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 Fe₃O₄ 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 Fe₃O₄ nanoparticle surface was modified using poly­(MPC) by surface-initiated atom-transfer radical polymerization. The density of poly­(MPC) was 0.16 chains/nm², 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 Fe₃O₄ 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 Ca²⁺ 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 Fe₃O₄ 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 Fe₃O₄. After poly­(MPC)-protected Fe₃O₄ 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 Fe₃O₄ 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 ¹H 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