molecular imprinting of bovine serum albumin on...
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e-mail: [email protected]
Molecular Imprinting of Bovine Serum Albumin on
Polypyrrolidone Magnetic Microparticles for Selective Recognition
of Proteins
Zlatan Denchev
i3N - Institute for Polymers and Composites, University of Minho,
Guimaraes, Portugal
SUNNY-ESF, Department of Chemistry Seminar, Sept. 28, 2017
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Outline2
1. Molecular Imprinting – definition, components, trends
2. Microencapsulation by anionic polymerization of lactams
3. Microencapsulation + Molecular Imprinting of BSA
4. Results and Discussion
5. Conclusions
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Molecular Imprinting Technology (MIT)3
MIT - making molecular locks to match specific molecular keys, i.e., construction of specific recognitions sitesin synthetic polymers.MIT employs templates, functional monomers, polymerization processes, and methods for selective elutionof the template.
Self assembly interactions: covalent, electrostatic/ionic, non-covalent, semi-covalent and coordinative
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Molecular Imprinting Technology (MIT)4
Main advantages of MIT: Structure predictability, recognition specificity and application versatility.
Main limitations of MIT:Functional monomers - mostly vinyl or acrylics;Polymerizations - mostly free radical with thermal/photochemical initiation.
Template types in MIT: Atoms, ions, molecules, macromolecules or ensembles of them, including
microorganisms.
Future Trends for MIT:Use of new monomers and polymerization techniques;Combination of MIT with other technologies;Smart, stimuli-responsive molecularly imprinted polymers (MIP);MIP with protein or enzyme templates.
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Molecularly Imprinted Polymers (MIP) -- Applications5
Selective membranes
Solid phase microextraction
Artificial antibodies
Chemical sensorsBiomimetic materials
Chromatographic mediaHeterogeneous catalysts
Immunosorbents
MIP
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Main ideas of this work6
1. Introducing a new group or functional monomers in MIT – lactams;
2. Introducing of activated anionic polymerization of lactams in MIT;
3. Combination of microencapsulation technology with MIT;
4. Molecular imprinting of model protein template;
5. Synthesis of smart MIP susceptible to external magnetic fields.
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Microencapsulation via AAROP of lactams 7
Starting mixture Viscous particles Hybrid micro/nano particles
Precipitationpolimerization
AAROP
ECL;
DDL,
2PD
APA6, PA12, PA4
Different payloads neat or mixturescan be used:
• Natural nanoclays -- CL15A, CL20A
• Carbon allotropes – CB, CNT, CNF,
fullerenes
• Metals – Cu, Al, Mg, Ag, Zn, Ag
• Magnetic particles – Fe, Fe3O4
The AAROP reaction
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AROP of Lactams - mechanism8
B COHN CON+Initiation:
+ CO
2
NC
1
OHN
slowCHN
O
C ON
3
CHN
O
C ON
aminic anion 4
Rate controlling step
CHN
O
C ON
4
rapid+CONH CON+
1 2Imide dimerN-(e-aminocaproyl)-caprolactam
5
CH2N
O
C ON
CONNR CO CONNHR CO N
COHN
+ +CO
NHR CO NH CO CON
PA6
Propagation:
AAP Catalytic system used:
Initiator: DILACTAMATE® ; Activator: BRUGGOLEN® C 20
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Morphology and magnetic properties of Microcapsules9
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Molecularly Imprinted Polymers - MIP10
Schematic representation of Non-covalent Molecular ImprintingMIP SAMPLES produced:PPD@BSAPPD-Fe-1%@BSAPPD-Fe3O4- 1%@BSAPPD-Fe3O4 -0.1%@BSA
andcorresponding NIP:
PPDPPD-Fe-1%PPD-Fe3O4 -1%PPD-Fe3O4 -0.1%
Functional monomer:2-pyrrolidone (PD)
Template:Bovine Serum Albumin (BSA)
HOOC
H2N
COOH
NH2
COOH
PD:BSA Self-assemblyby hydrogen bonds:
Materials used and processing
AAROP in the presence ofmagnetic nanoparticles: Fe ; Fe3O4 – 1 wt.%Catalytic system:DL+C2040ºC; no solvent.
Polymerization processConditions:
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BSA structure11
Three dimension image
of BSA molecule
(normal conditions).
α-helix structure in red;
Loops colored in green.
Side view
Front view
BSA
composed of 604 peptide units;
molecular weight of 66462 g/mol
consists of 55 – 65% α-helices, 21% β- sheet,
and the rest are turns.
pI 4.6-5
BSA secondary structure highly depends on pH:
pH 4.3 to 8.0, this is the so-called normal form.
pH below 4.3 --- unfolding starts, fast form with 45%
α-helix.
pH below 2.7 -- further unfolding to the expanded form
with 35%
pH above 8 -- the molecule adopts the basic form which
has 47% α-helix
BSA has hearth shapedmolecule – equilateral
triangle with side~8 nm and depth ~3nm
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PPD@BSA MIP/NIP SEM characterization12
PPD - NIP
PPD @BSA MIPbefore removingthe BSA template
PPD @BSA MIPafter removing
the BSA template
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PPD-Fe 1% @ BSA NIP/MIP SEM characterization13
Fe 1% NIP
Fe - 1% MIPBefore removingthe BSA template
Fe - 1% MIPAfter removing
the BSA template
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PPD-Fe3O4 1% @ BSA MIP/NIP SEM characterization14
Fe3O4 -1% NIP
Fe3O4 - 1% MIPBefore removingthe BSA template
Fe3O4 - 1% MIPAfter removing
the BSA template
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MIP/NIP characterization15
FTIR (ATR)
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MIP/NIP characterization16
TGA
TidMIP > Tid
NIP
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Low Temperature DSC analysis of in-pore constrained water17
PPD NIP/MIP PPD-Fe3O4 NIP/MIP
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BSA Adsorption kinetics of MIP/NIP samples18
0.1g MIP
2.5 ml 1wt.% BSA/H2O
1.0 ml B (PBS 0.1M pH 7)or (MES 0.1M pH 4)
MIP NIP
3h, 37ºC
Samples are removed after 0, 15, 30, 45, 60, 90, 120, 180 min incubation at 37ºC
0.1 ml of supernatant1.9 ml B
UV-VISAbsorbance at
280 nm ismeasured
Ab
sorb
ance
at
28
6 n
m
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PPD & PPD-Fe 1% MIP/NIP –Adsorption capacity Q, in MES Buffer19
Conditions: 0,1 g NIP/MIP; 1wt.% BSA; 3h incubation time; 37°C; 0.1 M pH 4.1 (MES buffer)
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PPD-Fe3O4 MIP/NIP –Adsorption capacity Q, in MES Buffer20
Conditions: 0,1 g NIP/MIP; 1wt.% BSA; 3h incubation time; 37°C; 0.1 M pH 4.1 (MES buffer)
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PPD&PPD-Fe 1% MIP/NIP - Adsorption capacity Q, in Phosphate Buffer 21
Conditions: 0,1 g NIP/MIP; 1wt.% BSA; 3h incubation time; 37°C; 0.1 M pH 7.2 (Phosphate buffer)
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PPD-Fe3O4 MIP/NIP - Adsorption capacity Q, in Phosphate Buffer 22
Conditions: 0,1 g NIP/MIP; 1wt.% BSA; 3h incubation time; 37°C; 0.1 M pH 7.2 (Phosphate buffer)
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PPD-Fe(PO4) MIP/NIP - Adsorption capacity Q 22
Conditions: 0,1 g NIP/MIP; 1wt.% BSA; 3h incubation time; 37°C
MES buffer 0.1 M pH 4.1 Phosphate buffer 0.1 M pH 7.2
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Evaluation of Imprinting Factor IF of PPD-MIP samples23
In pH 4,1 (MES buffer)
IF
In pH 7.2 (Phosphate buffer)
IF
IF = QMIP / QNIP
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Conclusions24
• Novel BSA molecularly imprinted magnetic responsive particles based onPA4 (2-polypyrrolidone) are synthesized via activated anionicpolymerization;
• The MIP particles are with controlled shape and sizes of 10-50 µm; themaximum dimensions of the imprinted cavities falls between 70-200 nm;
• All MIP samples showed superior adsorption capacity toward thetemplate BSA protein, as compared to the respective NIP samples, theimprovement factor varying between 30 and 320%;
• The adsorption capacity Q toward template protein is pH dependent, i.e.,much stronger in acidic than in basic media.
• Q can also depend on the surface treatment of the magnetic particles.
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Acknowledgements25
TSSiPRO NORTE-01-0145-FEDER-000015
Strategic projects UID/CTM/50025/2013 and LA25/2013-2014
SFRH/BSAB/130271/2017
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Еlina MarinhoClara RayaNadya DenchevaFilipa OliveiraJoana RompanteJoana BrazFilipa D. OliveiraShafagh Tochidi
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University of Minho
Thank you!
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University of Minho localization
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University of Minho
School of Natural Sciences
School of Engineering
Psychology School
Higher Institute of Education
Law School
School of Architecture
School of Economics and Management
School of Health Sciences
Founded:1974; Localization: Braga & Guimarães;Number of students: ca. 21.000 (incl. Post-graduation)
Higher School of Nursing
School of Social Sciences
School of Philology
School of Engineering
Department of BioengineeringDepartment of Civil ConstructionDepartment of Industrial ElectronicsDepartment of Mechanical EngineeringDepartment of Polymer Engineering (DEP)Department of Textile EngineeringDepartment of InformaticsDepartment of Production Systems Department of Informational Systems