Thomas Brandstetter

Simple One-Step Process for Immobilization of Biomolecules on Polymer Substrates Based on Surface-Attached Polymer Networks

For the miniaturization of biological assays, especially for the fabrication of microarrays, immobilization of biomolecules at the surfaces of the chips is the decisive factor. Accordingly, a variety of binding techniques have been developed over the years to immobilize DNA or proteins onto such substrates. Most of them require rather complex fabrication processes and sophisticated surface chemistry. Here, a comparatively simple immobilization technique is presented, which is based on the local generation of small spots of surface attached polymer networks. Immobilization is achieved in a one-step procedure: probe molecules are mixed with a photoactive copolymer in aqueous buffer, spotted onto a solid support, and cross-linked as well as bound to the substrate during brief flood exposure to UV light. The described procedure permits spatially confined surface functionalization and allows reliable binding of biological species to conventional substrates such as glass microscope slides as well as various types of plastic substrates with comparable performance. The latter also permits immobilization on structured, thermoformed substrates resulting in an all-plastic biochip platform, which is simple and cheap and seems to be promising for a variety of microdiagnostic applications.

Evaluation of methods to detect p53 mutations in ovarian cancer

Objective: The p53 status is increasingly regarded as a marker predictive of response to particular cancer therapies, but for this approach it is self-evident that the p53 status must be determined correctly. Methods: We have tested ovarian cancers with single-strand conformation polymorphism analysis (SSCP), immunohistochemical staining with DO-1 anti-p53 antibody (IHC), and yeast p53 functional assay (FASAY). Results: These techniques commonly used to detect p53 mutations showed important differences in their sensitivity. Of 53 tumors tested with three indirect techniques, 27 (50%), 33 (62%) and 41 (77%) were positive by SSCP, IHC, and FASAY, respectively. In a subset of 32 tumors strongly suspected of containing mutations, 25 (78%), 26 (81%), 29 (91%) and 30 (94%) were positive by SSCP, immunostaining, DNA sequencing and yeast assay, respectively. Conclusions: Under comparable routine conditions, the FASAY reached the highest sensitivity. Since no single technique detected all mutations, we recommend the use of at least two different techniques in situations where the p53 status will affect patient management.

A polymer-based DNA biochip platform for human papilloma virus genotyping

Genotyping of the human papilloma virus (HPV) is from a clinical point of view an important diagnostic task as some genotypes play a major role in the development of cervical carcinoma. So far PCR combined with blotting or in situ labelling is known to be the most accurate and sensitive method for detection and genotyping of HPV infection in clinical samples. However, specificity, cost-efficiency and sensitivity are not always satisfactory. A novel DNA biochip is described based on a plastic substrate, onto which small polymer droplets and single-stranded DNA are printed in the form of microarrays. Immobilisation of all compounds on the chip surface is achieved by a short UV-irradiation process, inducing photochemical reactions in the polymer. The chip designed for this study contains 36 probes for determining 12 common, different HPV genotypes. After isolation of the DNA, PCR and biochip read-out, the chip allows for genotyping of the most common virus strains, which, according to current prevalence studies, cover 85-95% of all infections. Following this approach as little as 10 virus copies can be detected within a short exposure time. Even using paraffin-embedded material and 10(4) copies per PCR are sufficient to allow rapid and reliable HPV genotyping.

Performance of a Polymer-Based DNA Chip Platform in Detection and Genotyping of Human Papillomavirus in Clinical Samples

ABSTRACT Human papillomavirus (HPV) plays a key role in the development of cervical and laryngeal cancers. The aim of our study was to compare the performance of a new hydrogel-based HPV genotyping biochip assay (Biochip) to a commercially available and CE-marked conventional PCR followed by reverse hybridization (GenID-PCR). One hundred twenty-three samples were available for the study. Of these samples, 101/123 were gynecological swabs, 8/123 were swabs or biopsy samples of genital warts, 7/123 were biopsy samples of otorhinolaryngeal lesions, 5/123 were samples of skin warts, and 2/123 were samples of orolabial abnormalities. These molecular methods for HPV genotyping showed comparable sensitivity and specificity. However, 19/123 of the results were discrepant. Specifically, Biochip showed better performance in the detection of multiple infections, especially when more than one high-risk genotype was present. Due to the different probe configurations used in the two assays, GenID-PCR achieves only group-specific detection of many HPV genotypes, whereas Biochip allows for specific identification. Overall, the newly developed HPV chip system (Biochip) proved to be a suitable tool for HPV detection and genotyping; it also proved to be superior for establishing HPV genotyping methods.

Sensitivity of microarray based immunoassays using surface-attached hydrogels

A promising pathway to improve on the sensitivity of protein microarrays is to immobilize the capture antibodies in a three dimensional hydrogel matrix. We describe a simple method based on printing of an aqueous protein solution containing a photosensitive polymer and the capture antibody onto a plastic chip surface. During short UV-exposure photocrosslinking occurs, which leads to formation of a hydrogel, which is simultaneously bound to the substrate surface. In the same reaction the antibody becomes covalently attached to the forming hydrogel. As the capture antibodies are immobilized in the three-dimensional hydrogel microstructures, high fluorescence intensities can be obtained. The chip system is designed such, that non-specific protein adsorption is strongly prevented. Thus, the background fluorescence is strongly reduced and very high signal-to-background ratios are obtained (SBR>6 for c(BSA)=1 pM; SBR>100 for c(BSA)>100 pM). The kinetics of antigen binding to the arrayed antibodies can be used to determine the concentration of a specific protein (for example the tumor marker β2-microglobulin) in solution for a broad range of analyte concentrations. By varying size and composition of the protein-filled hydrogel microstructures as well as adjusting the extent of labeling it is possible to easily adapt the surface concentration of the probe molecules such that the fluorescence signal intensity is tuned to the prevalence of the protein in the analyte. As a consequence, the signal tuning allows to analyze solutions, which contain both proteins with high (here: upper mg mL(-1) range) and with very low concentrations (here: lower μg mL(-1) range). This way quantitative analysis with an exceptionally large dynamic range can be performed.

Polysaccharide microarrays with a CMOS based signal detection unit

Microarray based test assays have become increasingly important tools in diagnostics for fast multi-parameter detection especially where sample volumes are limited. We present here a simple procedure to create polysaccharide microarrays, which can be used to analyze antibodies using an integrated, complementary metal-oxide-semiconductor (CMOS) based electric signal readout process. To accomplish this chips are used which consist of an array of silicon photodiodes and where different types of polysaccharides from the bacteria Streptococcus pneumoniae are printed on the (silicon dioxide) chip surface. Typical amounts of polysaccharide deposited in the printing process are around 12 attomol/spot. In a subsequent reaction step the polysaccharide microarrays were used for the measurement of IgG antibody concentrations in human blood sera using either chemiluminescence or fluorescence based detection. To understand the device performance the influence of surface density of the immobilized polysaccharide molecules and other parameters on the assay performance are investigated. The dynamic measurement range of the sensor is shown to reach over more than 3 decades of concentration and covers the whole physiologically relevant range for the analysis of antibodies against a large panel of pneumococcal polysaccharides.

Temperature and time-resolved total internal reflectance fluorescence analysis of reusable DNA hydrogel chips.

Total internal reflection fluorescence (TIRF) coupled with hydrogel-DNA droplet microarrays covalently bound on PMMA substrates presents a reusable, sensitive platform for evaluating DNA hybridization and for rapid biochip development. Hydrogel microarrays, which contain covalently bound DNA probes, are created via a simple printing and photocross-linking process. TIRF measurements of the arrays display robust reusability, show linear sensitivity down to 5 fmol of fluorescently labeled target DNA, and are sensitive to single basepair mismatches. Additionally, the ability to interrogate larger DNA is shown through studies with PCR amplification hybridization. We conclusively demonstrate an efficient, reproducible, low cost platform for DNA hybridization studies that could be used for fast high-throughput diagnostics as well as biochip development.

rhG-CSF affects genes involved in mitogen signalling and early gene expression in the ovarian cancer cell line HEY

The ovarian adenocarcinoma cell line HEY was used as an in vitro model to study the influence of recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) on epithelial tumours such as ovarian cancer. Serum‐starved cells were treated with rhG‐CSF in a time‐ and dose‐dependent manner. Cell proliferation, measured as cell division and DNA synthesis, was stimulated about 40% by rhG‐CSF. After harvesting, cells were examined for the presence of G‐CSF receptor (FACS analysis and RT‐PCR), as well as for expression of genes involved in mitogen signalling (ERKs, JNKs) and early gene expression (c‐jun). rhG‐CSF affected mitogen‐activated pathways and was receptor‐mediated if the G‐CSF receptor was present. After rhG‐CSF induction, Janus N‐terminal kinases (JNK 1 and 2) were simultaneously increased in the cytosol, up to 30‐fold as measured by Western blotting), whereas ERK 1 and 2 accumulated maximally by 2.5‐fold 1 hr after rhG‐CSF induction. c‐Jun was up‐regulated strongly by this cytokine at the translational level. Our data suggest that rhG‐CSF affects genes involved in mitogen signalling and early gene expression in solid tumours. We also noted the presence of G‐CSF receptor on ovarian cancer cell lines. Int. J. Cancer 75:847–854, 1998.

Universal nucleic acid sequence-based amplification for simultaneous amplification of messengerRNAs and microRNAs

A universal NASBA assay is presented for simultaneous amplification of multiple microRNA (miRNA) and messengerRNA (mRNA) sequences. First, miRNA and mRNA sequences are reverse transcribed using tailed reverse transcription primer pairs containing a gene-specific and an non-specific region. For reverse transcription of small miRNA molecules a non-specific region is incorporated into a structured stem-loop reverse transcription primer. Second, a universal NASBA primer pair that recognizes the tagged cDNA molecules enables a simultaneous, transcription-based amplification reaction (NASBA) of all different cDNA molecules in one reaction. The NASBA products (RNA copies) are detected by gene-specific DNA probes immobilized on a biochip. By using the multiplex reverse transcription combined with the universal NASBA amplification up to 14 different mRNA and miRNA sequences can be specifically amplified and detected in parallel. In comparison with standard multiplex NASBA assays this approach strongly enhances the multiplex capacity of NASBA-based amplification reactions. Furthermore simultaneous assaying of different RNA classes can be achieved that might be beneficial for studying miRNA-based regulation of gene expression or for RNA-based tumor diagnostics.

A lab-on-a-chip system for the development of complex assays using modular microfluidic components

For complex biological or diagnostic assays, the development of an integrated microfluidic device can be difficult and error-prone. For this reason, a modular approach, using individual microfluidic functional modules for the different process steps, can be advantageous. However often the interconnection of the modules proves to be tedious and the peripheral instrumentation to drive the various modules is cumbersome and of large size. For this reason, we have developed an integrated instrument platform which has generic functionalities such as valves and pumps, heating zones for continuous-flow PCR, moveable magnets for bead-based assays and an optical detection unit build into the instrument. The instrument holds a titerplate-sized carrier in which up to four microscopy-slide sized microfluidic modules can be clipped in. This allows for developing and optimizing individual assay steps without the need to modify the instrument or generate a completely new microfluidic cartridge. As a proof-of-concept, the automated sample processing of liquor or blood culture in microfluidic structures for detection of currently occuring Neisseria meningitidis strains was carried out. This assay involves the extraction of bacterial DNA, the fluorescent labeling, amplification using PCR as well as the hybridization of the DNA molecules in three-dimensional capture sites spotted into a microchannel. To define the assay sensitivity, chip modules were tested with bacteria spiked samples of different origins and results were controlled by conventional techniques. For liquor or blood culture, the presence of 200 bacteria was detected within 1 hour.