Protease amperometric sensor

Article abstract of DOI:10.1021/ac060253w

An amperometric biosensor for the detection of trypsin was developed. The latter was based on a two-layer configuration, namely, a polymer-glucose oxidase inner layer and a gelatin outer layer. In the presence of glucose, the enzyme layer produces H₂

Full text of DOI:10.1021/ac060253w

Anal. Chem. 2006, 78, 6327-6331
Protease Amperometric Sensor
Rodica E. Ionescu,† Serge Cosnier,*,† and Robert S. Marks*,‡,§
Laboratoire d’Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Mole´culaire de
Grenoble, FR CNRS 2607, Universite´ Joseph Fourier Grenoble I, BP 53, 38041 Grenoble, Cedex 9, France, Department of
Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, 84105 Beer-Sheva, Israel, and The National
Institute for Biotechnology in the Negev, and the Ilse Katz Center for Meso- and Nanoscale Science and Technology,
P.0. Box 653, 84105 Beer-Sheva, Israel
release from specialized cells on the intestinal mucosa. The CCK
then stimulates the pancreas to secrete more digestive enzymes
until the meal is digested and basal levels of free enzyme activity
return.2
Various types of pancreatitis such as chronic pancreatitis
(inflammation), acute pancreatitis (inflammation) and pancreatic
abscess (related to an infection) involve either irritation, inflamma-
tion, or infection of the pancreas. Trypsin and trypsinogen levels
are increased with some types of pancreatic disease such as acute
pancreatitis and cystic fibrosis. Unusually, low or normal levels
may be seen in chronic pancreatitis.
Acute pancreatitis is often defined as abdominal pain associated
with hyperamylasemia (or lipasemia) of greater than three times
above normal.3,4 Acute pancreatitis affects around 40/100 000 of
the Western general population, and its attacks are classified as
severe in 20-30% of the patients. This complex and ill-understood
common acute abdominal disorder lacks specific therapy, and its
clinical course and outcome are difficult to predict clinically at
the time of admission. Although conservative measures are usually
sufficient for the management of the large majority of cases of
acute pancreatitis that prove mild, early aggressive intervention
including enteral feeding, endoscopic retrograde cholangio-
pancreatography with sphincterotomy, broad spectrum antibiotics,
and intensive care unit monitoring are imperative in specific cases
of severe acute pancreatitis in order to reduce the accompanied
high morbidity and mortality.5 Unfortunately, the mortality rate
of acute pancreatitis can vary from 2 to 16% and in severe cases
from 7 to 47%.6
An amperometric biosensor for the detection of trypsin
was developed. The latter was based on a two-layer config-
uration, namely, a polymer-glucose oxidase inner layer
and a gelatin outer layer. In the presence of glucose, the
enzyme layer produces H2O2 and hence an amperometric
signal due to H2O2 electrooxidation was generated by
potentiostating the electrode at 0.6 V. The biosensor
detects the change in the increase in the maximum
current caused by the proteolytic digestion of gelatin,
which covers the platinum electrodes, thereby facilitating
a speedier access for the glucose substrate to the electrode
modified with both poly(pyrrole-alkylammonium) and
glucose oxidase molecules. Our biosensor detected low
trypsin concentrations down to 42 pM with a response
time of ∼10 min, making it a very sensitive device in the
detection of lower trypsin levels with such future putative
applications as the diagnosis of pancreatic diseases.
Any disorder in enzymatic expression can induce diseases.
Among different classes of enzymes, proteases are known to par-
ticipate in numerous physiological processes such as cell growth
and differentiation, cell-cell communication, and cell death. One
such protease, trypsin, is the most important digestive enzyme
produced by the pancreas. Trypsinogen (the precursor for trypsin)
is the only proenzyme that is specifically activated by a special
brush border enzyme (enterokinase) within the duodenum where
the conversion of trypsinogen to active trypsin is mediated by the
release of its 8-amino acid activation peptide, trypsinogen activation
peptide.1 Trypsin is the only enzyme involved in the digestive
enzyme activation cascade, which changes all of the other pan-
creatic proenzymes into their active forms within the intestine,
and then initiating autodigestion. Finally, trypsin plays a key role
in controlling pancreatic exocrine function. If free trypsin activity
in the duodenum diminishes because of a meal, then a trypsin
inhibitor, a trypsin-sensitive peptide, cholecystokinin (CCK) releas-
ing factor, avoids hydrolysis, accumulates, and stimulates CCK
To diagnose a pancreatic disorder, multiple clinical tests7 are
performed that can require more than 48 h to achieve reliability.8,9
Such tests use serum and urine samples where commonly high
concentrations of immunoreactive anionic trypsinogen failed to
(2) Whitcomb, D. C. Int. Congr. Ser. 2003, 1255, 49-60.
(3) Lankisch, P. G.; Burchard-Reckert S.; Lehnick D. Gut 1999, 44, 542-4.
(4) Byrne, M. F.; Mitchell, R. M.; Stiffler, H.; Jowell, P. S.; Branch, M. S.; Pappas,
T. N.; Tyler, D.; Baillie J. Can. J. Gastroenterol. 2002, 16, 849-54.
(5) Foitzik, T.; Klar, E.; Buhr, H. J.; Herfarth, C. Eur. J. Surg. 1995, 161, 187-
92.
* Corresponding authors. E-mail: rsmarks@bgumail.bgu.ac.il; serge.cosnier@
ujf-grenoble.fr. Tel: 972 8 647 71 82. Fax: 972 8 647 28 57.
† Universite´ Joseph Fourier Grenoble I.
(6) Tenner, S.; Sica, G.; Hughes, M.; Noordhoek, E.; Feng, S.; Zinner, M.;
Bankset, P. A. Gastroenterology 1997, 113, 899-903.
(7) Al-Bahrani, A. Z.; Ammori, B. J. Clin. Chim. Acta 2005, 362, 26-48.
(8) Ranson, J. H. C.; Rifkind, K. M.; Roses, D. F.; Fink, S. D.; Eng, K.; Spencer,
F. C. Surg. Gynecol. Obstet. 1974, 139, 69-81.
‡ Ben-Gurion University of the Negev.
§ The National Institute for Biotechnology in the Negev, and the Ilse Katz
Center for Meso- and Nanoscale Science and Technology.
(1) Rinderknecht, H. Dig. Dis. Sci. 1986, 31, 314-21.
(9) Imrie, C. W.; Benjamin, I. S.; Ferguson. J. C.; McKay, A. J.; Mackenzie, I.;
O’Neill, J.; Blumgart, L. H. Br. J. Surg. 1978, 65, 337-41.
10.1021/ac060253w CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/17/2006
Analytical Chemistry, Vol. 78, No. 18, September 15, 2006 6327

predict severe attacks of acute pancreatitis.10-14 By using radio-
immunoassay tests, serum trypsin levels have been estimated for
healthy patients (248 ( 94.9 µg/L), for patients with chronic renal
failure (1100 ( 548 µg/L), and for patients with acute pancreatitis
(1399 ( 618 µg/L).15
Figure 1. Structure of pyrrole-alkylammonium monomer.
The degradation of gelatin film has been used as a semiquan-
titative screening test for pancreatic disorders. However, such a
test cannot easily be calibrated to give an absolute measurement
of trypsin concentration. Therefore, other sensitive tests such as
affinity-based reactions, Bragg reflector devices that measure the
change of temperature, pressure,16 and humidity17 on gelatin-based
films were developed. Since all these techniques are time-
consuming and require specific laboratory-based instrumentation,
plus trained personal, a more rapid and precise tool is needed to
overcome such drawbacks.
One of the most promising areas in the development of precise
analytical techniques for the detection of various biological com-
pounds is based on the development of biosensors. Such biosen-
sors have been reported for protease (trypsin) detection. For
instance, hologram trypsin sensors18,19 and biosensors for collagen-
ase detection20,21 were developed. Unfortunately, these biosensors
are not sufficiently sensitive for the lowest trypsin detection limits
and have reached to date just below the nanomolar range.
The present paper reports on a successful quantitative test for
a protease (trypsin) picomolar detection by measuring the current
increase recorded from glucose oxidase (GOX)-poly(pyrrole-
alkylammonium)-modified platinum electrodes coated with a
gelatin film that was subjected to a proteolytic enzymatic action.
reacted with tosyl chloride (2.85 g, 15 mmol) in anhydrous
pyridine (3 mL). The mixture was stirred at 20 °C for 15 h, washed
with water, and extracted with dichloromethane. After evaporation,
the crude product was purified by chromatography (3.05 g, yield
74%). 11-Pyrrol-1-ylundecyl p-toluenesulfonate (1.5 g) was refluxed
for 15 h at 90 °C in ethanol (15 mL) with an excess of triethylamine
(11 mL). The solvent and excess of triethylamine were removed
under vacuum. Tosylate anions were then replaced by tetrafluoro-
borate anions on an ion-exchange column (Amberlite IRA 93)
leading to a brown oil (1.44 g, yield 65%). H1 NMR (250 MHz/
CD3Cl3): δ (ppm) 6.62 (s, 2H), 6.09 (s, 2H), 3.83 (t, 2H), 3.24 (m,
6H), 3.07 (m, 2H), 1.58 (m, 2H), 1.35-1.23 (m, 25H). LiClO4
(194711000) was obtained from Acros Organics. The 8% (w/v)
gelatin was dissolved in distilled water and heated to 50 °C, until
it became completely molten. The gelatin solution was prepared
anew for each experiment.
Apparatus. Cyclic voltammogram and electropolymerization
were performed with EG&G PAR, model 173 potentiostat equipped
with a model 175 universal programmer, and a model 179 digital
coulometer in conjunction with
a
Kipp and Zonen
BD 91 XY/t recorder. An electrochemical three-electrode cell
(Metrohm) was used. The amperometric measurements were
performed using a Tacussel PRG-DL potentiostat in conjunction
with a thermostated electrochemical cell at 20 °C. The work-
ing electrodes were platinum electrodes (i.d. ) 5 mm) systemat-
ically polished with 2-µm diamond paste (Mecaprex Press PM).
The reference electrode used was a saturated Ag-AgCl-KCl
electrode (Ag/AgCl) while a Pt wire was used as a counter
electrode.
Biosensor Preparation. The polymer-enzyme electrodes
were prepared according to a two-step procedure previously re-
ported by Cosnier.23 The 6 mM pyrrole-alkylammonium mono-
mer (brownish, oily suspension) was suspended in pure distilled
water and sonicated for 3 h to facilitate a total monomer solu-
bilization. The working electrodes (platinum disk) were modified
at room temperature by spreading over their surface an aqueous
mixture based on 15 µL of monomer solution and 200 µg of GOX
and then dried under vacuum atmosphere for 15 min. The
resulting “dry” modified electrodes were transferred into a cell
containing an aqueous 0.1 M LiClO4 solution. Electrochemical
polymerization of the adsorbed coating (GOX + pyrrole monomer)
was carried out by controlled potential electrolysis at 0.85 V versus
Ag/AgCl. Further, the enzyme-modified electrodes were coated
with 20 µL of 8% (w/v) gelatin solution and dried under vacuum
atmosphere for 20 min.
EXPERIMENTAL SECTION
Chemicals. Gelatin (type A, porcine skin, analytical grade,
G-2500), trypsin (from bovine pancreas, 9380 units/mg, T-4665),
GOX (from Aspergillus niger, type VII-S, 181 units/mg, G-7016),
and glucose (G-7528) were obtained from Sigma. (11-Pyrrol-1-
ylundecyl) triethylammonium tetrafluoroborate (Figure 1) was
synthesized in the following manner according to a previously
described protocol.22 11-Pyrrol-1-ylundecanol (2.6 g, 11 mmol) was
(10) Segal, I.; Chaloner, C.; Douglas, J.; John, K. D.; Zaidi, A.; Cotter, L.; Appelros,
S.; Borgstrosm, A.; Braganza, J. M. Am. J. Gastroenterol. 2002, 97, 883-
92.
(11) Appelros, S.; Lindgren, S.; Borgstrom, A. Eur. J. Surg. 2001, 167: 281-6.
(12) Yadav, D.; Agarwal, N.; Pitchumoni, C. S. Am. J. Gastroenterol. 2002, 97,
1309-18.
(13) Lempinen, M.; Kyl¨anpa¨a¨-Ba¨ck, M. L.; Stenman, U. H.; Puolakkainen, P.;
Haapiainen, R.; Finne, P.; Korvuo A.; Kemppainen, E. Clin. Chem. 2001,
47, 2103-7.
(14) Kyla¨np¨a¨a-Ba¨ck, M. L.; Kemppainen, E.; Puolakkainen, P. J. Pancreas 2002,
3, 34-48.
(15) Artigas, J. M.; Garcia, M. E.; Faure, M. R.; Gimeno, A. M. Postgrad. Med. J.
1981, 57, 219-22.
(16) Kersey, A. D.; Berkoff, T. A.; Morey, W. W. Opt. Lett. 1993, 18, 1370-2.
(17) Spoocer, R. C.; Al-Ramadhan, F. A.; Jones. B. E. Int. J. Optoelectron. 1992,
7, 449-52.
(18) Millington, R. B.; Mayes, A. G.; Blyth, J.; Lowe, C. R. Anal. Chem. 1995,
67, 4229-33.
(19) Millington, R. B.; Mayes, A. G.; Blyth J.; Lowe, C. R. Sens. Actuators, B:
RESULTS AND DISCUSSION
Electrochemical Characterization of Poly(pyrrole-alkyl-
ammonium)-GOX-Modified Electrodes. As reported previ-
ously,23 an original strategy of enzyme entrapment into electro-
generated polymers involved the immobilization of amphiphilic
Chem. 1996, 33, 55-5.
(20) Saum, A. G. E.; Cumming, R. H.; Rowell, F. J. Biosens. Bioelectron. 1998,
13, 511-8.
(21) Saum, A. G. E.;. Cumming, R. H.; Rowell, F. J. Biosens. Bioelectron. 2000,
15, 305-31.
(22) Coche-Guerente, L.; Deronzier, A.; Galland, B.; Labbe, P.; Moutet J. C.;
Reverdy, G. J. Chem. Soc. Chem. Commun. 1991, 381-6.
(23) Cosnier, S. Electroanalysis 1997, 9, 894-902.
6328 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006

Figure 3. Evolution of the biosensor response to glucose injection
(2 mM) as a function of gelatin degradation by trypsin (300 µg/mL)
over time. (a) Before gelatin coating; (b) after gelatin coating; there
after, different times soaking the biosensor in various trypsin solu-
tions: (c) 40 s; (d) 60 s; (e) 80 s; (f) 230 s; (g) 5 min; (h) 10 min; (i)
30 min. The biosensor was potentiostated at 0.6 V in stirred 0.1 M
PBS (pH 7).
Figure 2. Cyclic voltammogram of an electrode modified by a poly-
(pyrrole-alkylammonium) film containing 200 µg of GOX molecules
in H2O + 0.1 M LiClO4; scan rate, 0.1V‚s-1
.
monomers and enzymes together, by their adsorption on the
electrode surface before the electropolymerization. Taking advan-
tage of the poor solubility and strong adsorption properties of (11-
pyrrol-1-ylundecyl) triethylammonium tetrafluoroborate in water,
this amphiphilic pyrrole derivative was dispersed by ultrasoni-
cation and mixed with GOX enzymes, and the resulting mixture
was adsorbed on a platinum electrode. After the transfer of the
modified electrodes into an aqueous 0.1 M LiClO4 solution, free
of monomer, the oxidative electropolymerization of the adsorbed
coating at 0.85 V provides the entrapment of GOX molecules in
the “in situ” generated polypyrrole film. The electrochemical
characterization of the resulting electrodes with poly(pyrrole-
alkylammonium) film containing 200 µg of GOX molecules was
investigated by cyclic voltammetry in 0.1 M LiClO4 aqueous
solution. The cyclic voltammogram presents in the positive region,
a reversible peak system at 0.56 V reflecting the well-known
electroactivity of the polypyrrolic skeleton (Figure 2). This E1/2
value is in good agreement with those reported for conducting
functionalized polypyrroles, corroborating thus with the formation
of a polypyrrole film from the adsorbed biocoating. The apparent
As expected, the formation of an outer layer of gelatin generated
high steric hindrances toward the glucose diffusion to the
polymer-GOX layer since the current response decreased to 5%
of its initial value (Figure 3a). As a consequence, it was expected
that the detection of the protease activity could be carried out via
an increase in amperometric current due to the degradation of
the gelatin layer by the proteolytic digestion.
Detection of Protease Activity. The gelatin-coated enzyme
electrodes were immersed in a trypsin solution (300 µg‚mL-1
)
for different timed intervals and then transferred into 0.1 M PBS
to record the amperometric current response to glucose (2 mM).
Before current measurements were registered, the gelatin-
biosensor treated with trypsin solution was rinsed several times
with PBS solution. The gelatin-GOX biosensor current response
was initially recorded after 20 s of the proteolytic digestion treat-
ment began, followed by sets of the current measurements up to
30 min (Figure 3). The change in current intensity as a function
of time of trypsin incubation was compared to the initial current
response of the gelatin uncoated biosensor. As expected, the
removal of the gelatin layer with time induced initially an increase
in amperometric current that reached, after 1 min of trypsin incu-
bation, 64% of the current value observed for the gelatin uncoated
enzyme electrode. After this time, the enzymatic digestion of
gelatin was continued up to 30 min, inducing a progressive current
decrease down to 8% from the initial current response. This clearly
indicates a “toxic” effect of the trypsin on the entrapped GOX
enzyme molecules within the poly(pyrrole-alkylammonium) films.
Such an effect could be explained by the cleaving of the GOX
molecule at lysine and arginine residues by trypsin action. To
validate the role of trypsin on the immobilized GOX, a control
experiment was carried out with an uncoated enzyme electrode
incubated with trypsin solution (300 µg‚mL-1) for 30 min. As
expected, a marked decrease in current response to glucose (95%)
was observed demonstrating the possibility for trypsin to degrade
the GOX activity although these molecules were entrapped in a
polymer film. It should be noted that the polypyrrole film exhibited
long linear chains that are more permeable than a cross-linked
structure.24
surface coverage of polymeric film (Γ ) 5.88 × 10-8 mol‚cm-2
)
was determined from the charge recorded under the polypyrrole
oxidation wave, leading to an electropolymerization yield of 41%.
Since GOX catalyzes the aerobic oxidation of glucose with the
concomitant production of H2O2, the analytical performances of
the enzyme electrode for the determination of glucose were
investigated. The amperometric detection of glucose was assayed
in 0.1 M phosphate buffer (pH 7) by holding the modified
electrodes at 0.6 V in order to oxidize the enzymatically generated
H2O2 at the platinum underlying electrode. The calibration curve
was linear with the changing glucose concentration and reached
a plateau for glucose concentration above 22 mM. The sensitivity
of the biosensor (determined as the slope of the initial linear part
of the calibration curve) and its maximum current density at satu-
rating glucose condition were 23 mA‚M-1‚cm-2 and 102 mA‚cm-2
,
respectively. To quantify the effect of an additional gelatin layer
on the performance of the glucose biosensor, the amperometric
response of the enzyme electrode was recorded for 2 mM glucose.
This glucose concentration was chosen since it corresponds to
the linear part of the calibration curve where the biosensor
response is directly proportional to the substrate concentration.
(24) Cosnier, S.; Mousty, C.; Gondran, C.; Lepellec A. Mater. Sci. Eng.: C. In
press.
Analytical Chemistry, Vol. 78, No. 18, September 15, 2006 6329

Figure 4. Increase in current response of gelatin-GOX biosensors
to glucose (2 mM) in the presence of various trypsin concentrations
after 1 min of soaking in protease solutions. The increase was
reported as a ratio of the measured biosensor current response versus
the initial one in the absence of gelatin.
Figure 6. Protease (trypsin) calibration curve illustrated by the
increase in current response of gelatin-GOX biosensors to glucose
(2 mM) in the presence of various trypsin concentrations after 10 min
of soaking in protease solutions. Experimental conditions are as in
Figure 4.
40 ng/mL.18,19 In addition, these biosensors require large response
times (20 and 60 min) whereas our gelatin-GOX electrodes
exhibited a rapid response time ranging from 1 min for 300 µg/
mL trypsin and up to 10 min for 1 ng/mL trypsin. Furthermore,
non-biosensor-based protease assays such as a ion-selective elec-
trode detection,25 a Coomassie-stained gelatin agarose gel assay,26
a solid-phase protein cleavage assay,27 turbidity,28 and radiometry15
showed low limit of detection values at 1-10 µM protease,25 10 pg
of trypsin but the assay plates were incubated for 14 h within the
protease solution before reading,26 10 ng/mL protease,27 5 µg/
mL trypsin,28 and 248 ng/mL trypsin,15 respectively.
Figure 5. Influence of trypsin concentration on the time of gelatin
degradation necessary to obtain the maximum current response of
the biosensor to glucose (2 mM).
It should be noted that the repeatability of the amperometric
response of one gelatin-GOX electrode after a proteolytic process
in the presence of 300 µL/mL trypsin for 60 s was examined at a
glucose concentration of 2 mM. The RSD was 3.2% for four
successive current measurements. Moreover, the reproducibility
of the analytical response obtained from different modified
electrodes constructed by the same procedure was also investi-
gated. Eleven protease biosensors were tested independently for
the detection of a specific protease (trypsin) concentration (300
µL/mL) for a precise digestion time (60 s). A mean current
response of 5.7 µA to glucose (2 mM) was recorded with an RSD
of 4.8% (n ) 11). This demonstrates an efficient and reproducible
detection of the proteolytic digestion process, even though the
procedures used for enzyme immobilization and gelatin deposition
were done manually.
Biosensor Performances for the Determination of Trypsin.
To emphasize the high sensitivity of the gelatin-GOX biosensors
to the protease (trypsin) digestion, the effect of various concentra-
tions of trypsin (0.001 up to 300 µg/mL) in 0.1 M PBS on the
biosensor sensitivity was examined. Figure 4 shows a drastic
increase in amperometric current response to glucose (2 mM)
for concentrations ranging from 0.1 to 300 µg/mL. Although small
current differences are noticed between 1 and 100 ng, this
procedure cannot be used for the determination of extremely low
trypsin concentrations. To improve the biosensor sensitivity
toward protease, a proteolytic optimum time (POT) was defined.
The POT corresponds to the incubation time in trypsin necessary
to obtain the maximum current response, the latter being the best
compromise between the gelatin digestion and the GOX deactiva-
tion. Figure 5 depicts the evolution of POT as a function of trypsin
concentration. As expected, the lower the trypsin concentration
used, the longer the POT is. Therefore, the proteolytic optimum
time was found to range between 1 and 10 min, when using
300 µg/mL trypsin and 1ng/mL trypsin, respectively, for the
gelatin degradation. As a consequence, the determination of low
trypsin concentration was investigated by increasing the incuba-
tion time up to 10 min. For each trypsin concentration, three
freshly prepared gelatin-GOX biosensors were used and their
current response was recorded and averaged. A linear increase
of the current response with the logarithm of trypsin concentration
was observed between 1 and 250 ng/mL (Figure 6). The detection
limit of trypsin (1 ng/mL or 42 pM) is markedly more sensitive
than those reported for other biosensors, namely, 25 nM and
CONCLUSIONS
We describe herein an original and promising concept of
protease detection based on the suppression of a biosensor
response by steric hindrances and its reactivation by enzymatic
digestion of the blocking membrane. It is expected that this
extremely sensitive biosensor will be helpful for the easy and rapid
monitoring of patient health status.
(25) Hsia, C. Y.; Ganshaw, G.; Paech, C.; Murray, C. J. Anal. Biochem. 1996,
242, 221-7.
(26) Hagen, H. E.; Klager, S. L.; McKerrow, J. H.; Ham, P. J. Anal. Biochem.
1997, 251, 121-2.
(27) Koritsas, V. M.; Atkinson, H. J. Anal. Biochem. 1995, 227, 22-6.
(28) Trivedi, V. D.; Raman, B.; Ramakrishna, T.; Rao, C. M. J. Biochem. Biophys.
Methods 1999, 40, 49-55.
6330 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006

ACKNOWLEDGMENT
French Ministry of Foreign Affairs for supporting her stay at
the laboratory of S.C. through a Chateaubriand Postdoctoral
Fellowship.
The authors gratefully acknowledge the Arc-en-ciel/Keshet
joint program of the France Ministry of Foreign Affairs and the
Israel Ministry of Science as well as the North Atlantic Treaty
Organization (NATO) for the Collaborative Lingake Grant Award
981086. The authors also thank Tioga Gulon for the organic
synthesis of the amphiphilic pyrrole monomer. R.E.I. thanks the
Received for review February 8, 2006. Accepted July 18,
2006.
AC060253W
Analytical Chemistry, Vol. 78, No. 18, September 15, 2006 6331