Dieter Trau

Encapsulation of glucose oxidase microparticles within a nanoscale layer-by-layer film: immobilization and biosensor applications

We report on an immobilization strategy utilizing layer-by-layer encapsulated microparticles of enzymes within a nanoscale polyelectrolyte film. Encapsulation of glucose oxidase (GOD) microparticles was achieved by the sequential adsorption of oppositely charged polyelectrolytes onto the GOD biocrystal surface. The polyelectrolyte system polyallylamine/polystyrene sulfonate was used under high salt conditions to preserve the solid state of the highly water soluble GOD biocrystals during the encapsulation process. The resulting polymer multilayer capsule of about 15 nm wall thickness is permeable for small molecules (glucose), but non-permeable for macromolecules thus preventing the enzyme from leakage and at the same time shielding it from the outer environment e.g., from protease or microbial activity. Decrease of the buffer salt concentration leads to the dissolution of the enzyme under formation of mu-bioreactors. The spherical mu-bioreactors are bearing an extremely high loading of biocompound per volume. Encapsulated GOD was subsequently used to construct a biosensor by nanoengineered immobilisation of mu-bioreactor capsules onto an electrode surface. The presented approach demonstrates a general method to encapsulate highly soluble solid biomaterials and an immobilization strategy with the potential to create highly active thin and stable films of biomaterial.

Development of an amperometric flow injection immunoanalysis system for the determination of the herbicide 2,4-dichlorophenoxyacetic acid in water

An amperometric flow injection immunoanalysis (FIIA) system based on an immunoreactor with immobilized biocomponents on a silica surface has been developed for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). In the antigen coating mode the hapten was immobilized and monoclonal primary antibody against 2,4-D together with alkaline phosphatase (AP)-labelled secondary antibody were used as sensing elements in a titration assay. In the antibody coating mode a biotinylated monoclonal antibody was immobilized on the surface of the immunoreactor and a 2,4-D-AP-conjugate was used for detection. For electrochemical measurements p-aminophenol enzymatically generated from p-aminophenyl phosphate was oxidized at a carbon working electrode at +150 mV versus Ag/AgCl. The system enabled the determination of 2,4-D in drinking water samples in the range from 0.2 to 70 micrograms/l. The whole system was computer controlled with a measuring time of 12 min for one determination.

Amperometric immunosensor for the detection of 2,4-dichlorophenoxyacetic acid (2,4-D) in water

An amperometric immunosensor for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in water has been developed using sequential injection analysis techniques. The system is based on a rapid competitive enzyme immunoassay employing an alkaline phosphatase-labeled monoclonal antibody directed against the herbicide and an immunoreactor with 2,4-D immobilized via bovine serum albumin either to Eupergit in a column or directly to the surface of a glass capillary. The detection limit of the immunosensor at 0.1 μg 2,4-D/l without enrichment of the analyte makes automatic measurements of 2,4-D in drinking and ground water feasible.

Inwards Buildup of Concentric Polymer Layers: A Method for Biomolecule Encapsulation and Microcapsule Encoding†

Encoding by encapsulation: A polymeric shell fabrication approach combines biomolecule encapsulation with encoding. Striated polymeric shells, fabricated through an inwards diffusion of poly(allyla ...

Multiplex detection platform for tumor markers and glucose in serum based on a microfluidic microparticle array.

We present a multiplex detection platform based on a microfluidic microparticle array to detect proteins and glucose in serum simultaneously. Multiplex detection of proteins and glucose was performed using biofunctionalized microparticles arrayed on gel-based microstructures integrated in microfluidics. The microparticles immobilized on these microstructures showed high stability under microfluidic flow conditions. With arrays of antibody-coated microbeads, microfluidic quantitative immunoassays for two protein tumor markers, human chorionic gonadotropin (hCG) and prostate specific antigen (PSA) were performed in serum samples with detection limits bellow the cut-off values for cancer diagnosis. Parallel to the immunoassays, quantitative enzymatic assays for glucose in the physiological concentration range were performed. Multiplex detection was achieved by using a spatially encoded microarray. By patterning antibody-coated microbeads and enzyme-containing microparticles on a novel mixed structure array, we successfully demonstrated simultaneous immunoassays (binding based assay) for proteins and an enzymatic assay (reaction kinetic based assay) for glucose. Our microparticle arrays could be potentially used for the detection of multiple categories of biomolecules (proteins, small metabolites and DNA) for clinical diagnostics and other biological applications.

Reusable optical bioassay platform with permeability-controlled hydrogel pads for selective saccharide detection

A reusable optical bioassay platform using permeability-controlled hydrogel pads for selective saccharide detection has been developed. An optical glucose detection assay based on fluorescence resonance energy transfer (FRET) between dye-labeled dextran and Concanavalin A (ConA) was incorporated into hydrogel pads by entrapment. The hydrogel pads are constructed from hemispherical hydrogel attached onto hydrophobic surfaces of a microtiter plate. The resulted hemispherical hydrogel pads entrapping the sensing biological materials were further surface coated with polyelectrolyte multilayers through a Layer-by-Layer (LbL) self-assembly process to create a permeability-controlled membrane with nanometer thickness. The selective permeable LbL film deposited on the hydrogel surface allows small molecular weight analytes to diffuse into the hydrogel pads while the large molecular weight sensing biological molecules are immobilized. An encapsulation efficiency of 75% for the ConA/Dextran complex within the coated hydrogel pads was achieved and no significant leakage of the complex was observed. Glucose calibration curve with linear range from 0 to 10mM glucose was obtained. Selective permeability of the hydrogel pads has been demonstrated by measurement of saccharides with various molecular weights. The LbL hydrogel pads could selectively detect monosaccharides (glucose, MW=180) and disaccharides (sucrose, MW=342) while polysaccharides (dextran, MW approximately 70kDa) cannot diffuse through the LbL layer and are excluded. LbL hydrogel pads allow regeneration of the FRET system with good signal reproducibility of more than 90% to construct a reusable and reagentless optical bioassay platform.

NEW WAYS IN BIOANALYSIS ONE-WAY OPTICAL SENSOR CHIP FOR ENVIRONMENTAL ANALYSIS

An optical immunosensor system consisting of a disposable low-cost sensor chip including a fluidic system and a base unit for optical readout was developed. Near infrared (NIR)-fluorescence markers (Cy5) were excited by an evanescent wave generated on the surface of the sensor chip. The combination of both fluorescence measurements and evanescent wave excitation provides extremely sensitive detection and avoids any washing or separation steps. To demonstrate the feasibility of the system for environmental control assays for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were developed. Two different assay formats were applied to determine 2,4-D in the relevant concentration range. Due to the assay formats chosen a direct proportional relationship between analyte concentration and signal intensity was achieved. Within an assay time of 15 min only, the analyte 2,4-D could be determined in a linear concentration range covering three orders of magnitude.

Assembly of biomacromolecule loaded polyelectrolyte multilayer capsules by using water soluble sacrificial templates

In the quest for greater control over biomacromolecular loading and higher encapsulation efficiencies for biomacromolecule loaded microcapsules we devised a novel approach employing water soluble sacrificial templates. In traditional layer by layer (LbL) methods, aqueous solutions of polyelectrolyte salts in combination with water insoluble sacrificial template materials are used to prepare polyelectrolyte microcapsules that can be loaded with biomacromolecules. Here, we replaced the aqueous phase with pure aliphatic alcohols (Reversed-Phase) to greatly enhance the retention of biomacromolecular cargo close to 100% during microcapsule preparation in this Reverse-Phase Layer by Layer (RP-LbL) process. Formation of stable multilayered polyelectrolyte membranes onto water soluble template materials by sequential deposition of polystyrenesulfonic acid (PSS) and polyallylamine (PA) from pure 1-butanol is reported for the first time. The challenge to exert control over the biomacromolecule concentration within the template material and the resulting microcapsules was addressed by sacrificial template materials. Sacrificial template materials are water soluble and comprise of biomacromolecules embedded into a matrix of small molecular weight molecules such as glucose. Control over the concentration of biomacromolecules in the template material and microcapsules is conveniently exerted by adjusting weight ratios of bimacromolecules to sacrificial template material. This approach is envisioned to be applied alternatively to traditional polyelectrolyte microcapsule preparation techniques in cases where minute losses of expensive biomacromolecules are unfavorable or when accurate control over biomacromolecule concentration is important.

Self-Assembly of Polyamines as a Facile Approach to Fabricate Permeability Tunable Polymeric Shells for Biomolecular Encapsulation

In this article, the self-assembly of polyamines as a facile approach to fabricate permeability tunable polymeric shells for encapsulation of relatively low molecular weight (LM(w)) hydrophilic biomacromolecules (M(w) ≈ 4000 Da) is presented. The entire process is performed in organic solvents within 2 to 4 h to allow for nearly 100% encapsulation yield. The polymeric shells are fabricated by a two-step process: 1) The self-assembly of polyamines (nonionized poly(allylamine) (niPA) or branched nonionized polyethyleneimine (niPEI)) within porous agarose microbeads via an inwards buildup self-assembly process. 2) Stabilization of assembled polyamines either via covalent (cross-linkers) or ionic bonding (complex with nonionized poly(styrene sulfonic acid) (niPSS)). Stable and distinct polymeric shells are formed in both cases. The shell thickness is demonstrated to be tunable within a range of 1 to 14 μm; and as the inwards buildup self-assembly technique is not a self-limiting process, shells with broader thicknesses can be achieved. Also, it is demonstrated that the polymer density of the shell can be tuned. Depending on the fabrication parameters, the resulting polymeric shells have been demonstrated to have different permeability characteristics for relatively LM(W) dextran (M(W) ≈ 4000 Da). For example, niPEI shells are observed to have a higher permeability than niPA shells. Therefore, polymeric capsules can be fabricated via this facile approach for either retention of relatively LM(w) hydrophilic biomacromolecules or designed to passively or responsively release the biomacromolecule payload. This two-step shell fabrication process represent an alternative and facile approach for the fabrication of self-assembled polymeric shells in the fields of capsule-based reactors/sensors and drugs/gene delivery where relatively LM(w) macromolecules are concerned.

Utilizing microfluidics to synthesize polyethylene glycol microbeads for Förster resonance energy transfer based glucose sensing

Here, we utilize microfluidic droplet technology to generate photopolymerizeable polyethylene glycol (PEG) hydrogel microbeads incorporating a fluorescence-based glucose bioassay. A microfluidic T-junction and multiphase flow of fluorescein isothiocyanate dextran, tetramethyl rhodamine isothiocyanate concanavalin A, and PEG in water were used to generate microdroplets in a continuous stream of hexadecane. The microdroplets were photopolymerized mid-stream with ultraviolet light exposure to form PEG microbeads and were collected at the outlet for further analysis. Devices were prototyped in PDMS and generated highly monodisperse 72 ± 2 μm sized microbeads (measured after transfer into aqueous phase) at a continuous flow rate between 0.04 ml/h-0.06 ml/h. Scanning electron microscopy analysis was conducted to analyze and confirm microbead integrity and surface morphology. Glucose sensing was carried out using a Förster resonance energy transfer (FRET) based assay. A proportional fluorescence intensity increase was measured within a 1-10 mM glucose concentration range. Microfluidically synthesized microbeads encapsulating sensing biomolecules offer a quick and low cost method to generate monodisperse biosensors for a variety of applications including cell cultures systems, tissue engineering, etc.