated by comparing the response function
at R ) 0.36, which corresponds to 0.12 mM histidine, the normal
(40) Pau, C. P.; Rechintz, G. A. Anal. Chim. Acta 1 9 8 4 , 160, 141-147.
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D. Chem. Commun. 1 9 9 9 , 2011-2012.
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Analytical Chemistry, Vol. 74, No. 5, March 1, 2002 1091

Fig u re 3 . Basic photoinduced electron-transfer process in a porphyrin dimer system: (a) in a free state and (b) guest-species-bound.
the excited state, and the electron-transfer rates depend on the
donor-acceptor angle and distance.46 In the classic Marcus theory
for nonadiabatic electron transfer, the rate of electron transfer,
KeT, is given by47
2
KeT ) (π/ p2λkBT)1/ 2|V| exp[ -(∆G° + λ)2/ 4λkBT] (1)
where p is Planck’s constant divided by 2π, λ is the reorganization
energy, kB is the Boltzmann constant, T is the temperature, and
V is the coupling matrix element.
The fluorescence quenching of the porphyrin dimer can be
suppressed upon complexation with an ion or molecule and the
Fig u re 4 . Fluorescence emission spectra of the optode membrane
inhibiting of the electron transfer (Figure 3b). Fluorescence then
M1 in the presence of different concentrations of histidine at pH 8.0:
1, 0; 2, 0.004; 3, 0.008; 4, 0.02; 5, 0.04; 6, 0.08; 7, 0.2 ; 8, 0.4; and
can become the dominant decay pathway for the excited free-
base porphyrin. There are also other mechanisms for inhibiting
electron-transfer, such as conformational changes,24 micropolarity
modulations,48 or hydrogen bonding.49 The “guest” species induces
an increase in ionization/ oxidation.
Optode Membrane Response and P rinciple of Operation.
The optode membrane M1 was essentially weakly fluorescent
where exposed to plain buffer of pH 8.0. When the membrane
9, 2.0 mM (excitation wavelength, 423 nm).
was in contact with the aqueous buffer solution containing
histidine, an increase in fluorescence intensity was observed.
Figure 4 shows the fluorescence emission spectra of the optode
membrane M1 in the presence of histidine. As expected from the
original design, complexation of the porphyrin dimer with histidine
increases the fluorescence intensity of the membrane at two
bands, 657 and 719 nm, which correspond to the TPP emission.
The degree of fluorescence enhancement is related to histidine
concentration. At a histidine concentration of about 20 mM, the
fluorescence intensity of the optode membrane gradually leveled
off. Higher histidine concentrations resulted in no appreciable
(46) Osuka, A.; Maruyama K.; Mataga, N.; Asahi, T.; Yamazaki, I.; Tamai, N. J.
Am. Chem. Soc. 1 9 9 0 , 112, 4958-4959.
(47) Closs, G. L.; Calcaterra, L. T.; Green, N. T.; Penfield, K. W.; Miller, J. R. J.
Phys. Chem. 1 9 8 6 , 96, 3673-3683.
(48) Fromber, P.; Rieger, B. J. Am. Chem. Soc. 1 9 8 6 , 108, 5361-5362.
(49) D’Souza, F.; Deviprasad, G. R.; Hsieh, Y.-Y. Chem. Commun. 1 9 9 7 , 533-
534.
1092 Analytical Chemistry, Vol. 74, No. 5, March 1, 2002

changes. The fluorescence spectra of optode membrane M2 were
scarcely changed by exposing it to histidine solution, indicating
that the spectral changes are due to the interaction of the inner
metalloporphyrin and histidine.
The fluorescence enhancement of the optode membrane M1
by histidine could be attributed to the extraction of histidine from
aqueous solution into the membrane phase and the ligation
interaction of histidine with porphyrin cobalt(II). If the complex
equilibrium between histidine in the aqueous sample solution (aq)
and porphyrin dimer in the plasticized PVC membrane phase
(mem) will form an m-n complex, the overall equilibrium can
be represented as
K
mA(aq) + nB(mem) y
\
z AmBn(mem)
(2)
where A represents histidine and B represents the porphyrin
dimer. If the difference between the activities and concentrations
is neglected for simplification, the corresponding equilibrium
constant, K, can be expressed by the law of mass action,
Fig u re 5 . Response parameter values (R) at 423 nm/657 nm as a
function of the logarithm of histidine concentrations at pH 8.0.
Theoretical response of histidine as predicated by eq 5. The
experimental data were fitted to the equation with the different
complex ratios and equilibrium constant: (1) n:m ) 1:2, k ) 2.5 ×
108; (2) n:m ) 2:2, k ) 2.2 × 109; (3) n:m ) 1:1, k ) 1.5 × 104 (best
fit); (4) n:m ) 2:1, k ) 1.1 × 105 (9, experimentally observed data
points).
[AmBn](mem)
K ) kd×â )
(3)
[A]m(aq)[B]n(mem)
Characteristics of the Optode Membrane M1. The response
behavior for a given optical sensor depends significantly on the
membrane composition. Several membrane compositions were
investigated by selecting appropriate plasticizers and varying the
ratio of PVC, plasticizer, and the sensing material, porphyrin
dimer. A sensing membrane made of different plasticizers, such
as bis(2-ethylhexyl) sebacate(BOS), dibutyl phthalate, tricresyl
phosphate, and didecyl phthalate were prepared. Membranes
consisting of BOS show the best response to histidine. At each
porphyrin dimer concentration, maximum sensitivity was obtained
with a BOS/ PVC weight ratio of about 2:1. On the other hand,
the concentration of the sensing material incorporated within the
membrane influenced the magnitude of the sensor response and
its sensitivity. The fluorescence intensity of the optode membrane
increased with an increasing amount of the porphyrin dimer in
the membrane; however, more sensing material did not produce
a larger fluorescence signal change (F/ F0 - 1), where F0 and F
are the fluorescence intensities of the optode membrane in the
absence and presence of histidine. Taking the sensitivity and the
fluorescence intensity value of the bulk membrane into account,
the optimal porphyrin dimer immobilizing was 2.5 mg, 1.03 wt %
relative to the total amount of the membrane cocktail.
The fluorescence intensity of the optode membrane M1 in plain
buffer solution and that of the sensing membrane when exposed
to histidine-containing solution was influenced by the acidity of
the medium. Although a decrease in response was observed at
pH <6.0 or pH >10.0, at neutral or weak-base medium, the sensor
response to histidine was nearly independent of pH. The pH
dependence of the fluorescence intensity change (F/ F0 - 1) of
the sensing system for 0.08 mM histidine is shown in Figure 6.
The response reached a maximum value and remained a constant
between pH 7.0 and 9.0. In this working pH range, histidine exists
mainly in the neutral form, which is subjected to being extracted
into the organic film phase, forming the complex with the
metalloporphyrin, and causing fluorescence enhancement of the
where kd and â are the distribution coefficient and complex
formation constant; and [AmBn], [A], and [B] are the concentra-
tions of the respective species.
For the description of the response behavior of this optically
sensing membrane, it is useful to introduce the degree of reaction,
R, defined as the ratio between the free porphyrin dimer
concentration, [B], to the total concentration of the porphyrin
dimer, CB. R can be expressed as
F1 - F
F1 - F0
[B]
CB
R )
)
(4)
where F is the fluorescence intensity of the sensing membrane
actually measured at a defined histidine concentration, F0 is the
fluorescence intensity of the optode membrane in the plain buffer
solution, and F1 is the fluorescence intensity when the porphyrin
dimer is completely complexed by histidine. R is shown to be
dependent on the concentration of histidine in the aqueous sample
solution, [A], as follows.
Rn
1 - R
1
)
(5)
nKCBn-1[A]m
The relationship between R and [A], as expressed by eq 5, is
the basis of the quantitative determination of histidine concentra-
tion in aqueous solution by using this optode membrane. The
experimental data were fitted to eq 5 by changing the ratio of m
to n and adjusting the overall equilibrium constant, K. Figure 5
shows the fitted curves that represent the experimental data for
histidine. The curve referring to the 1:1 complex ratio and K )
1.5 × 104 is the best one fitted to the experimental data (Figure
5, solid line, 3). The best-fitting curve can serve as the calibration
curve for the determination of histidine.
Analytical Chemistry, Vol. 74, No. 5, March 1, 2002 1093

Fig u re 6 . Fluorescence response of the optode membrane M1 at
423 nm/657 nm as a function of pH (histidine concentration ,0.08 mM).
Fig u re 7 . Response function of the optode membrane on exposure
to aqueous amino acids measured at pH 8.0 and at a wavelength of
423 nm/657 nm: 1, methionine; 2, glycine; 3, tyrosine; 4, isoleucine;
5, tryptophan; 6, lysine; 7, arginine; 8, histidine. The solid lines were
calculated according to eq 5.
sensing membrane. The observed decreasing response at higher
pH values could be due to the formation of a metalloporphyrin-
OH complex.50 Lower pH leads to protonation and release of
histidine. In subsequent experiments, a pH 8.0 Tris/ HCl buffer
solution was used as an ideal experimental condition.
Ta b le 1 . S e le c t ivit y Co e ffic ie n t s (lo g Ko p t ) o f t h e
Op t o d e Me m b ra n e M1 fo r Am in o Ac id s a n d Co m m o n
Io n s in Co m p a ris o n t o His t id in e a t p H 8 .0
With the optimum conditions, the optode membrane M1
exhibited the highest sensitivity to histidine in the ∼0.0045-1.53
mM range. The limit of detection, which was determined by
alternately exposing the optode membrane to plain buffer and low
concentration of histidine (5 times each) and calculating the
histidine concentration corresponding to signal changes that are
6 times the standard deviation of the plain buffer signal, was found
to be 0.002 mM. The reproducibility and reversibility of the optode
membrane were evaluated by repetitively exposing it to 0.08 mM
histidine solution and Tris/ HCl buffer solution (pH 8.0). The mean
fluorescence intensity values with their confidence intervals were
found to be 38.33 ( 0.64 (n ) 5, 0.08 mM histidine) and 12.67 (
0.38(n ) 5, Tris/ HCl buffer solution). The response time of the
sensor depends on the thickness of the membrane, the flow rate
of the sample solution, and the change of the concentration of
histidine. When the thickness of the membrane reaches the order
of millimeters, the response time may reach 5 h. To prepare very
thin membranes, the spin-on technique was used, which allows
the production of very thin, homogeneous and reproducible PVC-
based membranes. The forward response time (going from lower
to higher histidine concentrations), t95(time needed for 95% of the
total signal change to occur) was within 6 min, whereas the time
for the reverse response was in the range of 2-4 min over the
entire concentration range. Additionally, the fluorescence intensity
of the optode membrane M1 at λex/ em ) 423/ 657 nm reduced by
about 8% during the determination of the sample solution over
three weeks. The decrease in the fluorescence intensity of the
membrane might be due to the leaching and the photodecompo-
sition of the membrane components when it was excited by
stronger incident lights.
interfering agent
log Kopt
interfering agent
log Kopt
arginine
lysine
tryptophan
isoleucine
tyrosine
glycine
-1.38
-1.93
-2.35
-2.62
-3.04
-3.52
-3.72
SCN-
sailcylate
I-
-1.43
-2.44
-2.65
-
ClO4
-3.06
-
NO2
-3.13
Br-
-3.34
methionine
Cl-
-3.39
-
H2PO4
SO4
<-3.70
<-3.90
2-
membrane to other amino acids, the sensing membrane was
subjected to different concentrations of histidine, arginine, lysine,
tryptophan, isoleucine, tyrosine, glycine, and methionine sepa-
rately for a contact time of 6 min. In Figure 7, the membrane
responses, R, at pH 8.0 are plotted as functions of the concentra-
tion logarithms of histidine and other amino acids. The curve-
fitting for the experimental points was calculated from eq 5
assuming a 1:1 complex ratio. The amino acid optical selectivity
coefficients, log Kopt values (logarithm of the relative selectivities
between the interfering amino acids (or anions) obtained by
calculating the horizontal distance between the amino acids
response curves or anions response curves (not given) at the point
of R ) 0.36)39 are summarized in Table 1. Table 3 also shows the
recoveries of histidine in the mixture of some potential interfering
agents in human serum (see Table 2). As shown in Figure 7 and
Tables 1 and 3, the porphyrin-dimer-based histidine-sensitive
optode membrane shows excellent selectivity over the several
amino acids and common anions. The selectivity of the present
sensor for amino acids is histidine . arginine > lysine >
tryptophan > isoleucine > tyrosine > glycine > methionine. The
order of selectivity of some the common anions is as follows:
Probably the most important characteristic feature of a sensor
is its response to the species to be measured over that to other
species present in the solution. To define the response of the
-
-
histidine . SCN- > salicylate > I- > ClO4 > NO2 > Cl-
>
-
Br- > H2PO4 > SO42-. This selectivity patterns deviates from
(50) Chaniotakis, N. A.; Chasser, A. M.; Meyerhoff, M. E.; Groves, J. T. Anal.
Chem. 1 9 8 8 , 60, 185-188.
the Hofmeister selectivity pattern that is based solely on lipo-
1094 Analytical Chemistry, Vol. 74, No. 5, March 1, 2002

Ta b le 2 . Co m p o s it io n s o f S im u la t e d S e ru m a
compd
concn (mM)
compd
glycine
aspartic acid
phenylalanine
alanine
concn (mM)
tyrosine
arginine
lysine
histidine
isoleucine
methionine
tryptophan
0.08
0.22
0.25
0.12
0.11
0.032
0.07
0.14
0.09
0.15
0.45
15
NaHCO3
NaCl
100
a
All amino acids are L or DL isomers.
Ta b le 3 . Re c o ve ry S t u d ie s o f His t id in e fro m S yn t h e t ic
S e ru m S a m p le s
concn, mM
Fig u re 8 . Effect of histidine concentration on the absorption of the
porphyrin dimer at pH 8.0: (1) 0.0, (2) 0.08, (3) 0.2, (4) 0.4, (5) 0.8
mM.
sample
added
found, meana ( SDb
recovery, (%)
1
2
3
4
5
0.02
0.10
0.20
0.50
0.80
0.016 ( 0.0006
0.103 ( 0.0031
0.198 ( 0.0082
0.483 ( 0.01
97.2
103.4
99.1
96.5
105.4
the extinction coefficient and shifts slightly to longer wavelength
(431 nm) as the concentrations of histidine were increased, as
shown in Figure 8. This is characteristic for the histidine
coordination with the central metal.53 The Soret band reflects the
valence state of the central metal. The coordination of histidine
with the central cobalt(II) results in the delocalization of the cobalt-
(II) d orbitals, which causes the red-shift of the Soret band. Figure
8 also shows that the numbers of the absorption peaks do not
change after porphyrin cobalt(II) coordinated with histidine. This
indicates that after histidine is coordinated with the central metal,
the symmetry group of the metallopropyrin-histidine complex
agrees with the C2ν symmetry group of the metalloporphyrin,54
and one histidine is coordinated with one central cobalt atom in
the process of ligation interaction.
Determination of Synthetic Biological Samples. The high
degree of histidine selectivity of the optode membrane makes it
potentially useful for monitoring concentration levels of histidine
in biological samples. To investigate the possibility of applying
the sensor for the quantization of histidine, recovery studies were
conducted using a synthetic serum containing ∼0.02-0.8 mM
histidine. The composition of the synthetic serum is listed in Table
2. The concentration of each component of the serum was chosen
to match its normal level reported in human serum.40 Acceptable
recoveries were obtained, as shown in Table 3. The results suggest
that application of the constituents of the synthetic serum sample
do not interfere in any way with the detection of histidine.
Therefore, it is feasible to apply the proposed sensor to quanti-
tatively determine the concentration of histidine in serum samples.
0.843 ( 0.011
Mean of three determinations. b Standard deviation.
a
philicty. Furthermore, this pattern indicates the existence of a
selective interaction between the metalloporphyrin and some
specific anions which is consistent with the data reported previ-
ously.18 As can be seen, the selectivity coefficients obtained with
the porphyrin-dimer-based histidine optode systems fulfill the
selectivity requirements for the histidine assay in physiological
fluids. The origin of the fluorescence response of the porphyrin
dimer toward histidine is likely due to direct and relatively strong
interaction of the imidazole moiety of histidine as an axial ligand
of cobalt(II).51 Since the π-symmetry orbits of the low-spin Co(II)
(d7) ion are completely filled, this would suggest that the five-
membered imidazole rings are better π acceptors from Co(II) than
are aliphatic amines and pyridines of other amino acids,33 which
act to remove much of the steric interaction with the hydrogen
bonds of the adjacent carbons.
Interaction Mechanism of Metalloporphyrin with Histi-
dine. Although it has been supposed that there is an interaction
between metalloporphyrin and amino acids, the interaction mech-
anism has not been studied in depth so far. The change of the
UV-vis spectra of the porphyrin dimer in the visible region when
in contact with a histidine-containing solution might shed some
light on the interaction mechanism of metalloporphyrin with amino
acids. Normal metalloporphyrin electronic absorption spectra
show three prominent bands in the visible region. The most
intense is the B, or Soret, band, which usually occurs near 419
nm for organic media of metallo-TPP derivatives. The other two
intense bands are the Q and â bands, which are usually found
between 500 and 600 nm in the same solvent.52
CONCLUSION
We have reported here for the first time the synthesis and
application of a new bisporphyrin-based fluorescence sensor for
histidine. The design of the approach consists of meso-tetraphen-
ylporphyrin cobalt(II) as a molecular probe and a covalent
attachment of meso-tetraphenylporphyrin as a fluorescent reporter.
The response mechanism of the approach is based on intramo-
lecular photoinduced electron transfer and the coordination action
The interaction of the porphyrin dimer with histidine was
investigated at room temperature by measuring the UV-vis
spectra of a series of solutions in which the concentration of
histidine was varied. The visible peak at 417 nm decreased by
(53) Nappa, M.; Valentine, J. S. J. Am. Chem. Soc. 1 9 7 7 , 99, 5799-5780.
(54) Hoffman, A. B.; Collins, D. M.; Day, V. W.; Fleischer, E. B.; Srivastatva, T.
S.; Hoard, J. J. Am. Chem. Soc. 1 9 7 2 , 94, 3620-3626.
(51) Walker, F. A. J. Am. Chem. Soc. 1 9 7 3 , 95, 1154-1159.
(52) Nappa, M.; Valentine, J. S. J. Am. Chem. Soc. 1 9 7 8 , 100, 5075-5080.
Analytical Chemistry, Vol. 74, No. 5, March 1, 2002 1095

between the central metal and amino acids. Under certain
conditions, the coordination ability determines the selectivity
sequence of amino acids to be sensed. The dye has been described
that can be applied in optical sensor membrane to determine
histidine in aqueous solution. The recognition process is based
on favorable extraction of histidine into the bulk organic mem-
brane and complexation with the inner metallopophyrin moiety
and inhibiting the electron transfer process, which leads to
increased fluorescence of the membrane. The optode membrane
is easy to prepare and is selective to histidine over several amino
acids and common anions. Another unique feature of the proposed
sensor for histidine is that it responds directly to histidine itself
rather than indirectly monitoring the products from derivatization
procedures or enzymatic reactions.
ACKNOWLEDGMENT
This work was supported by the National Outstanding Youth
Foundation of China (29875110), Joined Research Fund for
Oversea Chinese and Hong Kong Youth Scholar of the National
Nature Science Foundation of China (20028506), Nature Science
Foundation of Hunan Province (00JKY1011), and the Key Project
Foundation of China Education Ministry (2000-156).
Received for review April 4, 2001. Accepted November 7,
2001.
AC010386B
1096 Analytical Chemistry, Vol. 74, No. 5, March 1, 2002