A multiparametric fluorescence assay for screening aptamer-protein interactions based on microbeads.

For improving aptamer-ligand binding we have developed a screening system that defines optimal binding buffer composition. Using multiplex assays, one buffer system is needed which guarantees the specific binding of all aptamers. We investigated nine peer-reviewed DNA aptamers.

Fluorescence-encoded poly(methyl metharcylate) nanoparticles for a lateral flow assay detecting IgM autoantibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) belongs to the most often occurring autoimmune diseases in the world. For serological diagnosis, IgM auto-antibodies directed against the Fc portion of IgG referred to as rheumatoid factor are used as biomarkers. The autoantibody detection is usually done by ELISA.

Hydrogels and Their Role in Biosensing Applications.

Hydrogels play an important role in the field of biomedical research and diagnostic medicine. They are emerging as a powerful tool in the context of bioanalytical assays and biosensing. In this context, this review gives an overview of different hydrogels and the role they adopt in a range of applications.

Lifetime encoding in flow cytometry for bead-based sensing of biomolecular interaction.

To demonstrate the potential of time-resolved flow cytometry (FCM) for bioanalysis, clinical diagnostics, and optically encoded bead-based assays, we performed a proof-of-principle study to detect biomolecular interactions utilizing fluorescence lifetime (LT)-encoded micron-sized polymer beads bearing target-specific bioligands and a recently developed prototype lifetime flow cytometer (LT-FCM setup). This instrument is equipped with a single excitation light source and different fluorescence detectors, one operated in the photon-counting mode for time-resolved measurements of fluorescence decays and three detectors for conventional intensity measurements in different spectral windows. First, discrimination of bead-bound biomolecules was demonstrated in the time domain exemplarily for two targets, Streptavidin (SAv) and the tumor marker human chorionic gonadotropin (HCG).

Spatial Separation of Microbeads into Detection Levels by a Bioorthogonal Porous Hydrogel for Size-Selective Analysis and Increased Multiplexity.

Multiplex detection techniques are emerging within the fields of life science research and medical diagnostics where it is mandatory to analyze a great number of molecules. The detection techniques need to be highly efficient but often involve complicated and expensive fabrication procedures. Here, we present the immobilization and geometric separation of fluorescence-labeled microbeads for a multiplex detection in k levels.

Quantification of Aldehydes on Polymeric Microbead Surfaces via Catch and Release of Reporter Chromophores.

Aldehyde moieties on 2D-supports or micro- and nanoparticles can function as anchor groups for the attachment of biomolecules or as reversible binding sites for proteins on cell surfaces. The use of aldehyde-based materials in bioanalytical and medical settings calls for reliable methods to detect and quantify this functionality. We report here on a versatile concept to quantify the accessible aldehyde moieties on particle surfaces through the specific binding and subsequent release of small reporter molecules such as fluorescent dyes and nonfluorescent chromophores utilizing acylhydrazone formation as a reversible covalent labeling strategy.

Precise supramolecular control of surface coverage densities on polymer micro- and nanoparticles.

We report herein the controlled surface functionalization of micro- and nanoparticles by supramolecular host-guest interactions. Our idea is to exploit the competition of two high-affinity guests for binding to the surface-bound supramolecular host cucurbit[7]uril (CB7). To establish our strategy, surface azide groups were introduced to hard-sphere (poly)methylmethacrylate particles with a grafted layer of poly(acrylic acid), and a propargyl derivative of CB7 was coupled to the surface by click chemistry.

Switchable Oligo(glycidyl ether) Acrylate Bottlebrushes "Grafted-from" Polystyrene Surfaces: A Versatile Strategy toward Functional Cell Culture Substrates.

Thermoresponsive brushes based on linear poly(glycidyl ether)s (PGEs) have already shown to be functional coatings for cell sheet fabrication. In here, we introduce a method to functionalize polystyrene (PS) tissue culture substrates with thermoresponsive coatings comprising glycidyl ether-based bottlebrush architectures. Utilizing the UV-induced "grafting-from" approach, thermoresponsive oligo(glycidyl ether) acrylate (OGEA) macromonomers were polymerized from PS substrates under bulk conditions.

Bioorthogonal in Situ Hydrogels Based on Polyether Polyols for New Biosensor Materials with High Sensitivity.

Both noncovalent and covalent encapsulations of active biomolecules, for example, proteins and oligonucleotides, for a new biosensor matrix in an in situ bioorthogonal hydrogel formation via a strain-promoted azide-alkyne cycloaddition reaction were investigated. Unspecific interaction between the gel and the biomolecules as well as protein denaturation was prevented by the bioorthogonal gel components, which ensure a uniform aqueous environment in the hydrogel network. No leaching of the active biomolecules was observed.

En route to traceable reference standards for surface group quantifications by XPS, NMR and fluorescence spectroscopy.

The fluorine content of polymer particles labelled with 2,2,2-trifluoroethylamine was reliably quantified with overlapping sensitivity ranges by XPS and solid-state NMR. This provides a first step towards reference materials for the metrological traceability of surface group quantifications. The extension of this concept to fluorescence spectroscopy is illustrated.

Scope and limitations of surface functional group quantification methods: exploratory study with poly(acrylic acid)-grafted micro- and nanoparticles.

The amount of grafted poly(acrylic acid) on poly(methyl methacrylate) micro- and nanoparticles was quantified by conductometry, (13)C solid-state NMR, fluorophore labeling, a supramolecular assay based on high-affinity binding of cucurbit[7]uril, and two colorimetric assays based on toluidine blue and nickel complexation by pyrocatechol violet. The methods were thoroughly validated and compared with respect to reproducibility, sensitivity, and ease of use. The results demonstrate that only a small but constant fraction of the surface functional groups is accessible to covalent surface derivatization independently of the total number of surface functional groups, and different contributing factors are discussed that determine the number of probe molecules which can be bound to the polymer surface.

Quantification of surface functional groups on polymer microspheres by supramolecular host-guest interactions.

We introduce a method to determine the number of accessible functional groups on a polymer microsphere surface based on the interaction between the macrocyclic host cucurbit[7]uril (CB7) and a guest reacted to the microsphere surface. After centrifugation, CB7 in the supernatant is quantified by addition of a fluorescent dye. The difference between added and detected CB7 affords the number of accessible surface functional groups.

Simple colorimetric method for quantification of surface carboxy groups on polymer particles.

We present a novel, simple, and fast colorimetric method to quantify the total number of carboxy groups on polymer microparticle and nanoparticle surfaces. This method exploits that small divalent transition metal cations (M(2+) = Ni(2+), Co(2+), Cd(2+)) are efficiently bound to these surface functional groups, which allows their extraction by a single centrifugation step. Remaining M(2+) in the supernatant is subsequently quantified spectrophotometrically after addition of the metal ion indicator pyrocatechol violet, for which Ni(2+) was identified to be the most suitable transition metal cation.