15 research outputs found
Label-Free Specific Detection and Collection of C‑Reactive Protein Using Zwitterionic Phosphorylcholine-Polymer-Protected Magnetic Nanoparticles
In this study, poly[2-methacryloyloxyethyl
phosphorylcholine (MPC)]-protected
Fe<sub>3</sub>O<sub>4</sub> nanoparticles were prepared and used for
the label-free specific detection and collection of an acute inflammation
marker, C-reactive protein (CRP), in a simulated body fluid. The Fe<sub>3</sub>O<sub>4</sub> nanoparticle surface was modified using poly(MPC)
by surface-initiated atom-transfer radical polymerization. The density
of poly(MPC) was 0.16 chains/nm<sup>2</sup>, and the colloidal stability
of the nanoparticles in aqueous media and human plasma was effectively
improved by surface modification. The size of the as-prepared poly(MPC)-protected
Fe<sub>3</sub>O<sub>4</sub> nanoparticles was ∼200 nm. After
coming into contact with CRP, the nanoparticles aggregated as CRP
comprises five subunits, and each subunit can bind to a phosphorylcholine
group with two free Ca<sup>2+</sup> ions. The change in the nanoparticle
size exhibited a good correlation with the CRP concentration in the
range of 0–600 nM. A low limit of detection of 10 nM for CRP
was observed. The particles effectively reduced the adsorption of
nonspecific proteins, and the change in the nanoparticle size with
CRP was not affected by the coexistence of bovine serum albumin at
a concentration 1000 times greater than that of CRP. Nanoparticle
aggregates formed using CRP were dissociated using ethylenediamine-<i>N</i>,<i>N</i>,<i>N</i>′,<i>N</i>′-tetraacetic acid, disodium salt, thereby
regenerating poly(MPC)-protected Fe<sub>3</sub>O<sub>4</sub> nanoparticles.
In addition, CRP was collected from aqueous media using an acidic
buffer solution and human plasma. CRP-containing aqueous solutions
were treated with poly(MPC)-protected Fe<sub>3</sub>O<sub>4</sub>.
After poly(MPC)-protected Fe<sub>3</sub>O<sub>4</sub> nanoparticles
were separated using a neodymium magnet and centrifugation, the concentration
of CRP in the media dramatically decreased. In stark contrast, the
concentration of albumin present in the test solution did not change
even after treatment with the nanoparticles. Therefore, nanoparticles
specifically recognize CRP from complex biological fluids. Although
inhibition tests in the presence of 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine liposomes or free poly(MPC)
were also carried out, the binding of poly(MPC)-protected Fe<sub>3</sub>O<sub>4</sub> to CRP was not affected by these inhibitors. In conclusion,
poly(MPC)-brush-bearing magnetic nanoparticles can serve not only
as reliable materials for detecting and controlling the levels of
CRP in simulated body fluids but also as diagnostic and therapeutic
materials
Well-defined protein immobilization on photo-responsive phosphorylcholine polymer surfaces
<p>In this study, we propose a new polymer substrate that is able to covalently couple intended proteins and reduce nonspecific protein fouling. Poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-ran-<i>N</i>-methacryloyl-(L)-tyrosinemethylester (MAT)] [P(MPC/MAT)] was synthesized by free-radical polymerization. The photooxidation of the MAT unit in the copolymer was observed under ultraviolet (UV) light at 254 nm. P(MPC/MAT) was spin-coated on silicon (Si) and gold substrates. Without UV irradiation of the polymer-coated surface, P(MPC/MAT) physisorbed on the substrates, and the thickness of the polymer layer was less than 10 nm, regardless of the polymer concentration in the coating solution. In contrast, when the polymer-coated surface was irradiated with UV light, the thickness of the polymer layer could be controlled by changing the polymer concentration of the coating solution. Competitive protein adsorption on P(MPC/MAT) was studied. Bovine serum albumin was first contacted with the surface and later challenged with bovine fibrinogen. On bare gold and Si substrates, a large amount of albumin was adsorbed, and the competitive adsorption of albumin and fibronectin was observed. In contrast, the non-UV-irradiated P(MPC/MAT) surface effectively reduced protein adsorption. Interestingly, on the UV-irradiated P(MPC/MAT) surface, the primary protein preferably bonded, and significantly less secondary protein was adsorbed compared to primary protein. Cell adhesion was also tested on the substrate to clarify the effects of proteins existing on the substrates. On the bare Si surface, many adherent cells were observed, regardless of the protein pretreatment. On the non-UV-irradiated P(MPC/MAT) surface, cell adhesion was effectively reduced along with protein adsorption. Cell adhesion on the UV-irradiated P(MPC/MAT) surface depended strongly on the type of protein that was initially in contact with the surface. We concluded that the desired proteins could be immobilized on the photo-activated P(MPC/MAT) surface while preserving their function. Moreover, competitive protein exchange and multilayer adsorption hardly occurred on the surface.</p
Preparation of Biointeractive Glycoprotein-Conjugated Hydrogels through Metabolic Oligosacchalide Engineering
In
the current study, synthetic hydrogels containing metabolically
engineered glycoproteins of mammalian cells were prepared for the
first time and selectin-mediated cell adhesion on the hydrogel was
demonstrated. A culture of HL-60 cells was supplemented with an appropriate
volume of aqueous solution of <i>N</i>-methacryloyl mannosamine
(ManMA) to give a final concentration of 5 mM. The cells were then
incubated for 3 days to deliver methacryloyl groups to the glycoproteins
of the cells. A transparent hydrogel was formed via redox radical
polymerization of methacryloyl functionalized glycoproteins with 2-methacryloyloxyethyl
phosphorylcholine and a cross-linker. Conjugation of the glycoproteins
into the hydrogel was determined using Coomassie brilliant blue (CBB)
and periodic acid–Schiff (PAS) staining. The surface density
of P-selectin glycoprotein ligand-1 (PSGL-1) on the hydrogels was
also detected using gold-colloid-labeled immunoassay. Finally, selectin-mediated
cell adhesion on hydrogels containing glycoproteins was demonstrated.
Selectin-mediated cell adhesion is considered an essential step in
the progression of various diseases; therefore, hydrogels having glycoproteins
could be useful in therapeutic and diagnostic applications
Stabilization of DNA Structures with Poly(ethylene sodium phosphate)
The structure and stability of biomolecules
under molecular crowding
conditions are of interest because such information clarifies how
biomolecules behave under cell-mimicking conditions. The anionic surfaces
of chromatin, which is composed of DNA strands and histone complexes,
are concentrated in cell nuclei and thus generate a polyanionic crowding
environment. In this study, we designed and synthesized an anionic
polymer, poly(ethylene sodium phosphate) (PEP·Na), which has
a nucleic acid phosphate backbone and created a cell nucleus-like
environment. The effects of molecular crowding with PEP·Na on
the thermodynamics of DNA duplexes, triplexes, and G-quadruplexes
were systematically studied. Thermodynamic analysis demonstrated that
PEP·Na significantly stabilized the DNA structures; e.g., a free
energy change at 25 °C for duplex formation decreased from −6.6
to −12.8 kcal/mol with 20 wt % PEP·Na. Thermodynamic parameters
further indicated that the factors for the stabilization of the DNA
structures were dependent on sodium ion concentration. At lower polymer
concentrations, the stabilization was attributed to a shielding of
the electrostatic repulsion between DNA strands by the sodium ions
of PEP·Na. In contrast, at higher polymer concentrations, the
DNA structures were entropically stabilized by volume exclusion, which
could be enhanced by electrostatic repulsion between phosphate groups
in DNA strands and in PEP·Na. Additionally, increasing PEP·Na
concentration resulted in increasing enthalpy of the DNA duplex but
decreasing enthalpy of DNA G-quadruplex, indicating that the polymers
also promoted dehydration of the DNA strands. Thus, polyanionic crowding
affects the thermodynamics of DNA structures via the sodium ions,
volume exclusion, and hydration. The stabilization of DNA by the cell
nucleus-like polyanionic crowding provides new information regarding
DNA structures and allows for modeling reactions in cell nuclei
Antimicrobial Silver Nanoclusters Bearing Biocompatible Phosphorylcholine-Based Zwitterionic Protection
Infection
is one of the most serious issues in medical treatments
leading to the development of several antimicrobial agents. In particular,
silver ions released from silver substrates is well-known as a reliable
antimicrobial agent that either kills the microorganisms or inhibits
their growth. Unfortunately, many reports have shown that silver-based
antimicrobial agents are toxic for human cells as well. To improve
the biocompatibility of silver antimicrobial agents, we have synthesized
thiol-terminated phosphorylcholine (PC-SH)-protected silver nanoclusters
(PC–AgNCs) via strong thiol–metal coordination with
controlled ultrasmall size of the clusters. A change in plasmon-like
optical absorption was studied to affirm the successful synthesis
of small thiolated AgNCs through the absorption spectra that become
molecular-like for the AgNCs. We observed that PC–AgNCs were
spherical with an average diameter of <2 nm. The ultrasmall size
clusters were exceedingly immobilized by the PC-SH on the surface,
resulting in excellent biocompatibility and antibacterial activity
simultaneously. The biocompatible, antimicrobial PC–AgNCs exhibit
interesting advantages compared with other silver antimicrobial agents
for medical applications
Low-Temperature Processable Block Copolymers That Preserve the Function of Blended Proteins
Low-temperature
processable polymers have attracted increasing
interest as ecological materials because of their reduced energy consumption
during processing and suitability for making composites with heat-sensitive
biomolecules at ambient temperature. In the current study, low-temperature
processable biodegradable block copolymers were synthesized by ring-opening
polymerization of l-lactide (LLA) using polyphosphoester
as a macroinitiator. The polymer films could be processed under a
hydraulic pressure of 35 MPa. The block copolymer films swelled in
water because the polyphosphoester block was partially hydrated. Interestingly,
the swelling ratio of the films changed with temperature. The pressure-induced
order-to-disorder transition of the block copolymers was characterized
by small-angle X-ray scattering; a crystallinity reduction in the
block copolymers was observed after application of pressure. The crystallinity
of the block copolymers was recovered after removing the applied pressure.
The Young’s modulus of the block copolymer films increased
as the LLA unit content increased. Moreover, the modulus did not change
after multiple processing cycles and the recyclability of the block
copolymers was also confirmed. Finally, polymer films with embedded
proteinase K as a model protein were prepared. The activity of catalase
loaded into the polymer films was evaluated after processing at different
temperatures. The activity of catalase was preserved when the polymer
films were processed at room temperature but was significantly reduced
after high-temperature processing. The suitability of low-temperature
processable biodegradable polymers for making biofunctional composites
without reducing protein activity was clarified. These materials will
be useful for biomedical and therapeutic applications
Clickable and Antifouling Platform of Poly[(propargyl methacrylate)-<i>ran</i>-(2-methacryloyloxyethyl phosphorylcholine)] for Biosensing Applications
A functional copolymer platform,
namely, poly[(propargyl methacrylate)-<i>ran</i>-(2-methacryloyloxyethyl
phosphorylcholine)] (PPgMAMPC),
was synthesized by reversible addition–fragmentation chain-transfer
polymerization. In principle, the alkyne moiety of propargyl methacrylate
(PgMA) should serve as an active site for binding azide-containing
molecules via a click reaction, i.e., Cu-catalyzed azide/alkyne cycloaddition
(CuAAC), and 2-methacryloyloxyethyl phosphorylcholine (MPC), the hydrophilic
monomeric unit, should enable the copolymer to suppress nonspecific
adsorption. The copolymers were characterized using Fourier transform
infrared (FTIR) and <sup>1</sup>H NMR spectroscopies. Thiol-terminated,
PPgMAMPC-SH, obtained by aminolysis of PPgMAMPC, was immobilized on
a gold-coated substrate using a “grafting to” approach
via self-assembly. Azide-containing species, namely, biotin and peptide
nucleic acid (PNA), were then immobilized on the alkyne-containing
copolymeric platform via CuAAC. The potential use of surface-attached
PPgMAMPC in biosensing applications was shown by detection of specific
target molecules, i.e., streptavidin (SA) and DNA, by the developed
sensing platform using a surface plasmon resonance technique. The
copolymer composition strongly influenced the performance of the developed
sensing platform in terms of signal-to-noise ratio in the case of
the biotin–SA system and hybridization efficiency and mismatch
discrimination for the PNA–DNA system
Development of a Novel Antifouling Platform for Biosensing Probe Immobilization from Methacryloyloxyethyl Phosphorylcholine-Containing Copolymer Brushes
The immobilization of thiol-terminated poly[(methacrylic
acid)-<i>ran</i>-(2-methacryloyloxyethyl phosphorylcholine)]
(PMAMPC-SH) brushes on gold-coated surface plasmon resonance (SPR)
chips was performed using the “grafting to” approach
via self-assembly formation. The copolymer brushes provide both functionalizability
and antifouling characteristics, desirable features mandatorily required
for the development of an effective platform for probe immobilization
in biosensing applications. The carboxyl groups from the methacrylic
acid (MA) units were employed for attaching active biomolecules that
can act as sensing probes for biospecific detection of target molecules,
whereas the 2-methacryloyloxyethyl phosphorylcholine (MPC) units were
introduced to suppress unwanted nonspecific adsorption. The detection
efficiency of the biotin-immobilized PMAMPC brushes with the target
molecule, avidin (AVD), was evaluated in blood plasma in comparison
with the conventional 2D monolayer of 11-mercaptoundecanoic acid (MUA)
and homopolymer brushes of poly(methacrylic acid) (PMA) also immobilized
with biotin using the SPR technique. Copolymer brushes with 79 mol
% MPC composition and a molecular weight of 49.3 kDa yielded the platform
for probe immobilization with the best performance considering its
high S/N ratio as compared with platforms based on MUA and PMA brushes.
In addition, the detection limit for detecting AVD in blood plasma
solution was found to be 1.5 nM (equivalent to 100 ng/mL). The results
have demonstrated the potential for using these newly developed surface-attached
PMAMPC brushes for probe immobilization and subsequent detection of
designated target molecules in complex matrices such as blood plasma
and clinical samples
Improvement of Antifouling Properties of Polyvinylidene Fluoride Hollow Fiber Membranes by Simple Dip Coating of Phosphorylcholine Copolymer via Hydrophobic Interactions
We
present a simple surface modification method for improving the
antifouling properties of polyvinylidene fluoride (PVDF) hollow fiber
membranes for water treatment. Membranes were dip coated in a block
copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and butyl
methacrylate (BMA) (poly(MPC-co-BMA)) aqueous solution. Membranes
coated with poly(MPC-co-BMA) at various coating concentrations exhibited
higher antifouling properties than bare and MPC homopolymer-coated
membranes, while showing higher water permeabilities after fouling.
Fluorescence observation revealed the effect of coating concentration
on poly(MPC-co-BMA) distribution within the hollow fiber membranes.
The results of quartz crystal microbalance measurements showed that
almost no bovine serum albumin was adsorbed onto the poly(MPC-co-BMA)
coating, whereas it was highly adsorbed onto bare and MPC homopolymer
coatings. We quantified the amount of poly(MPC-co-BMA) on the membrane
before and after cleaning, using fluorescence microscopy. The poly(MPC-co-BMA)
coating layer used in the hydrophobic interaction between BMA moieties
and the PVDF membrane surface was quite stable
Fiber-Optic Bio-sniffer (Biochemical Gas Sensor) Using Reverse Reaction of Alcohol Dehydrogenase for Exhaled Acetaldehyde
Volatile organic compounds (VOCs)
exhaled in breath have huge potential
as indicators of diseases and metabolisms. Application of breath analysis
for disease screening and metabolism assessment is expected since
breath samples can be noninvasively collected and measured. In this
research, a highly sensitive and selective biochemical gas sensor
(bio-sniffer) for gaseous acetaldehyde (AcH) was developed. In the
AcH bio-sniffer, a reverse reaction of alcohol dehydrogenase (ADH)
was employed for reducing AcH to ethanol and simultaneously consuming
a coenzyme, reduced form of nicotinamide adenine dinucleotide (NADH).
The concentration of AcH can be quantified by fluorescence detection
of NADH that was consumed by reverse reaction of ADH. The AcH bio-sniffer
was composed of an ultraviolet light-emitting diode (UV-LED) as an
excitation light source, a photomultiplier tube (PMT) as a fluorescence
detector, and an optical fiber probe, and these three components were
connected with a bifurcated optical fiber. A gas-sensing region of
the fiber probe was developed with a flow-cell and an ADH-immobilized
membrane. In the experiment, after optimization of the enzyme reaction
conditions, the selectivity and dynamic range of the AcH bio-sniffer
were investigated. The AcH bio-sniffer showed a short measurement
time (within 2 min) and a broad dynamic range for determination of
gaseous AcH, 0.02–10 ppm, which encompassed a typical AcH concentration
in exhaled breath (1.2–6.0 ppm). Also, the AcH bio-sniffer
exhibited a high selectivity to gaseous AcH based on the specificity
of ADH. The sensor outputs were observed only from AcH-contained standard
gaseous samples. Finally, the AcH bio-sniffer was applied to measure
the concentration of AcH in exhaled breath from healthy subjects after
ingestion of alcohol. As a result, a significant difference of AcH
concentration between subjects with different aldehyde dehydrogenase
type 2 (ALDH2) phenotypes was observed. The AcH bio-sniffer can be
used for breath measurement, and further, an application of breath
analysis-based disease screening or metabolism assessment can be expected
due to the versatility of its detection principle, which allows it
to measure other VOCs by using NADH-dependent dehydrogenases