Fluorescein O-methacrylate Research Articles

Hemin-catalyzed SI-RAFT polymerization for thrombin detection

Ultrasensitive protein detection has considerable potential for early diagnosis and screening of diseases. Here, based on Hemin-catalyzed surface-initiated reversible addition-fragmentation chain transfer (SI-RAFT) polymerization, this work developed an assay for ultrasensitive fluorescent biosensing of proteins and established a thrombin fluorescent sensor. First, the amino-modified aptamer 1 was immobilized on Fe3O4 magnetic beads, and then thrombin was added to form the aptamer 1-thrombin bioaffinity complex. Another thiol-modified aptamer 2 binds to bound thrombin to form a sandwich structure. Fluorescein O-methacrylate was used as the monomer, acetylacetone (ACAC) as the initiator, and Hemin as the catalyst. Through Hemin-catalyzed SI-RAFT polymerization, a large number of thrombin molecules are labeled with fluorescein O-methacrylate, resulting in severalfold amplification of the fluorescent signal. By detecting the fluorescent signal in thrombin from 1.0 fM to 0.1 nM, the proposed super sandwich strategy can detect thrombin at 0.98 fM, which is lower than the detection limit of most other methods.

Development of a Potassium-Ion-Responsive Star Copolymer with Controlled Aggregation/Dispersion Transition.

Stimuli-responsive star polymers are promising functional materials whose aggregation, adhesion, and interaction with cells can be altered by applying suitable stimuli. Among several stimuli assessed, the potassium ion (K+), which is known to be captured by crown ethers, is of considerable interest because of the role it plays in the body. In this study, a K+-responsive star copolymer was developed using a polyglycerol (PG) core and grafted copolymer arms consisting of a thermo-responsive poly(N-isopropylacrylamide) unit, a metal ion-recognizing benzo-18-crown-6-acrylamide unit, and a photoluminescent fluorescein O-methacrylate unit. Via optimization of grafting density and copolymerization ratio of grafted arms, along with the use of hydrophilic hyperbranched core, microsized aggregates with a diameter of 5.5 μm were successfully formed in the absence of K+ ions without inducing severe sedimentation (the lower critical solution temperature (LCST) was 35.6 °C). In the presence of K+ ions, these aggregates dispersed due to the shift in LCST (47.2 °C at 160 mM K+), which further induced the activation of fluorescence that was quenched in the aggregated state. Furthermore, macrophage targeting based on the micron-sized aggregation state and subsequent fluorescence activation of the developed star copolymers in response to an increase in intracellular K+ concentration were performed as a potential K+ probe or K+-responsive drug delivery vehicle.

Ion-responsive fluorescence resonance energy transfer between grafted polyacrylic acid arms of star block copolymers

Interactions between polyacrylic acid (PAA) arms of star block copolymers through metal ions were investigated by fluorescence resonance energy transfer (FRET). Dendritic polyester (DPE) core was grafted with block copolymer arms of oligo (ethylene glycol) methyl ether acrylate (OEGA) and PAA. Fluorescein o-methacrylate (FMA) or methacryloxyethyl thiocarbamoyl rhodamine B (MTR) was introduced via copolymerization to the terminated PAA blocks. The unique copolymer structure enabled FRET between PAA chains without causing precipitation, which inevitably occurs for linear PAA. Addition of metal ions such as Zn2+, Ca2+, or Na+ induced FRET, the order of which was Zn2+>Ca2+≫Na+. FRET intensity was also correlated with concentration of each ion. Furthermore, in situ FRET measurement during the hydrogel formation of the star block copolymers was performed by mixing the star block copolymers and Zn2+.

Ratiometric Fluorescent pH Probes Based on Glycopolymers.

Effectively detecting pH changes plays a critical role in exploring cellular functions and determining physiological and pathological processes. A novel ratiometric pH probe based on a glycopolymer, armored with properties of serum-stability, tumor-targeting, and pH monitoring, is designed. Random copolymers of 2-(methacrylamido) glucopyranose and fluorescein O-methacrylate are first synthesized by reversible addition fragmentation chain transfer polymerization. Acryloxyethyl thiocarbamoyl rhodamine B is then attached to the polymer chain to prepare ratiometric fluorescent pH probes via a thiol-ene reaction. The synthesized polymeric probes are characterized by NMR, gel permeation chromatography, UV-vis spectroscopy, and transmission electron microscopy, and the fluorescence responses are examined in phosphate buffer at different pHs. The cytotoxicity and confocal imaging experiments of the probes are detected using HeLa cells, demonstrating a low toxicity and superior biocompatibility for detecting pH changes in bioapplications.

Clickable poly(ionic liquid)s for modification of glass and silicon surfaces

Covalent attachment of poly(ionic liquid)s (PILs) by click chemistry on glass or silicon (Si) surfaces was performed. Poly[1-(4-vinylbenzyl)-3-butylimidazolium bis(trifluoromethylsulfonyl)imide] (polyVBBI+Tf2N−), and copolymers of polyVBBI+Tf2N− with fluorescein O-methacrylate were synthesized by conducting an atom transfer radical polymerization (ATRP) from initiators containing azide or thioacetate groups. The azide- and thiol-terminated PILs were then successfully grafted onto alkyne and alkene modified glass/Si wafers by thermal azide–alkyne cycloaddition and photoinitiated thiol-ene click reactions, respectively. The modified surfaces were characterized by contact angle measurements and ellipsometry. The fluorescent PIL functionalized surfaces showed strong fluorescence under UV irradiation. This procedure of tethering PILs to substrates also provides an easy way to change the surface hydrophilicity by replacing the anions in the grafted PILs. The present approach could be readily applied for surface modifications with other types of PILs or their copolymers to achieve different functionalities on various surfaces.

Disposable Fluorescence Optical pH Sensor for Near Neutral Solutions

The design, development and performance evaluation of a fluorescence-based pH sensor for on-line measurements is presented. The pKa of the sensing element has been calculated to be 7.9, thus the sensor is suitable for measurement of near neutral solutions. The sensor consists of a low-cost disposable polymer sensing probe, in contact with the solution under test, interrogated by an optoelectronic transduction system. The pH sensitive dye is based on fluorescein O-methacrylate, which has been covalently linked to a hydrogel matrix, realized through the use of HEMA (2-hydroxyethyl methacrylate), HDDA (1,6-hexanediol diacrylate) and PEGDA (polyethylene glycol diacrylate). The optical interrogation setup, together with the electronics, has been developed to acquire and process the fluorescence signal. The sensor works over a pH range between 6.5 and 9.0. In the range between 7.0 and 8.0, the sensor shows a linear behavior with a maximum linearity error of 5%. Thanks to the good performance of the sensing element and transduction system, the short term drift of the reading (measured over 40 min) is lower than 0.15%. The measuring system also exhibits good performance in terms of response time and reproducibility.

Synthesis and characterization of multifunctional Fe3O4/poly(fluorescein O-methacrylate) core/shell nanoparticles

Multifunctional fluorescent and superparamagnetic Fe3O4/poly(fluorescein O-methacrylate) [Fe3O4/poly(FMA)] nanoparticles with core/shell structure were synthesized via surface-initiated polymerization. First, polymerizable double bonds were introduced onto the surface of Fe3O4 nanoparticles via ligand exchange and a condensation reaction. A fluorescent monomer, FMA, was then polymerized to the double bonds at the surface via free-radical polymerization, leading to form a fluorescent polymer shell around the superparamagnetic Fe3O4 core. The resultant Fe3O4/poly(FMA) nanoparticles were characterized by Fourier transform infrared, nuclear magnetic resonance, and X-ray diffraction spectroscopy to confirm the reactions. Transmission electron microscopy images showed that the Fe3O4/poly(FMA) nanoparticles have a spherical and monodisperse core/shell morphology. Photoluminescence spectroscopy and superconducting quantum interference device magnetometer analyses confirmed that the Fe3O4/poly(FMA) nanoparticles exhibited fluorescent and superparamagnetic properties, respectively. In addition, we demonstrated the potential bioimaging application of the Fe3O4/poly(FMA) nanoparticles by visualizing the cellular uptake of the nanoparticles into A549 lung cancer cells.

Multi-functional graphene as an in vitro and in vivo imaging probe

A strategy has been developed for the synthesis of multi-functional graphene (MFG) using green synthetic approach and explored its biomedical application as a promising fluorescent marker for in vitro and in vivo imaging. In- situ microwave-assisted reduction and magnetization process was adopted to convert the graphene oxide into magnetic graphene within 1 min, which was further covalently modified to build a polyacrylic acid (PAA) bridge for linking the fluorescein o-methacrylate (FMA) to yield MFG with water-dispersibility (∼2.5 g/l) and fluorescence property (emission maximum at 526 nm). The PAA bridges also functions to prevent graphene-induced fluorescence quenching of conjugated FMA. The extent of reduction, magnetization, and functionalization was confirmed with TEM, AFM, Raman, XPS, FT-IR, TGA, and SQUID measurements. In vitro cytotoxicity study of HeLa cells reveal that MFG could stand as a biocompatible imaging probe with an IC 50 value of ∼100 μg/ml; whereas in vivo zebrafish study does not induce any significant abnormalities nor affects the survival rate after microinjection of MFG. Confocal laser scanning microscopy images reveals that MFG locates only in the cytoplasm region and exhibits excellent co-localization and biodistribution from the head to tail in the zebrafish. Our results demonstrate the applicability of graphene based fluorescence marker for intracellular imaging and, more significantly, as well as whole-animal imaging. Hence, MFG could preferentially serve as a dual functional probe in biomedical diagnostics.

PH-Responsive Fluorescent Molecular Bottlebrushes Prepared by Atom Transfer Radical Polymerization

Fluorescein O-methacrylate was copolymerized with n-butyl acrylate by atom transfer radical polymerization in a “grafting from” reaction with a multifunctional linear macroinitiator to form pH responsive fluorescent bottlebrushes. The brush-like structure of the synthesized macromolecules was confirmed through molecular imaging by atomic force microscopy. NMR spectroscopy showed that the fluorescent units were successfully incorporated into the polymer. The synthesized bottlebrushes displayed highly fluorescent properties under basic conditions, yet showed no fluorescence under neutral or acidic conditions. The fluorescence could be turned on and off by changing the pH of the solution. These bottlebrush molecules could have potential applications in molecular imaging.

Rapid Cellular Internalization of Multifunctional Star Polymers Prepared by Atom Transfer Radical Polymerization

Poly(ethylene glycol) (PEG) star polymers containing GRGDS (Gly-Arg-Gly-Asp-Ser) peptide sequences on the star periphery were synthesized by atom transfer radical polymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMA), GRGDS modified poly(ethylene glycol) acrylate (GRGDS-PEG-Acryl), fluorescein o-methacrylate (FMA), and ethylene glycol dimethacrylate (EGDMA) via an "arm-first" method. Star polymers were approximately 20 nm in diameter, as measured by dynamic light scattering and atomic force microscopy. Conjugation of FMA to the stars was confirmed by fluorescence microscopy, and successful attachment of GRGDS segments to the star periphery was confirmed by (1)H NMR spectroscopy. Both fluorescent PEG star polymers with and without peripheral GRGDS peptide segments were cultured with MC3T3-E1.4 cells. These star polymers were biocompatible with ≥ 90% cell viability after 24 h of incubation. Cellular uptake of PEG star polymers in MC3T3-E1.4 cells was observed by confocal microscopy. Rapid uptake of PEG star polymers with GRGDS peptides (∼ 100% of FITC-positive cells in 15 min measured by flow cytometry) was observed, suggesting enhanced delivery potential of these functional star polymers.

Polymer-Modified Gadolinium Metal-Organic Framework Nanoparticles Used as Multifunctional Nanomedicines for the Targeted Imaging and Treatment of Cancer

Novel nanoscale theragnostic devices were successfully prepared through attachment of well defined, multifunctional polymer chains to gadolinium (Gd) metal-organic framework (MOF) nanoparticles. Copolymers of poly(N-isopropylacrylamide)-co-poly(N-acryloxysuccinimide)-co-poly(fluorescein O-methacrylate) were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization. The succinimide functionality was utilized as a scaffold for attachment of both a therapeutic agent, such as methotrexate, and a targeting ligand, such as H-glycine-arginine-glycine-aspartate-serine-NH(2) peptide. Employment of a trithiocarbonate RAFT agent allowed for reduction of the polymer end groups to thiolates providing a means of copolymer attachment through vacant orbitals on the Gd(3+) ions at the surface of the Gd MOF nanoparticles. These versatile, nanoscale scaffolds were shown to be biocompatible and have cancer cell targeting, bimodal imaging, and disease treatment capabilities. This unique method provided a simple yet versatile route of producing polymer-nanoparticle theragnostic materials with an unprecedented degree of flexibility in the construct, potentially allowing for tunable loading capacities and spatial loading of targeting/treatment agents, while incorporating bimodal imaging capabilities through both magnetic resonance and fluorescence microscopy.

Preparation of Fluorescent Magnetic Nanodiamonds and Cellular Imaging

Magnetic nanodiamonds were prepared via solid-state microwave arcing of a nanodiamond-ferrocene mixed powder in a focused microwave oven. High-resolution transmission electron microscope (HRTEM) images show that a magnetic nanodiamond is composed of iron nanoparticles encapsulated by graphene layers on the surface of nanodiamonds. Fluorescence property was introduced onto magnetic nanodiamonds by chemical modification of magnetic nanodiamonds via surface grafting of poly(acrylic acids) and fluorescein o-methacrylate. Fluorescent magnetic nanodiamonds are water soluble with a solubility of approximately 2.1 g/L. Cellular-imaging experiments show that fluorescent magnetic nanodiamonds could be ingested by HeLa cells readily in the absence of agonist (i.e., folate) moieties on the surface of nanodiamonds.