A Novel N-Substituted Pyrrole Based Surface Modification for Biosensing

Article abstract of DOI:10.1002/pssa.201800030

Immobilization of proteins, mostly antigens, antibodies or enzymes on a solid surface, is one of the most important steps in the development of every biosensor. Different chemical methods as well as site-directed strategies have been developed in order to obtain highest functionality and stability for the sensoric device. In the present work, a novel method for the immobilization of biomolecules on a surface plasmon resonance (SPR) device is described. New N-substituted pyrroles are synthesized and deposited on a gold surface using electrochemical polymerization. To evaluate the established four different surfaces, the binding behavior between a fusion protein consisting of the lectin concanavalin A (ConA) as well as streptavidin (Sav) binding domains and the sugar mannan, which has been chemically coupled to the poly-pyrrole (PPy) surface is investigated. A pyrrole-N-C₁₆/pyrrole co-polymer surface as well as a pyrrol-1-yl hexanoic acid/pyrrole (PHCP) surface give best results.

Full text of DOI:10.1002/pssa.201800030

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ORIGINAL PAPER
N-Substituted Pyrroles
www.pss-a.com
A Novel N-Substituted Pyrrole Based Surface
Modification for Biosensing
Fabian Rüger, Jens Schäfer, Udo Bakowski, Michael Keusgen, and Doru Vornicescu*
In the present work, a novel method for
Immobilization of proteins, mostly antigens, antibodies or enzymes on a solid
surface, is one of the most important steps in the development of every
biosensor. Different chemical methods as well as site-directed strategies have
been developed in order to obtain highest functionality and stability for the
sensoric device. In the present work, a novel method for the immobilization of
biomolecules on a surface plasmon resonance (SPR) device is described. New
N-substituted pyrroles are synthesized and deposited on a gold surface using
electrochemical polymerization. To evaluate the established four different
surfaces, the binding behavior between a fusion protein consisting of the lectin
concanavalin A (ConA) as well as streptavidin (Sav) binding domains and the
sugar mannan, which has been chemically coupled to the poly-pyrrole (PPy)
surface is investigated. A pyrrole-N-C /pyrrole co-polymer surface as well as a
surface modification as a platform technol-
ogy for further immobilization of biomo-
lecules on Surface Plasmon Resonance
(
SPR) gold layers is described. In order to
achieve maximum versatility, simple N-
substituted pyrrole (NSP) building blocks
were designed bearing functional groups
like carboxylic acids. The obtained pyrrole/
NSP co-polymers should be polymerized
via electro-polymerization. After the depo-
sition, established assays like sugar-lectin
binding assays should be introduced in
order to test the obtained SPR surfaces. The
utilization of the lectin-sugar pairs, com-
bined with the Sav-biotin strategy, will take
a central role in the here presented
1
6
pyrrol-1-yl hexanoic acid/pyrrole (PHCP) surface give best results.
[3,4]
experimental layout.
A
recently
designed fusion protein consisting of two
complementary binding sites will be used
1
. Introduction
for functional testing of the obtained
surfaces (Figure 1). The first binding domain of the fusion protein
is concanavalin A (ConA), a lectin derived from Canavalia ensiformis,
and the second one is streptavidin (Sav), a protein obtained from
Streptomyces avidinii. ConA binds specifically to polysaccharides like
mannan and dextran. The binding between ConA and polysacchar-
idescanbecleavedbytheadditionofmethylα-D-mannopyranosideas
Immobilization of antigens, antibodies or enzymes on a solid
surface is an important step in the development of protein-based
[
1]
biosensor. A rather convenient method is the immobilization
of streptavidin (Sav), which can bind to biotinylated proteins by
[
2]
self-organization. This strategy is rather convenient because
Sav is stable under various chemical conditions and can be
immobilized chemically or by adsorption. Also, proteins can be
biotinylated under smooth conditions.
[5,6]
a 10% solution. This allows a smooth regeneration of the sensor
surface.Asalreadystatedabove,theSavbindingdomaincanbebound
to biotinylated proteins, DNA, and further biological recognition
First attempts to immobilize biomolecules on solid surfaces
were undertaken with aldehydes to achieve a certain level of
cross linking. Epoxides and active esters could be used in the
same manner. With these methods high surface loadings could
be achieved. As a disadvantage of these strategies, most of the
proteins will be inactivated. Therefore, smoother methods are
required.
[7]
elements. In contrast to the ConA-mannan, the Sav-biotin interface
is very stable and can be only cleaved under harsh conditions.
2. Experimental Section
Allusedchemicals wereofp.agrade orthehighestavailablepurity.
They were purchased from Sigma-Aldrich Chemie GmbH
(
Mu ̈n chen, Germany), Tokyo Chemical Industry Co. Ltd. (Tokyo,
Japan), Carl Roth GmbH & Co. KG (Karlsruhe, Germany) and
CANDOR Bioscience GmbH (Wangen, Germany).
F. Rüger, Prof. M. Keusgen, Dr. D. Vornicescu
Institute of Pharmaceutical Chemistry
Philipps-Universität Marburg
Marbacher Weg 6, 35032 Marburg, Germany
E-mail: doru.vornicescu@staff.uni-marburg.de
2
.1. Synthesis of Different Pyrrole Monomers
Dr. J. Schäfer, Prof. U. Bakowski
Department of Pharmaceutics and Biopharmaceutics
Philipps-Universität Marburg
The amine compound (18.0 mmol), together with sodium acetate
(
(
18.0 mmol, 1.476 g, 1eq) and tetraheptyl-ammoniumbromide
50 mg, ꢀ1 mol.%) are added to a mixture of water (20 mL) and
Robert-Koch-Straße 4, 35037 Marburg, Germany
acetic acid (99%, 12.5 mL). After addition of toluene (25 mL), the
two-phasic mixture was brought to a mild reflux and kept there
for 5 min. 2,5-Dimethoxy-tetrahydrofurane (18.9 mmol, 2.498 g,
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/pssa.201800030.
DOI: 10.1002/pssa.201800030
2
.449mL, 1.05eq) was added within 15 min and the reaction
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acetone for 5 min, washed with H O followed
by treatment with 6 M NaOH for 2 min.
The so pre-washed prisms were incubated
in a solution of 0.1 M KOH þH
2 2
O (30%)
ꢂ
(
ratio 1:1) for 30 min at 70–80 C and washed
2
with H O followed by acetonitrile. The freshly
cleaned prisms were used as anode during
electro-polymerization, the cathode was an
identical gold coated prism. The distance
between the electrodes was kept at 15 mm.
Several different mechanisms were postu-
lated for the formation of poly pyrroles on the
surface of the anode. One of the most widely
accepted proposals is given in Figure 3 and
[
11]
described inamodified form by Gourlay et al.
Figure 1. Schematic cross section through the layers which are subject of this investigation.
mixture was heated under reflux for further 6 h. After cooling to
room temperature and separation of the organic toluene phase,
the aqueous layer was extracted with toluene (two times with
Toimprove thequality ofthelayers,co-polymers
of plain pyrrole and NSP were generated
Figure 4). This procedure led to relatively even and stable poly-
pyrrole films. The following composition was used for the two-
electrode cell: 0.1 M tBu NBF , N-substituted pyrrole (0.05 M) and
(
4
4
5
0 mL). The pooled organic phases were washed with a small
pyrrole (0.05 M) in MeCN (99.5%).
amount of water (three times with 20 mL) and dried over
anhydrous sodium sulphate.
Evaporation of the solvent under reduced pressure, followed
by column chromatography gave the desired products in yields
between 67 and 95%. Stationary phase: silica gel 60 0.04–
The voltage was kept at 0.9 V using a laboratory voltage source
during deposition, the current was monitored until a stable value
was achieved. The exposition time depended on the used
pyrroles and took between 5 and 20 min. After removal of the
chip from the cell, it was rinsed with acetonitrile, EtOH and
0
.063 mm, mobile phase: cyclohexane ꢁ ethyl acetate mixtures:
water to remove traces of monomers and tBu
steps with ethyl acetate, methanol and H O followed by adjacent
drying at room temperature until further use.
4 4
NBF . Washing
For pyrrol-N-C16 and pyrrol-N-C 35:1; 4-(1H-pyrrol-1-yl) phenol
18
2
1
0:1 and for 6-(1H-pyrrol-1-yl) hexanoic acid 5:1.
All N-substituted pyrroles used in this work were synthesized
[8–10]
using a modified Clauson Kaas reaction (Figure 2).
2
.3. SPR Measurements
2
.2. Production of the Poly Pyrrole (PPy) Coated SPR Gold
After preparation of the gold coated glass prisms as described above,
SPR measurements were performed in order to test through the
binding behavior of the fusion protein ConA-Sav. The main focus
was on which PPy surface supports the binding of analyte to the
system best. The SPR method allows observation in real time of the
Surfaces by Electro-Polymerization
SPR gold (50 nm) covered prisms, on which the polymer layer
should be deposited, were cleaned by the following procedure
prior to electro-polymerization: The prisms were rinsed with
Figure 2. Clauson Kaas reaction yielding different N-substituted pyrroles (NSP’s).
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Figure 3. General scheme of electro-polymerization of pyrrole building blocks.
binding process between the ConA-Sav fusion protein and the
2.4. AFM Measurements
mannan coated chip free of labelling or other kind modifications of
[
12]
the binding partners1. An 8-channel cuvette based device from the
To evaluate the surface morphology of the pyrrole coated surface,
atomic force microscopy (AFM) measurements were performed at
different stages of coating. Chips were carefully taken out of the SPR
company Plasmonic Biosensor in Wallenfels Germany (Plasmonic
SOne ) served as device for the measurements.
1
The SPR device detects changes in refractive index occurring
within the few hundred nanometers above the gold surface (called
devices, rinsed with water and dried. Measurements were done with
TM
a vibration dampened JPK Nano Wizard I
(JPK Instruments,
TM
‘evanescent field’) when biomolecules in solution bind to their
Berlin, Germany) equipped with a TopViewOptic in an acoustic
enclosure, AFM cantilevers HQNSC14/AlBS (Micromasch,
Estonia) with a resonance frequency of 160 kHz and a nominal
force constant of 5N/m were chosen for all the measurements. To
avoid damaging the surfaces, intermittent contact (air) mode was
chosen. The scan speed was proportional to the scan size.
partners which already had been immobilized onto the sensorsurface
in a preliminary step. The response signal is measured in pixel
corresponding to the reflection angle shift and plotted against time on
[8]
the display. It describes the occurring interactions in real-time.
After recording the baseline with PBS buffer (30 mmol L
phosphate, 120 mmol L NaCl; pH 7.3), mannan (1.0 mg mL
ꢁ
ꢁ1
1
ꢁ
1
)
The determination of the RMS (root mean square) roughness
Rq of the surface was done with JPK Data Processing Software of
was added on the PPy coated SPR chip by use of mix mode. A
2
blocking step was performed by adding of Smart Blocking
the height images (trace). The variable zi defines the vertical
1
Solution (CANDOR) followed by five washing steps with the
same buffer covering the vacancies and removing the
unbounded material. According to the measurement setup,
the addition of ConA-Sav fusion protein (400 μg mL dissolved 3. Results and Discussion
in PBS buffer) onto the chip surface, followed by washing with
deviation to an averaged ideal surface.
ꢁ
1
3
.1. Synthesis of Different Pyrrole Monomers
PBS buffer was performed. The production and characterization
[12]
of the ConA-Sav is described by Dassinger et al.
Different N-substituted pyrroles could be successfully synthe-
sized from different rather inexpensive starting materials using
To regenerate the surface and remove the specifically bound
fusion protein, methyl α-D-mannopyranoside 10% (w/v) diluted
in PBS buffer was added. All adding/washing steps were
2
,5-dimethoxy-tetrahydrofurane and different amino com-
ꢂ
pounds. The obtained yields were between 67 and 95% (Table 1).
The standard reaction time was set to 6 h, but also shorter times
performed at 22.0 C by using the ‘mix-mode’ setting. Display
and processing of the data were performed by using of Origin
1
Pro 8.0 software (Origin Lab, USA).
Table 1. Yields of some NSP’s synthesized using Clauson Kaas
reaction.
Type
Pyrrole structure
Yield [%]
95
a)
A
b)
B
67
c)
C
73
82
d)
D
Figure 4. Potential structure of a pyrrole/6-(1H-pyrrol-1-yl) hexanoic acid
co-polymer (PHCP).
a)
b)
c)
d)
For analytical data, see supporting information: S1; S4; S2; S3.
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Figure 7. SPR-based pyrrole-N-C16 pyrrole co-polymer studies: 1.
Figure 5. SPR-based mannan-ConA-Sav binding on PHCP co-polymer
ꢁ
1
ꢁ1
addition of mannan (1 mg mL ); 2. blocking step with Candor; 3.
studies: 1. addition of mannan (1 mg mL ); 2. blocking step with Candor;
ꢁ
1
ꢁ
1
addition of ConA-Sav fusion protein (300 μg mL ).
3
. addition of ConA-Sav fusion protein (300 μg mL ).
successfully adapted on pyrrole/6-(1H-pyrrol-1-yl) hexanoic acid
co-polymer (PHCP) (Table 1 (Type A)), pyrrole- N-C16 pyrrole co-
polymer (Table 1 (Type C)) as well as pyrrole/4-(1H-pyrrol-1-yl)
phenol pyrrole co-polymer (Table 1 (Type D)) surfaces. Because no
significantdifferenceinthestructureasalsohowitbehavesduring
the SPR measurements, the results for pyrrole- N-C18 pyrrole co-
polymer (Table 1 (Type B)) were not shown.
seem to be possible in the case of aliphatic amines with low steric
hindrance. Disappearance of the amine compound spot (thin
layer chromatography) is a good endpoint indication for the
reaction. The obtained NSP’s showed purities of above 90% prior
to column chromatography and could be used directly for
electro-polymerization without further purification.
3
.2. Production of the Poly pyrrole (PPy) Coated SPR Gold
3
.3. SPR Measurements
Surfaces by Electro-Polymerization
The evaluation of the established PPy coated SPR surfaces was
done by a functional test according to Figure 1. Firstly, different
PPy-films were treated with a mannan solution, followed by the
addition of the ConA-Sav fusion protein (Figures 5–8). After
coating with mannan, a blocking step with Candor-buffer has
been introduced. This step is necessary to guarantee that after
After cleaning the prisms, the gold surface of the SPR sensor was
modified by electro polymerising a 1:1 mixture of NSP’s and
pyrrole (U: 0, 9V, t: 15 min.). This procedure leads to the formation
of a visible, slightly grey, thin layer on the gold surface. Tests using
an established mannan/ConA-Sav fusion protein assay were
Figure 6. SPR-based covalently coupled mannan-ConA-Sav binding on
PHCP co-polymer studies: 1. surface activation with EDC; 2. addition of
ꢁ
1
mannan (1 mg ml ); 3. blocking step with Candor; 4. addition of ConA-
Figure 8. SPR-based 4-(1H-pyrrol-1-yl)phenol pyrrole co-polymer studies:
ꢁ1
ꢁ1
©
Sav fusion protein. (300 μg ml ); 5. regeneration with 10% methyl α-D-
mannopyranoside.
1. addition of mannan (1 mg mL ); 2. blocking step with Candor ; 3.
ꢁ
1
addition of ConA-Sav fusion protein (300 μg mL ).
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Table 2. SPR signal increase after addition of the fusion protein to
different mannan treated PPy surfaces as given in Table 1.
addition of the fusion protein only a SPR-signal for a specific
binding is visible.
Figure 5 displays the results for immobilization of the PHCP
co-polymer followed by mannan coating. Interestingly, the
loading seems to be rather high and a specific binding event
could be monitored after addition of ConA-Sav. This led to the
assumption that there is a chance of hydrogen bridge or ionic
interactions between the PHCP and the mannan probably
accompanied by van der Waal’s interactions. The formed layer
seems to be stable.
NSP used
Signal increase (pixel)
A
100
170
233
165
153
A þ EDC
B
C
D
In the next step, The carboxylic acid functionality of the PHCP
co-polymer was chemically activated by 1-ethyl-3-[3-dimethyla-
minopropyl] carbodiimide hydrochloride (EDC) and the mannan
has been added after activation. In the experiment, the mannan
should be covalently bound to the PHCP co-polymer.
The corresponding SPR measurement including on-chip
activation is shown in Figure 6. The binding of the fusion protein
to the covalently immobilized polysaccharide gave reason for the
assumption that the chemical reaction has been successful,
leading to a thinner more evenly ordered layer of mannan.
Moreover, the obtained SPR signals were much higher as in the
case of the experiment shown in Figure 5.
Table 3. RMS roughness (root-mean-squared), Ra roughness and
scanned area of the three surfaces B, C, and D as given in Figure 9.
Type of surface
RMS [nm]
Ra [nm]
Scanned area [μm]
B
C
D
2.560
4.237
7.323
1.996
3.014
6.099
1 ꢃ 1
2.5 ꢃ 2.5
2.5 ꢃ 2.5
As shown in Figure 6, also a regeneration step with methyl α-
D-mannopyranoside has been performed.
Regeneration is possible by applying a sugar
solution whose binding to the fusion protein is
thermodynamically favored compared to man-
[13,14]
nan.
A relative high regeneration rate
(
43%) of the mannan surface could be
achieved. A complete regeneration could not
be achieved because of steric hindrance. The
methyl α-D-mannopyranoside must reach the
binding site of the ConA to allow cleavage of
the mannan-ConA binding. This appears to be
difficult in a heterogeneous system deposited
on a solid surface.
As a further experiment, ConA was immo-
bilized on pyrrole-N-C16 co-polymer surface by
unspecific hydrophobic interactions (Figure 7).
Interestingly, obtained results were nearly
comparable to those given in Figure 6. The
advantage of this method is, that it is very
simple and should work for nearly all types of
proteins. Also in this case, liberation of the
ConA-Sav fusion protein from the chip surface
by washing with a methyl α-D-mannopyrano-
side solution was performed, but only insuffi-
cient regeneration could be achieved (data not
shown). As explained above, steric hindrance
could be the reason for the low regeneration
rate. Modification of the gold chip surface with
a 4-(1H-pyrrol-1-yl)phenol pyrrole co-polymer
shows high binding performance towards the
fusion protein, although not as unpolar and
without a long alkyl chain, the surface seems to
bind mannan in a similar fashion to pyrrole-N-
C16 and pyrrole-N-C18 co-polymers (Figure 8).
Figure 9. AFM height images (cantilever HQ:NSC14AlBS) of modified chips: a) overview of
pure gold surface without modification; b) detailed image of pure gold surface without
modification indicating smooth surface (roughness 2.56 nm); c) surface modified with pyrrole
and mannan; d) surface modified with pyrrol-N-C16 pyrrole co-polymer and mannan þ ConA- The substantially higher binding rate of ConA-
Sav (increased roughness of mannan and ConA-Sav clusters visible).
Sav could be explained with a higher ratio of
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“
exposed” mannan. The binding rates for the fusion protein on
applicable to many different proteins, allows immobilization by
self-organization, as well as a swift regeneration of the surface.
The final step of the entire assay as shown in Figure 1 would
be the immobilization of a biotinylated protein, typically a
biotinylated antibody capturing the analyte. This has been also
tested in these studies, but until now only with little success
because of inactivation of the antibody. The reason for this
phenomenon is not clear in the moment. The reason can be
insufficient washing steps, inappropriate buffer systems or
denaturation of antibodies by contact with uncoated chip areas.
The solution for this problem is a main topic of ongoing
research.
different mannan coated PPy surfaces are shown in Table 2.
3
.4. AFM Measurements
After the SPR measurements, chips were carefully removed out
of the SPR device and analysed by AFM as depicted in Figure 8.
An area of 2.5 ꢃ 2.5 μm was observed.
In general, the surface showed a somewhat rough, but
uniform coating. Results of the roughness study are given in
Table 3. The surface after mannan coating showed structures of
about 50 nm with round or oval shapes. Due to the polymeric
structure of mannan, these shapes do fit rather well to its
polysaccharide structure.
Supporting Information
After loading with the fusion protein (43 kDa), the surface
looks quite similar. As it is visible in Figure 8(d), a fine supra-
structure can be observed. This could be caused by the ConA-Sav
fusion protein. Generally, the loading seems to be rather
homogeneous. However, because of the roughness of the
mannan-loaded surface (Figure 8(c)), the deposition step needs
further improvement. For example, by optimization of the
concentration of the mannan solution during the loading step,
additionally the mix-mode of the SPR device could be modified.
Supporting Information is available from the Wiley Online Library or from
the author.
Conflict of Interest
The authors declare no conflict of interest.
Keywords
4
. Conclusion
atomic force microscopy (AFM), electro polymerization, N-substituted
pyrroles, poly-pyrroles (PPy), surface plasmon resonance (SPR)
Four different PPy surfaces could be successfully established.
Different gold SPR chips, modified with the same PPy, showed very
similar binding characteristics. The so produced PPy films adhered
strongly to the gold layer and are stable within a broad pH range.
Widely used dextran surfaces show a negative charge above pH 3.5,
Received: January 15, 2018
Revised: March 26, 2017
Published online:
[15]
this could lead to unspecific interactions with proteins. By using
different NSPs the net charge of the layer can be controlled. Binding
inhibitorscovalently tothePPy couldeliminate theriskofunspecific
interactions. The rough surface evaluated by AFM measurements
could be optimized towards a smoother surface by carefully
improving and modifying the electro-polymerization as well as
concentrations of the monomeric pyrroles in solution. Real time
monitoring of the electrochemical polymerization process with the
SPR technique is something that could be adopted for a complete
control of the whole process. The following immobilization steps
have some potential for optimization. Out of these surfaces, the
pyrrole-N-C16 co-polymer surface as well as the PHCP co-polymer
surface with and without chemical activation gave sufficient results
in a binding assay with the ConA-Sav fusion protein.
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[
4
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allows for regaining the analyte.
[
There are further variations of the method thinkable. A very
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2þ
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(S 5) which, after treatment with Ni or Co salts, is suitable
1
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