Uwe Schedler

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. The fluorophore labeling approach was modified to exclude artifacts due to fluorescence quenching, but absolute quantum yield measurements still indicate a major uncertainty in routine fluorescence-based surface group quantifications, which is directly relevant for biochemical assays and medical diagnostics. Comparison with results from protein labeling with streptavidin suggests a porous network of poly(acrylic acid) chains on the particle surface, which allows diffusion of small molecules (cutoff between 1.6 and 6.5 nm) into the network.

Nucleic acid detection based on the use of microbeads: a review

AbstractMicrobead-based technologies represent elegant and versatile approaches for highly parallelized quantitative multiparameter assays. They also form the basis of various techniques for detection and quantification of nucleic acids and proteins. Nucleic acid-based methods include hybridization assays, solid-phase PCR, sequencing, and trapping assays. Microbead assays have been improved in the past decades and are now important tools in routine and point-of-care diagnostics as well as in life science. Its advances include low costs, low workload, high speed and high-throughput automation. The potential of microbead-based assays therefore is apparent, and commercial applications can be found in the detection and discrimination of single nucleotide polymorphism, of pathogens, and in trapping assays. This review provides an overview on microbead-based platforms for biosensing with a main focus on nucleic acid detection (including amplification strategies and on selected probe systems using fluorescent labeling). Specific sections cover chemical properties of microbeads, the coupling of targets onto solid surfaces, microbead probe systems (mainly oligonucleotide probes), microbead detection schemes (with subsections on suspension arrays, microfluidic devices, and immobilized microbeads), quantification of nucleic acids, PCR in solution and the detection of amplicons, and methods for solid-phase amplification. We discuss selected trends such as microbead-coupled amplification, heterogeneous and homogenous DNA hybridization assays, real-time assays, melting curve analysis, and digital microbead assays. We finally discuss the relevance and trends of the methods in terms of high-level multiplexed analysis and their potential in diagnosis and personalized medicine. Contains 211 references. Figureᅟ

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. We demonstrate that the difference between added and detected M(2+) is nicely correlated to the number of surface carboxy groups as determined by conductometry, thereby affording a validated measure for the trueness of this procedure. The variation coefficient of ~5% found in reproducibility studies underlines the potential of this novel method that can find conceivable applications for the characterization of different types of poly(carboxylic acid)-functionalized materials, e.g., for quality control by manufacturers of such materials.

Surface engineering: molecularly imprinted affinity membranes by photograft polymerization

Commercial polymer microfiltration membranes were surface-modified with a graft copolymer of a functional monomer and a crosslinker in the presence of a template (triazine-herbicide). As result, membranes covered with a thin layer of imprinted polymer (MIP) selective to the template were obtained. The influence of the polymerization conditions on membrane recognition properties was studied by membranes

New continuous polymeric surfaces for spot-synthesis, combinatorial chemistry, compound libraries, and high-throughput applications

Solid phase synthesis, especially spot-synthesis on cellulose membranes is well established for facile preparation of large arrays of biomolecules, e.g. peptides, nucleic acids, peptoides and the subsequent solid and solution phase, screening. Despite many good properties of cellulose membranes for some applications this material has a number of limitations such as low chemical and mechanical stability and high concentration of reactive, chemical different hydroxyl groups causing side reactions. Therefore a novel continuous polymeric material, porous membranes and non-porous materials, was developed based on the chemical and photochemical surface functionalization by grafting of flexible polymer chains carrying the functional and reactive groups for the syntheses. Several classes of compounds e.g. peptides, nucleic acids, peptoides, glucoconjugates and small organic molecules were synthesized using the new continuous polymeric surface.