1. Can hydrogel contribute to metabolomics?
A.M. Moschovi1,3, S.N. Yannopoulos2, V. Dracopoulos3,4, M. Klapa 1
forth|iceht
1 Metabolic Engineering & Systems Biology Lab,
2 Advanced Amorphous Materials and Nanomaterials Lab,
3 Ionic Liquids & Molten Salts Lab, 4 Microscopy & X-ray Diffraction Lab
Foundation for Research & Technology-Hellas, Institute of Chemical Engineering Sciences (FORTH/ICE-HT), Patras, GREECE
References :
1. Bolisay et al. Biomaterials 27 (2006) 4165–4168
2. P. Parmpi et al., Biomaterials 25 , 2004, 1969–1973
Acknowledgments:
Metabolomics is the high-throughput bioanalytical platform for the simultaneous
quantification of the relative concentration of free small metabolites in biological
systems. Metabolomics is presently at its standardization phase, during which data
acquisition and analysis protocols need to be optimized regarding performance and
resolution.
Introduction
Gas Chromatography(GC)-Mass Spectrometry(MS)
metabolomics will remain an integral component of
the metabolomics laboratory, due to distinct
advantages over the other utilized techniques.
However, a significant issue of GC-MS metabolomics is that the majority of measured
metabolite peaks have yet to be identified. Moreover, even identifiable peaks
corresponding to molecules of similar chemical structure, thus of similar GC retention
times and MS fragmentation patterns, cannot be easily differentiated. The phenomenon
affects the quantification of sugars (e.g. glucose, galactose) and their pyranoses, sugar-
alcohols and sugar-phosphates, accurate quantification of which is of importance in
many applications, as key intermediates of primary metabolism. Finally, the significantly
higher concentration of a metabolite (most commonly glucose) could affect the
derivatization and quantification of other molecules, thus its selective filtering is
desirable.
Hydrogels are insoluble crosslinked polymer network structures composed of hydrophilic
co- or homo-polymers which exhibit the ability to absorb significant amount of water.
Molecular imprinting (MI) in hydrogels is a technique in which functional groups of the
hydrogel are allowed to form a network around a template molecule. After the removal of
the template molecule, cavities with specific recognition sites and size are generated for
the preferential binding of the target over similar molecules.1 These materials are
candidates for molecular recognition, drug delivery, highly specific catalysis, quantitative
analysis and nanofiltration in conjunction with chromatographic techniques. We propose
to combine the recognition capabilities of MI polymer hydrogels with GC-MS
metabolomics to increase the resolution of the metabolic profiles. Initially, we will use a
MI hydrogel based on a crosslinked poly(allylamine) PAA∙HCl polymer with and/or
without D-glucose-6-phosphate imprinting, which has been reported as selective for
glucose over fructose1. Preliminary results on gelation dynamics and structure for the
bulk polymer have indicated their potential contribution to the monosaccharide
separation capabilities of the MI hydrogel.
Molecular Imprinting
Hydrogels are insoluble crosslinked polymer network
structures composed of hydrophilic polymers which
exhibit the ability to absorb significant amount of water.
Molecular imprinting
involves the formation of a complex between a
functional monomer and a template molecule
(poly-sacharides, viruses, proteins )1 with specific
chemical structure and functionality
(shape/ size/ functional groups).
Trends in Biotechnology, December 2010, vol 28, No12
After the template is
removed, the
product is a hetero-
polymer matrix with
specific recognition
capacities.
Monomer NaOH Crosslinker Template
200 100 26 3
Glucose specific HG
The binding capacity and
selectivity of molecular
imprinted Poly(allylamine)
hydrochloride PAA·HCl
polymer with the following
stoichiometry has been tested
in glucose and fructose
solutions.2,3
The presence of the template
during the synthesis procedure
resulted in the formation of
cavities with specific properties
.
The PAA·HCl pore before and after removing the template.
%
template
Glucose
binding(g/g)
Fructose
binding(g/g)
GPS-Na
0.50 0.58±0.02 0
1.00 0.54±0.03 0
1.5 0.50±0.01 0
No imprint 0.20±0.01 0
GPS-Ba 1.5 0,593±0.003 0.110±0.019
No
template
0.139±0.015 0.105±0.003
GPS-Ba 1.5 0.601±0.032 0.84±0.02
No
template
0.132±0.02 0.079±0.011
Non imprinted hydrogels exhibited
glucose selectivity and binding capacity
lower than the imprinted ones.
ConceptSome biological samples such as blood serum
and leaf extracts need special treatment due to
poor resolution in accurate quantification of
molecules having similar chemical structure and
the high concentration of some metabolites
(mainly glucose) which affects the derivatization
and quantification of other metabolites.
20 22 24 26 28
0
300000
600000
Intenisty
(counts)
t (min)
The latter problem can be
bypassed by selectively
filtrating the sample from the
metabolite in excess, before
the derivatization process in a
pre-column step, or by
separating metabolites with
similar structure using an
imprinted hydrogel prepared
with the target molecule as
the template.
Imprinted
Non- imprinted
Different kind of molecules(D-fructose, D-glucose, L-
glucose and D-gluconamide) have been tested as targets
for glucose-imprinted PAA·HCl hydrogel compared to the
non-imprinted polymer binding capabilities.4
It has been shown that the binding capacity of hydrogel was
not significantly enhanced by the imprinting.
Template D-Fructose D-glucose L-glucose D-gluconamide
none 0.308±0.019 0.649±0.038 0.548±0.045 0.795±0.026
Thus, we need to examine the role of the bulk in the binding capacities of the imprinted hydrogel for different targets
and investigate the reproducibility of the hydrogel synthesis process and binding capabilities for specific targets within
complex biological samples.
Previous Work
Gel synthesis
As prepared
SEM
XRD
DLS
0 20 40 60 80 100 120
3,5
4,0
4,5
5,0
5,5
6,0
6,5
7,0
7,5
RH
time(min)
10
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
0,0
0,3
0,6
0,9
g
(2)
(t)-1
time (msec)
1min
18min
45min
65min
71min
74min
76min
90min
120min
10
-3
10
-2
10
-1
10
0
10
1
10
2
10
3
10
4
10
5
76min
74min
a.u.
t(msec)
120min
90min
71min
65min
45min
18min
1min
At the beginning of the
synthesis, three different
processes take place,
attributed to the monomers.
During the gelation at 60-
70min, a new process
appears corresponding to
aggregates and clusters
formation.
Later on the gelation, a new
process appears attributed
to the network formed in the
structure.
According to FTIR- ATR
spectra vibrational changes
are observed at 80-90min,
after the crosslinker was
added.
Conclusions
I. Gelation begins after 60-65 minutes at static
conditions at 25oC; process is highly reproducible.
II. NaCl crystals are formed from the neutralization
of the HCl groups of the monomer with the
NaOH.
III. After washing, NaCl crystals are totally removed
and pores are formed in the bulk of the HG.
IV. After the washing treatment, a small amount of
the template GPS-Ba is trapped in the porosity of
the HG.
V. Non imprinted HG are expected to exhibit binding
capabilities for small metabolites due to the
porosity formed by the NaCl crystals.
Future work
• Sugar binding capacity and selectivity of
imprinted and non-imprinted hydrogels will be
studied in standard composition & complex
biological mixtures (eg blood serum, leaf extracts)
• Physicochemical properties, structure and binding
capacities of imprinted hydrogels will be studied
with Dynamic Light Scattering, Infrared
Spectroscopy, GC-MS profiling.
Dynamic Light Scattering
Infrared Spectroscopy
Binding test
The binding properties of the bulk of the hydrogel were
investigated with glucose, fructose and ribitol aqueous solutions
and their mixtures.
The concentration of the binding solution was 50mg/ml.
Samples were pepared for the GC-MS measurements according
to Ref. 5
•Dissolution of the polymer in H2O (25% w/v)
•Addition of the template
•Neutralization of the amine groups with NaOH
•Addition of the crosslinker EPI
•Gelation
•Washing with NaOH to remove unreacted reagents
•Removal of the NaOH excess with H2O
•Dry at 50oC
No selectivity was observed for the template-free hydrogel
3. Wizeman W.J. et al. Biomaterials 22, 2001, 1485-1491
4. Fazal et al. Bioorganic & Medicinal Chemistry Letters 17, 2007, 235-238
5. Kanani et al. J of Chromatography B, 871(2008) 191-201 National Strategic Reference Framework
FUNDING:
Template GPS-Ba washed(NaOH/H2O)Template free /washed (H2O) Template free /washed(NaOH/H2O)
Template free
As prepared Dried
Imprinted
As prepared Washed
NaCl crystals and template
molecules are removed after
washing treatment for imprinted
and template free hydrogels.
200nm
200nm
2μm10μm20μm
2μm
100μm
2μm
Template and NaCl
removal enable the
formation of porosity
in the bulk