High-throughput roll-to-roll production of polymer biochips for multiplexed DNA detection in point-of-care diagnostics.

Roll-to-roll UV nanoimprint lithography has superior advantages for high-throughput manufacturing of micro- or nano-structures on flexible polymer foils with various geometries and configurations. Our pilot line provides large-scale structure imprinting for cost-effective polymer biochips (4500 biochips/hour), enabling rapid and multiplexed detections. A complete high-volume process chain of the technology for producing structures like µ-sized, triangular optical out-couplers or capillary channels (width: from 1 µm to 2 mm, height: from 200 nm up to 100 µm) to obtain biochips (width: 25 mm, length: 75 mm, height: 100 µm to 1.5 mm) was described. The imprinting process was performed with custom-developed resins on polymer foils with resin thicknesses ranging between 125-190 µm. The produced chips were tested in a commercial point-of-care diagnostic system for multiplexed DNA analysis of methicillin resistant Staphylococcus aureus (e.g., mecA, mecC gene detections). Specific target DNA capturing was based on hybridisation between surface bound DNA probes and biotinylated targets from the sample. The immobilised biotinylated targets subsequently bind streptavidin-horseradish peroxidase conjugates, which in turn generate light upon incubation with a chemiluminescent substrate. To enhance the light out-coupling thus to improve the system performance, optical structures were integrated into the design. The limits-of-detection of mecA (25 bp) for chips with and without structures were calculated as 0.06 and 0.07 µM, respectively. Further, foil-based chips with fluidic channels were DNA functionalised in our roll-to-roll micro-array spotter following the imprinting. This straightforward approach of sequential imprinting and multiplexed DNA functionalisation on a single foil was also realised for the first time. The corresponding foil-based chips were able to detect mecA gene DNA sequences down to a 0.25 µM concentration.

Advanced Photonic Sensors Based on Interband Cascade Lasers for Real-Time Mouse Breath Analysis.

A multiparameter gas sensor based on distributed feedback interband cascade lasers emitting at 4.35 µm and ultrafast electro-spun luminescence oxygen sensors has been developed for the quantification and continuous monitoring of 13CO2/12CO2 isotopic ratio changes and oxygen in exhaled mouse breath samples. Mid-infrared absorption spectra for quantitatively monitoring the enrichment of 13CO2 levels were recorded in a miniaturized dual-channel substrate-integrated hollow waveguide using balanced ratiometric detection, whereas luminescence quenching was used for synchronously detecting exhaled oxygen levels. Allan variance analysis verified a CO2 measurement precision of 1.6‰ during a 480 s integration time. Routine online monitoring of exhaled mouse breath was performed in 14 mechanically ventilated and instrumented mice and demonstrated the feasibility of online isotope-selective exhaled breath analysis within microliters of probed gas samples using the reported combined sensor platform.

Synthesis and characterization of naphthalimide-functionalized polynorbornenes.

ABSTRACT: Highly fluorescent and photostable (2-alkyl)-1H-benzo[de]isoquinoline-1,3(2H)-diones with a polymerizable norbornene scaffold have been synthesized and polymerized using ring-opening metathesis polymerization. The monomers presented herein could be polymerized in a living fashion, using different comonomers and different monomer ratios. All obtained materials showed good film-forming properties and bright fluorescence caused by the incorporated push-pull chromophores. Additionally, one of the monomers containing a methylpiperazine functionality showed protonation-dependent photoinduced electron transfer which opens up interesting applications for logic gates and sensing.

Gold nanoparticles in the engineering of antibacterial and anticoagulant surfaces.

Simultaneous antibacterial and anticoagulant surfaces have been prepared by immobilization of engineered gold nanoparticles onto different kinds of surfaces. The gold nanoparticle core is surrounded by a hemocompatible, anticoagulant polysaccharide, 6-O chitosan sulfate, which serves as reduction and stabilizing agent for the generation of gold nanoparticles in a microwave mediated reaction. The particle suspension shows anticoagulant activity, which is investigated by aPTT and PT testing on citrated blood samples of three patients suffering from congenital or acquired bleeding disorders. The amount of nanoparticles deposited on the surfaces is quantified by a quartz crystal microbalance with dissipation unit. All gold containing surfaces exhibit excellent antimicrobial properties against the chosen model organism, Escherichia coli MG 1655 [R1-16]. Moreover, blood plasma coagulation times of the surfaces are increased after deposition of the engineered nanoparticles as demonstrated by QCM-D.

Triggering protein adsorption on tailored cationic cellulose surfaces.

The equipment of cellulose ultrathin films with BSA (bovine serum albumin) via cationization of the surface by tailor-made cationic celluloses is described. In this way, matrices for controlled protein deposition are created, whereas the extent of protein affinity to these surfaces is controlled by the charge density and solubility of the tailored cationic cellulose derivative. In order to understand the impact of the cationic cellulose derivatives on the protein affinity, their interaction capacity with fluorescently labeled BSA is investigated at different concentrations and pH values. The amount of deposited material is quantified using QCM-D (quartz crystal microbalance with dissipation monitoring, wet mass) and MP-SPR (multi-parameter surface plasmon resonance, dry mass), and the mass of coupled water is evaluated by combination of QCM-D and SPR data. It turns out that adsorption can be tuned over a wide range (0.6-3.9 mg dry mass m(-2)) depending on the used conditions for adsorption and the type of employed cationic cellulose. After evaluation of protein adsorption, patterned cellulose thin films have been prepared and the cationic celluloses were adsorbed in a similar fashion as in the QCM-D and SPR experiments. Onto these cationic surfaces, fluorescently labeled BSA in different concentrations is deposited by an automatized spotting apparatus and a correlation between the amount of the deposited protein and the fluorescence intensity is established.

Cationically rendered biopolymer surfaces for high protein affinity support matrices.

The use of cationic biopolymer surfaces for high protein binding affinity matrices is described. As model proteins, fluorescently labeled bovine serum albumins (FITC-BSA, TRITC-BSA) have been employed. The amount of proteins on such cationically rendered surfaces was quantified by QCM-D. In addition, flexible, transparent, patterned COP slides have been prepared and loaded with proteins ranging from 15 pM to 15 µM TRITC-BSA.

A luminescence lifetime-based capillary oxygen sensor utilizing monolithically integrated organic photodiodes.

A novel optical sensor device monolithically integrated on a glass capillary is presented. Therefore, we took advantage of the ability to fabricate organic optoelectronic devices on non-planar substrates. The functionality of the concept is demonstrated by realizing an integrated oxygen sensor based on luminescence decay time measurement.

Enabling luminescence decay time-based sensing using integrated organic photodiodes.

The use of organic photodiodes (OPDs) for measuring phosphorescent lifetimes of optochemical oxygen sensors is described. Phosphorescent indicators with lifetimes ranging from ∼5 to 60 µs have been studied using light-emitting diodes as the excitation source and organic photodiodes integrated into the sensor substrate for detection. A measurement system using an adjusted electronic circuitry to detect photocurrents in the nanoampere range is presented. The response behaviour of the organic photodiodes has been characterized, and it was found that a forward (positive) bias had to be applied in order to decrease the response time of the OPDs to a range suitable for phosphorescence decay time measurements. A modulation cutoff frequency of ∼100 kHz has been determined, corresponding to a response time of the organic photodiodes of 1.6 µs. Two sensor dyes have been characterized regarding their lifetimes upon exposure to 0-20% oxygen, and it was shown that results comparable to literature data and inorganic photodetectors can be achieved.

2D crystalline protein layers as immobilization matrices for the development of DNA microarrays.

There is a growing demand for functional layers for the immobilization of (bio)molecules on different kinds of substrates in the field of biosensors, microarrays, and lab-on-a-chip development. These functional coatings should have the ability to specifically bind (bio)molecules with a high binding efficiency, while showing low unspecific binding during the following assay. In this paper we present rSbpA surface layer proteins (S-layer proteins) as a versatile immobilization layer for the development of DNA microarrays. S-layer proteins show the ability to reassemble into two-dimensional arrays on solid surfaces and their functional groups, such as carboxylic groups, are repeated with the periodicity of the lattice, allowing for immobilization of other (bio)molecules. Different fluorescently labeled amino functionalized DNA oligomers were covalently linked to the S-layer matrices to allow the characterization of DNA binding on S-layers. Hybridization and dissociation of DNA-oligomers were studied on S-layer coated slides, revealing low levels of unspecific adsorption of DNA on S-layer based immobilization matrices. In the following the principle was transferred to a DNA microarray design showing successful spotting and hybridization on whole microarray slides. Besides common laser scanning for fluorescence detection, S-layer based microarrays were evaluated with a compact, low cost platform for direct fluorescence imaging based on surface plasmon enhanced fluorescence excitation. It could be shown that S-layer protein layers are promising as immobilization matrices for the development of biosensors and microarrays.

Filter-free integrated sensor array based on luminescence and absorbance measurements using ring-shaped organic photodiodes.

An optical waveguiding sensor array featuring monolithically integrated organic photodiodes as integrated photo-detector, which simplifies the readout system by minimizing the required parts, is presented. The necessity of any optical filters becomes redundant due to the proposed platform geometry, which discriminates between excitation light and sensing signal. The sensor array is capable of measuring luminescence or absorption, and both sensing geometries are based on the identical substrate. It is demonstrated that background light is virtually non-existent. All sensing and waveguide layers, as well as in- and out-coupling elements are assembled by conventional screen-printing techniques. Organic photodiodes are integrated by layer-by-layer vacuum deposition onto glass or common polymer foils. The universal and simple applicability of this sensor chip is demonstrated by sensing schemes for four different analytes. Relative humidity, oxygen, and carbon dioxide are measured in gas phase using luminescence-based sensor schemes; the latter two analytes are also measured by absorbance-based sensor schemes. Furthermore, oxygen and pH in aqueous media were enabled. The consistency of calibration characteristics extending over different sensor chips is verified.

Functional polysaccharide conjugates for the preparation of microarrays.

A method for the immobilization of functional molecules on cellulose surfaces was developed. The irreversible deposition of the water-soluble polyelectrolyte carboxymethyl cellulose (CMC) on solid cellulose surfaces was used as a basis for this immobilization. CMC was modified using aminofluorescein (AMF) as a model compound for a functional molecule. The carbodiimide mediated coupling efficiency of AMF to CMC was studied in detail, and the functional conjugates were isolated. A quartz crystal microbalance with dissipation was employed to study the immobilization of the functionalized CMC onto cellulose model films in situ. The influence of the carbodiimide concentration, the degree of substitution, and the molecular weight of CMC on the immobilization process was investigated. Atomic force microscopy was used to characterize the changes in the surface morphology of the modified cellulose films. Finally, microspotted arrays of AMF-CMC conjugates were prepared with the knowledge obtained from the basic interaction studies. The successful deposition of AMF-CMC conjugates onto cellulose surfaces was proven by fluorescence microscopy. The conjugation of functional molecules to CMC and the subsequent deposition of these products on cellulose can be seen as a versatile method to immobilize these molecules for applications in the field of microarrays and other sensor surfaces. It offers the possibility to introduce new properties on a variety of cellulosic materials.

Wettability and surface composition of partly and fully regenerated cellulose thin films from trimethylsilyl cellulose.

The wettability and surface free energy (SFE) of partly and fully regenerated cellulose model surfaces from spin coated trimethylsilyl cellulose were determined by static contact angle (SCA) measurements. In order to gain detailed insight into the desilylation reaction of the surfaces the results from SCA measurements were compared with data from other surface analytical methods, namely thickness measurements, X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance infrared spectroscopy (ATR-IR). Additionally, the influence of ultra high vacuum treatment (UHV) during XPS measurements on the water wettability and surface morphology of regenerated cellulose thin films was investigated. The wetting of polar and non-polar liquids increased with prolonged regeneration time, which is reflected in the higher SFE values and polarities of the films. After UHV treatment the water SCA of partly regenerated films decreases, whereas fully regenerated cellulose shows a higher water SCA. Therefore it is assumed that volatile desilylation products tend to adsorb on partly regenerated films, which strongly influences their wettability.

Optical oxygen sensors based on Pt(II) porphyrin dye immobilized on S-layer protein matrices.

This paper describes the development of planar and fiber optic oxygen sensors utilizing surface layer (S-layer) proteins as immobilization matrix for oxygen sensitive dyes. S-layer proteins have the intrinsic capability to reassemble into two-dimensional arrays in suspension and at interfaces. Due to their crystalline character the distribution of functional groups, such as carboxylic groups, is repeated with the periodicity of the lattice and thus allows the reproducible and geometrically distinct binding of functional molecules. For the development of oxygen sensors an oxygen sensitive Pt(II) porphyrin dye was covalently bound to the S-layer matrix. Measurement of the oxygen concentration was performed by phase modulation fluorimetry. Setups comprising low cost optoelectronic components like LEDs and silicon photodiodes were constructed. For both sensor setups (planar and fiber optic) variations in the oxygen concentrations resulted in distinct and reproducible changes in luminescence lifetime and intensity. The luminescence quenching efficiency of these sensors was found to be 1.5-1.9 (expressed as the ratio of signal under nitrogen and air) which compares well to other sensor systems using luminophores embedded in polymer matrices. These results demonstrated the application potential of S-layers as immobilization matrices in the development of (bio-)sensors.

A planar waveguide optical sensor employing simple light coupling.

An optical sensor concept utilizing the sensing layer as the light propagating layer and a new method to couple light into a planar waveguide is presented. The concept enables simple manufacturing by coating or printing techniques and the integration of organic (plastic) opto-electronic components.

Oscillating streaming potential measurement system for macroscopic surfaces.

A method and instrumentation is described capable of streaming potential measurements of various macroscopic surfaces. It differs from other approaches due to the creation of an oscillatory flow of electrolyte solutions through or alongside the sample. This technique offers a wide range of applied flow frequency and amplitude resulting in a fast and highly accurate measurement. This enables the streaming potential detection at rather high ionic strength and in a short time regime, which allows the monitoring of adsorption processes. Streaming potential and applied pressure are measured simultaneously, together with the specific conductivity of the bulk solution, pH value, and temperature. Combining these data, the zeta potential (zeta) for many different material types (fibers, films, foils, granules, and particles) can be calculated. The apparatus comprises reliable and robust measurements, simple handling, a high degree of automation, and advanced software control. With this setup, automated pH and concentration dependent zeta-potential measurements are possible for a variety of analytes and adsorbing species (e.g., ionic strength, surfactants, polyelectrolytes, and proteins); time-resolved measurements are facilitated down to the seconds time scale. The device allows the necessary sample preparation and equilibration outside the instrument using exchangeable sample holders. This offers the opportunity of high sample throughput.

Surface thermodynamic properties of polyelectrolyte multilayers.

Multilayer architectures of polyelectrolytes fabricated by the layer-by-layer technique (LbL) on pretreated polymeric and inorganic substrates were studied by contact angle measurements. Poly(diallyldimethylammonium chloride), PDADMAC, and poly(sodium 4-styrenesulfonate), PSS, were used as polyelectrolytes. Contact angle data were used to calculate the van der Waals and Lewis acid-base components of the surface tension of the investigated surfaces. Knowledge of these quantities provides valuable information on surface composition, coating density, and possible interactions of the surface with other substances. Unusual wetting behavior of PDADMAC layers upon prewetting of the multilayer surfaces was found and described in terms of surface tension changes. A model of polymer chain rearrangement upon wetting was proposed to explain this behavior.