5 research outputs found
CoreāShell Molecularly Imprinted Polymer Nanoparticles as Synthetic Antibodies in a Sandwich Fluoroimmunoassay for Trypsin Determination in Human Serum
We describe the application
of a fluorescently labeled water-soluble coreāshell molecularly
imprinted polymer (MIP) for fluorescence immunoassay (FIA) to detect
trypsin. <i>p</i>-Aminobenzamidine (PAB), a competitive
inhibitor of trypsin, was immobilized in the wells of a microtiter
plate enabling the capture of trypsin in an oriented position, thus
maintaining its native conformation. Fluorescent MIP nanoparticles,
which bound selectively to trypsin, were used for quantification.
The MIP was prepared by a multistep solid-phase synthesis approach
on glass beads functionalized with PAB, orientating all trypsin molecules
in the same way. The coreāMIP was first synthesized, using
a thermoresponsive polymer based on <i>N</i>-isopropylacrylamide,
so as to enable its facile liberation from the immobilized template
by a simple temperature change. The shell, mainly composed of allylamine
to introduce primary amino groups for postconjugation of fluorescein
isothiocyanate (FITC), was grafted in situ on the coreāMIP,
whose binding cavities were still bound and protected by the immobilized
trypsin. The resulting coreāshell MIP was endowed with a homogeneous
population of high-affinity binding sites, all having the same orientation.
The MIP has no or little cross-reactivity with other serine proteases
and unrelated proteins. Our MIP-based FIA system was successfully
applied to detect low trypsin concentrations spiked into nondiluted
human serum with a low limit of quantification of 50 pM, which indicates
the significant potential of this assay for analytical and biomedical
diagnosis applications
EGFR Inhibition by Curcumin in Cancer Cells: A Dual Mode of Action
Epidermal
Growth Factor Receptor (EGFR) is an important target
of anticancer therapy. Nowadays, the search for new molecules inhibiting
this receptor is turning toward natural substances. One of the most
promising natural compounds that have shown an anti-EGFR activity
is curcumin, a polyphenol found in turmeric. Its effect on the receptor
kinase activity and on the receptor autophosphorylation has been already
described, but the mechanism of how curcumin interacts with EGFR is
not fully elucidated. We demonstrate that the mode of action of curcumin
is dual. This polyphenol is able to inhibit directly but partially
the enzymatic activity of the EGFR intracellular domain. The present
work shows that curcumin also influences the cell membrane environment
of EGFR. Using biomimetic membrane models, we show that curcumin insertion
into the lipid bilayer leads to its rigidification. Single particle
tracking analyses performed in the membrane of A431 cancer cells confirmed
that this effect of curcumin on the membrane slows down the receptor
diffusion. This is likely to affect the receptor dimerization and,
in turn, its activation
Toward a Universal Method for Preparing Molecularly Imprinted Polymer Nanoparticles with Antibody-like Affinity for Proteins
We describe a potentially universal,
simple and cheap method to
prepare water-compatible molecularly imprinted polymer nanoparticles
(MIP-NPs) as synthetic antibodies against proteins. The strategy is
based on a solid phase synthesis approach where glass beads (GBs)
are functionalized with a metal chelate, acting as a general affinity
ligand to attract surface-bound histidines present on proteins. This
configuration enables an oriented immobilization of the proteins,
upon which thermoresponsive MIP-NPs are synthesized. The GBs play
the role of both a reactor and a separation column since, after synthesis,
the MIP-NPs are released from the support by a simple temperature
change, resulting in protein-free polymers. The resulting MIP-NPs
are endowed with improved binding site homogeneity, since the binding
sites have the same orientation. Moreover, they are stable (no aggregation)
in a buffer solution for prolonged storage time and exhibit apparent
dissociation constants in the nanomolar range, with little or no cross-reactivity
toward other proteins
Tracking Hyaluronan: Molecularly Imprinted Polymer Coated Carbon Dots for Cancer Cell Targeting and Imaging
War
against cancer constantly requires new affinity tools to selectively
detect, localize, and quantify biomarkers for diagnosis or prognosis.
Herein, carbon nanodots (CDs), an emerging class of fluorescent nanomaterials,
coupled with molecularly imprinted polymers (MIPs), are employed as
a biocompatible optical imaging tool for probing cancer biomarkers.
First, N-doped CDs were prepared by hydrothermal synthesis using starch
as carbon source and l-tryptophan as nitrogen atom provider
to achieve a high quantum yield of 25.1 Ā± 2%. The CDs have a
typical size of ā¼3.2 nm and produce an intense fluorescence
at 450 nm upon excitation with UV light. A MIP shell for specific
recognition of glucuronic acid (GlcA) was then synthesized around
the CDs, using the emission of the CDs as an internal light source
for photopolymerization. GlcA is a substructure (epitope) of hyaluronan,
a biomarker for certain cancers. The biotargeting and bioimaging of
hyaluronan on fixated human cervical cancer cells using CD core-MIP
shell nanocomposites is demonstrated. Human keratinocytes were used
as noncancerous reference cells and indeed, less staining was observed
by the CD-MIP
Dual-Oriented Solid-Phase Molecular Imprinting: Toward Selective Artificial Receptors for Recognition of Nucleotides in Water
We
describe the synthesis of water-soluble molecularly imprinted
polymer nanoparticles (MIP-NPs) as a new artificial host receptor
for the recognition of adenosine monophosphate (AMP), used herein,
as a model nucleotide. MIP-NPs were prepared by solid-phase synthesis
on glass beads (GB) using, for the first time, immobilized FeĀ(III)-chelate
as an affinity ligand to orientate the AMP via its phosphate group.
A polymerizable thymine monomer which can induce complementary base-pairing
with the adenine moiety of the nucleotide was synthesized and incorporated
in the polymerization mixture to constrain the AMP in a dual-orientated
configuration. The MIP-NPs were remarkably selective toward AMP as
they did not bind other nucleotides, GMP, UMP, and CMP. This strategy
of using the phosphate group of AMP as a hinge enables unhindered
pairing of the nucleobase with its corresponding complementary base
monomer and can be extended to the preparation of specific MIP receptors
for other key nucleotides in aqueous conditions