Polyacrylamide gels with selective recognition of the tetrameric molecular form [629244]

Polyacrylamide gels with selective recognition of the tetrameric molecular form
of human growth hormone
Rimantas Kublickas1*, Carsten Werner2, Brigitte Voit2
1) Kaunas University of Technology, Department of Food S cience and Technology,
Radvilenu pl. 19, LT -50254 Kaunas , Lithuania
2) Leibniz Institute of Polymer Research Dresden, Hohe Strasse 6, 01069 Dresden,
Germany

*Corresponding author, e -mail: [anonimizat] , Tel. +3706007754 5, Fax:
+370 373001 52.
Abstract . Networks of polyacrylamide were studied for the possibility of imprinting
of the oligomeric form of human growth hormone. The tetrameric molecular form of
human growth hormone was molecularly imprinted for the first time. The results
show that approximately 50 -70 % (w/w) of the templates (depending on
polymerization conditions) could be extracted from the molecularly imprinted
acrylamide polymers. The resulting “gel antibodies” against this form of human
growth hormone in the form of gra nules of polyacrylamide were compared with
granules of non -imprinted polymer. The selectivity of the artificial gel antibodies
was studied. Investigation of the binding to imprinted polymer of the template
hormone, other molecular forms of the hormone and other proteins shows the
selectivity of the developed artificial gel antibodies.

Keywords : polymer gels, molecularly imprinted polymers , human growth hormone
1. Introduction
Human growth hormone (hGH ) like many bioactive substances, is a heterogeneous
polypeptide [1]. This hormone plays an important role in somatic growth through its
effects on the metabolism of proteins, carbohydrates, and lipids. Growth hormone
with different biological activities occurs in the human body as several structural
isoforms. The main molecular form is the so -called monomeric hGH with a
molecular weight of 22 kDa, consisting of 191 amino acids. However, there are also
molecular forms (isoforms) consisting of two or more ( up to five) monomeric hGH
units and it is very difficult to separate the usually obtained mixtures. There are no

natural antibodies enough specific for recognizing these differently sized forms of
the hormone.
Molecular imprinting is a recent promising tec hnique for the fabrication of
biomimetic polymeric recognition sites with a selective affinity for a target molecule
(such as a drug, protein or biomacromolecule), which is attracting rapidly increasing
interest and many potential applications in the pharm aceutical and biotechnological
field [2-8]. Imprinted materials may constitute an alternative to natural recognition
elements. In molecular imprinting, recognition sites are tailor -made in situ by self –
assembly of suitable monomers and the templates follow ed by copolymerization with
cross -linkers to form a polymer network in the presence of the template (s. Scheme
1). The templates are subsequently washed from the molecularly imprinted polymer
(MIP), leaving recognition sites complementary in the positionin g of functional
groups and in shape. The method creates materials resembling the binding sites of
receptors and antibodies.

Scheme 1 . General scheme of molecular imprinting technology (T stands for
template)
Therefore the imprinting of macromolecules is a challenging research field [7].
Conventional molecular imprinting technology allows the synthesis in organic
solvents of MIPs selective toward relatively low molecular weight compounds.
Proteins mainly work in aqueous solutions in biological systems and many
researchers have attempted to find the optimal conditions in protein imprinting [8].
Synthesis in aqueous media of chemically and mechanically stable MIPs that can
recognize biomolecules such as peptides and proteins is still a great challenge [8].
Stellan Hjertén for the first time successfully synthesized highly selective artificial
gel antibodies specific against proteins using acrylamide and methylene
bisacrylamide as monomers and aqueous buffer as a solvent by the molecular
imprinting technique [9 ]. The method was to prepare the gel granules in the presence
of a protein of interest [9,10], where the protein was mixed and polymerized with a

monomer solution (a buffer containing acrylamide, methylene bisacrylamide,
N,N,N’,N’-tetramethylethylenediamin e and ammonium persulfate). It was shown that
human growth hormone and some other proteins can also be adsorbed specifically on
crosslinked polyacrylamide gel particles prepared by Hjerten’s method using
entrapment and molecular imprinting [10].
Some human peptide hormones and other larger peptides were successfully
molecularly imprinted in aqueous media and show sufficient recognition (and
selectivity) with respect to the molecularly imprinted polymer [4, 11 -15]. Here, the
monomeric hGH and dimeric hGH wer e used for the entrapment and recognition.
Typically, polyacrylamide gels are selective because the size and shape of the cavity
originally occupied by the substrate is retained by high crosslinking density, but in
case of Hjertén ’s method this does not ap ply [10].
Many experiments with protein templates and various functional monomers show
that the incorporation of functional groups with either negative or positive charge
into the polyacrylamide gel leads also to high selectivity for the recognition of th e
protein [12 -15]. In our previous work [13] for the first time it was shown that
monomeric and dimeric hGH can successfully be molecularly imprinted into
polyacrylamide hydrogels with ionic functional groups (and dimeric hGH was
molecularly imprinted for the first time). Significant imprinting efficiency was
detected using ultraviolet absorbance. The selectivity study shows excellent results
for selective binding when the analyte and the template had been identical and a very
low binding of other proteins . Natural antibodies usually only reflect the
concentration of hGH and do not discriminate the structural differences between
hGH dimer and monomer [1, 15].
Recently Ghasemzadeh et al. [14, 15] used a molecular imprinting approach to
synthesize artificial gel antibodies against hGH dimer and monomer. The gel
antibodies could specifically adsorb these growth hormone forms used for their
preparation. It was concluded that these two sets of artificial gel antibodies might be
useful for discriminating between d imeric and monomeric hGH in clinical samples.
So far, no investigation of the molecular imprinting of bigger (oligomeric) molecular
forms of hGH exists.
The aim of this work was the synthesis of molecularly imprinted polyacrylamide
hydrogels with or witho ut ionizable groups in the presence of the tetrameric form of

hGH. Furthermore, the binding of the template hormone, other molecular forms of
the hormone and other proteins to the imprinted polymer was investigated.
Such molecularly imprinted polymers have possible future applications in the fields
of quantitative sorbent assays (as artificial antibodies) or chromatography.
2. Experimental
2.1. Materials
The monomeric human growth hormone with the molecular weight of 22 kDa, dimeric
and oligomeric (three -five globular units) molecular forms of hGH were prepared and
characterized as described earlier [16]. Bovine serum albumine, ≥99% (Sigma),
human serum albumin, ~99% (Sigma), lysozyme (from chicken egg white), ~95%
(Sigma), acrylamide, ≥99%, (Sigma), 2 -(dimethylamino)ethyl methacrylate, ≥98%
(Aldrich), methacrylic acid, ≥99% (Aldrich), N,N’-methylene bisacrylamide, ≥99%,
(Merck), N,N,N’,N’-tetramethylethylenediamine (TEMED) (BioRad), potassium
persulfate, 99.99% (Aldrich), sodium chloride, ≥99.5% (Sigma -Aldrich), 1 M NaOH
solution (Sigma) were used as obtained.
Deionized water was produced by a Millipore water system. PBS buffer solution (pH
6.2) was prepared from 18.4 ml disodium hydrogen phosphate 1/15 M solution
(Merck) and 81.6 ml potassium dihydrogen p hosphate 1/15 M solution (Merck).
2.2. Instruments
Ultraviolet absorbance (by 280 nm) measurements were performed using a UV -VIS
spectrophotometer Genesys 10UV. Every measurement was repeated three times.
2.3. Preparation of molecularly imprinted polymers by Hjertén ’s method
(MIP -H)
In a typical polymer synthesis PBS buffer solution (1/15 M, pH 6.2) was used.
Acrylamide 0.288 g, various amount of N,N’-methylene bisacrylamide ( MBA ) (was
varied from 0.006 g up to 0.012 g) and hGH 0.015 g (tetrameric form of hGH or
c.a. 1:1:1 mixture of trimeric, tetrameric and pentameric forms of hGH) were mixed
with 5 ml of PBS in a Schlenk tube at 25◦C and purged with nitrogen for 15 min.
After purging, potassium persulfate [5% (w/w) aqueous solution] 0.05 ml and
TEMED [5% (w/w) aqueous solution] 0.05 ml (3.23. 10 –5 mol) was added to the
solution to start the polymerization. After the polymerization proceeded for 30 min,

the solution mixture was wet -sieved through a 100 -mesh sieve. The result ant gels
(MIP -H-tetra in case of tetrameric form of hGH or MIP -H-mix in case of mixture of
trimeric, tetrameric and pentameric forms of hGH) were washed with 200 ml
deionized water three times, 200 ml 10% acetic acid and 2% SDS solution three
times, 200 ml deionized water three times. UV spectra for the wash liquid were taken
to determine the amount of hGH extracted. The gels were lyophilized and sieved to
obtain particles of 100 -150 μm size which were stored at ambient temperature.
2.4. Preparation of mole cularly imprinted polymers with ionized functional
groups (MIP)
In a typical polymer synthesis, 2 -(dimethylamino)ethyl methacrylate 0.4824 ml
(0.00286 mol) and methacrylic acid 0.2428 ml (0.00286 mol) were mixed with 3.55
ml of PBS buffer solution (1/15 M, pH 6.2). The solution was adjusted to pH 6.2 by
1 M NaOH. Then, acrylamide 0.2035 g (0.00286 mol), various amount of N,N’-
methylene bisacrylamide ( MBA ) (was varied from 0.05 g up to 0.1 g) (0.0003245
mol up to 0.000649 mol) and hGH form 0.06 g (2.727 10-6 mol) were added to the
solution in a Schlenk tube at 25 ◦C and purged with nitrogen for 15 min. The
concentration of hGH (tetrameric form of hGH or mixture (c.a. 1:1:1) of trimeric,
tetrameric and pentameric forms of hGH) in the solution was 1.29% (w/w). After
purging, potassium persulfate [1.5% (w/w) aqueous solution] 0.25 ml (1.39. 10-5 mol)
and TEMED [3.75% (w/w) aqueous solution] 0.1 ml (3.23. 10 –5 mol) was added to
the solution to start the polymerization. After the polymerization proceeded for 30
min, the solution mixture was wet -sieved through a 100 -mesh sieve. The resultant
gels (MIP -tetra in case of tetrameric form of hGH or MIP -mix in case of mixture of
trimeric, tetrameric and pentameric forms of hGH) were washed with 200 ml
deionized water thr ee times, 200 ml NaCl solution (0.1 M) three times and 200 ml
deionized water three times. UV spectra for the wash liquid were taken to determine
the amount of hGH extracted. The gels were lyophilized and sieved to obtain
particles of 100 -150 μm size which were stored at ambient temperature.

2.5. Investigation of molecularly imprinted polymers

After extraction of the template the hGH -tetramer -imprinted gel particles were added
to a solution in which a certain amount of hGH tetramer (50 % of the amount of

template removed from MIP) was dissolved. The amount of the lyophilized gel was
0.05 ± 0.001 g. The concentration of hGH (tetramer and other molecular forms of
hGH) or other test -proteins in test solution was 0.129 % (w/w). The liquid
supernatant was separated from the gel after the test tube was centrifuged at 1000
rpm during 5 min. All experiments were reproduced three times.
3. Results and discussion
3.1. Synthesis of the imprinted polymers
The imprinted functionalized p olyacrylamide hydrogels (MIP) were prepared by free
radical polymerization of acrylamide, 2 -(dimethylamino)ethyl methacrylate and
methacrylic acid (1:1:1 ratio) together with the crosslinker N,N’-methylene
bisacrylamide in buffer solution in the presence of the selected molecular form of the
hGH. In case of Hjertén ’s method (MIP -H) only acrylamide and crosslinker N,N’-
methylene bisacrylamide was used. A highly effective redox initiation system was
used to allow polymerization by both methods at the same room temperature for the
same short time (30 min.). The template is enough stable under these conditions [1].
3.2. The removal of the template
After the polymerization was completed, the template had to be removed by
intensive washing. The volume change in the MIP hydrogel is very dependent on the
ion strength of the solution [13]. To remove the template molecules from the MIP as
effectively as p ossible, the MIP particles were rinsed therefore by changing the ion
strength of the solution. The removal of the template from the imprinted particles
was confirmed by quantitative measurements of the UV absorbance at 280 nm of the
NaCl solution used for washing the hGH from the particles. A part of template
(approximately 40 -50 % in case of MIP -H-tetra and 30 -40 % in case of MIP -tetra)
could not be extracted from the gel and thus, was fixed to MIP (Table 1). Increasing
of intensity of washing procedure ca nnot solve this problem. As possible reasons for
that one can assume that some hGH molecules are entrapped so strongly in the cross –
linked hydrogel that it could not be removed. The second possible reason – some free
radicals attacked these hGH molecules l eading to covalent binding to the gel – can be
excluded because of previous experience with imprinting of monomeric and dimeric
hGH forms [13]. The amount of template which could not be extracted from the gel

(MIP -tetra) was significantly higher than in th e case of imprinting experiments of
monomeric and dimeric hGH (it was only 9 -10 %) [13] using the same
polymerization conditions. This can be explained by the complicated and not so rigid
structure of this hGH form.

Table 1. Efficiency of the template removal from the prepared MIPs

MIP Amount of
MBA in
polymerization
mixture, g From MIP
extracted
template in % Fixed to MIP
template in %
MIP-H-tetra
MIP-H-tetra
MIP-H-tetra
MIP-H-mix
MIP-H-mix
MIP-H-mix
MIP-tetra
MIP-tetra
MIP-tetra
MIP-mix
MIP-mix
MIP-mix 0.006 ± 0.0001
0.009 ± 0.0001
0.012 ± 0.0001
0.006 ± 0.0001
0.009 ± 0.0001
0.012 ± 0.0001
0.05 ± 0.0001
0.075 ± 0.0001
0.1 ± 0.0001
0.05 ± 0.0001
0.075 ± 0.0001
0.1 ± 0.0001 60.2 ± 0.5
59.1 ± 0.4
50.1 ± 0.5
24.2 ± 0.3
24.9 ± 0.3
23.9 ± 0.4
70.2 ± 0.6
69.5 ± 0.5
60.1 ± 0.5
30.6 ± 0.5
29.9± 0.4
28.9± 0.5 39.8 ± 0.5
40.9 ± 0.4
59.9 ± 0.5
75.8 ± 0.3
75.1 ± 0.3
76.1 ± 0.4
29.8 ± 0.6
30.5 ± 0.5
39.9 ± 0.5
69.4 ± 0.5
70.1 ± 0.4
71.1 ± 0.5

A big part of mixture -template (approximately 75 % in case of MIP -H-mix and 70 %
in case of MIP -mix) could not be extracted from the gel. MIPs prepared with the
mixture of three molecular forms of hGH as template cannot be considered as
potential artificia l antibodies against molecular forms of hGH. On the other hand,
these results (and the results obtained in our previous work [13]) can show us that
only one single molecular form of hGH can serve as possible template for molecular
imprinting.
3.3. Recognit ion of hGH templates
After extraction of the template the imprinted gel ( tetrameric hGH as template and
Hjerten’s method of imprinting ) particles were added to a solution in which a certain
amount (50 % of the amount of template removed from MIP) of test -hGH-form (or
its mixture of trimeric, tetrameric and pentameric hGH ) was dissolved. The amount
of the lyophilized gel was 0.05 g. The concentration of hGH in test solution was
0.129 % (w/w). The liquid supernatant was separated from the gel after the test t ube
was centrifuged at 1000 rpm during 5 min. The amount of hGH adsorbed onto the

gel particles was calculated by subtracting the quantity of hGH in the supernatant
from that in the original solution. The same experiment was carried out with MIP
with ioniz ed functional groups using the test -hGH form or its mixture.
Table 2. Specifically bound amount of hGH (B) on the MIPs prepared by Hjerten’s
method (the same test protein (or mixture) was used as the template)
MIP Amount of
MBA in
polymerization
mixture, g B in % after
2 h B in % after 18
h
MIP-H-tetra
MIP-H-tetra
MIP-H-tetra
MIP-H-mix
MIP-H-mix
MIP-H-mix 0.006 ± 0.0001
0.009 ± 0.0001
0.012 ± 0.0001
0.006 ± 0.0001
0.009 ± 0.0001
0.012 ± 0.0001 40.3 ± 0.5
49.1 ± 0.4
42.1 ± 0.5
5.2 ± 0.2
5.4 ± 0.2
5.0 ± 0.3 49.7 ± 0.5
54.9 ± 0.5
50.4 ± 0.5
5.8 ± 0.3
5.6 ± 0.2
5.4 ± 0.3

Non-imprinted polymers ( NIPs ) were synthesized the same way as the MIP -H-tetra
or MIP -tetra (by all three different concentrations of MBA in polymerization
mixture) and after polymerization the NIPs were treated exactly the same way as the
appropriate MIPs. The non -specific binding of hGH (tetrameric hGH or mixture of
three molecular forms of hGH) to the NIP particles was also measured and found to
be only 1.0 ± 0.1 % for polym erization by Hjerten’s method and 0.9 ± 0.1 % for
polymerization of MIPs with ionized functional groups (by all three different
concentrations of MBA in polymerization mixture).
Table 3 . Specifically bound amount of hGH (B) on the MIPs with ionized functi onal
groups
MIP Amount of
MBA in
polymerization
mixture, g B in % after
2 h B in % after 18
h
MIP-tetra
MIP-tetra
MIP-tetra
MIP-mix
MIP-mix
MIP-mix 0.05 ± 0.0001
0.075 ± 0.0001
0.1 ± 0.0001
0.05 ± 0.0001
0.075 ± 0.0001
0.1 ± 0.0001 38.4 ± 0.5
45.2 ± 0.4
40.2 ± 0.4
6.6 ± 0.4
5.9± 0.4
5.8± 0.4 43.6 ± 0.5
50.9 ± 0.4
46.4 ± 0.5
7.4 ± 0.4
7.1 ± 0.4
6.8 ± 0.3

Similar low non -specific binding of hGH was found in our previous experiments
with monomeric and dimeric forms of hGH and NIP with ionized functional groups
[13]. By subtracting the amount of hGH adsorbed onto the non -imprinted reference

gel particles from that adsorbed onto the hGH -imprinted polymer particles, the
amount of specifically bound hGH (B) was calculated and transferred to % from
original concentration values (Table 2 and Table 3).
All MIPs prepared with the mixture of three molecular forms of hGH as template
show very low value of specifically binding B. The MIPs prepared using single
molecular form of hGH (t etramer) show much higher B value. It shows that single
molecular form (as template) can give MIPs with good specific binding.
3.4. Selectivity of the imprinted polymers
The MIPs with best recognition of template protein was used for the investigation of
cross -reactivity of the MIPs with other hGH forms and other proteins.
To confirm the selectivity of the imprinted polymer, monomeric and dimeric
molecular forms of hGH and other proteins such as bovine serum albumin ( BSA ),
human serum albumin ( HSA ), or lysozyme ( LSZ) were dissolved in the solutions,
and the same experiments as those for the template hGH form were carried out using
the same concentration of the protein. Tabl e 4 represents the results of investigation
of the adsorption onto MIPs after 18 h in % from the original solution.
Table 4. Selectivity of the imprinted polymers MIP -H-tetra and MIP -tetra in
adsorption experiments using different analytes
Test protein Adsorption onto MIP after 18 h in
% from the original solution
hGH form Other protein MIP-H-tetra MIP- tetra
–
–
–
tetrameric
hGH
dimeric hGH
monomeric hGH BSA
HSA
LSZ
–
–
– 0.9  0.1
1.6  0.2
0.5  0.1
54.9 ± 0.5
5.5  0.1
3.9  0.1 1.1  0.1
1.4  0.1
0.6  0.1
50.9 ± 0.4
6.4 ± 0.1
5.1  0.1

The other proteins, however, show ed a very low tendency to bind to the MIPs with
values i n the range of 0.5 -1.6. Two of the weakly adsorbing proteins – BSA and HSA
– have approximately the same isoelectric point as hGH (pH 5.1 or 5.2). This can be
considered as a good proof for an effective imprinting mechanism and not just plain
adsorption capacity of the hydrogel s. The crossreactivity of the monomeric hGH (up
to 5.1  0.1) and dimeric hGH (up to 6.4  0.1) to the MIPs is enough low. These
MIPs can be considered as artificial antibodies against tetrameric form of hGH. Such
molecularly imprinted hydrogels can provid e selective recognition of this molecular
form of human growth hormone and, thus, solve problems related to the absence of
the natural antibodies that can specifically recognize this molecular form of hGH.
Furthermore, the proposed imprinted polymers have general advantages as compared
to immobilized natural antibodies like robustness and longer lifetime.
4. Conclusions
The tetrameric molecular form of the human growth hormone was molecularly
imprinted into polyacrylamide hydrogels with or without ionic fun ctional groups. A
part of template (approximately 40 -50 % in case of hydrogel without ionic functional
groups and 30 -40 % in case of other hydrogel) could not be extracted from the gel.
The hydrogels with best recognition of the template showed selective b inding when
the analyte and the template had been identical and a very low binding of other
proteins. The monomeric and dimeric molecular forms of hGH showed low cross –
reactivity within the imprinted hydrogels. Thus, the reported polyacrylamide
hydrogels c an be considered as artificial antibodies against the tetrameric form of the
hormone.

Acknowledgements . The research was supported in part by Alexander von
Humboldt -Stiftung.

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