Advanced microarray technologies for clinical diagnostics

摘要: DNA microarrays become increasingly important in the field of clinical diagnostics. These microarrays, also called chips, are small solid substrates, typically having a maximum surface area few cm2, onto which many spots arrayed pre-determined pattern. Each these contains multiple copies one oligonucleotide, capture probe, has sequence complementary to single specific target molecule. A microarray assay is based on preferential binding between molecule with its probe present surface. Provided appropriate conditions, two stranded molecules sequences form double during process hybridization. When labeled e.g. fluorophore, fluorescent pattern achieved upon incubation sample provides information presence sample. The key advantage their large degree parallelism per due simultaneous reaction probes and sequences. This thesis focuses low density arrays that limited hundred for diagnostic use, rapid detection constrained set One major application lies identification pathogens infectious diseases. So, high containing as millions different allows screening much higher number targets simultaneously, usually applied studying gene expression studies, beyond scope this thesis. transfer microarray-based assays from research lab into clinic taken more time than anticipated it faces technical regulatory challenges. especially related quality control data reproducibility caused by lack fundamental understanding biochemical processes involved. In thesis, challenges addressed investigated, potential routes improvement identified. work technology order enable faster implementation practice. Inkjet printing suitable manufacturing microarrays. It is, however, an open-loop does therefore not provide any feedback manufactured E.g. missing unacceptable since could lead potentially false negative results. Moreover, generally being considered complexity products Clinical Laboratory Improvement Amendments (CLIA), or risk applications Food Drug Administration (FDA). Therefore measures diagnostics needed. chapter 2, closed-loop inkjet system equipped optical droplet investigate failure mechanisms described. was found all analyzed, 1.6 % jetting failed at some point time. 14 cases, failures have been detected advance changing velocities jetting. Real analysis characteristics can yield process. improved production explained 2 basis experiments carried out results experimental chapters 3-5. Many chemistries exist graft immobilization hybridization separate hampers use Immobilization rates oligonucleotides tails amine-functionalized surfaces using 254 nm UV-light studied. chemistry attractive because ease-of-use, robustness cost. method developed enables systematic study subsequent independently. efficiencies greatly influenced both UV dose composition, well length attached oligonucleotides. Hybridization mainly oligonucleotides, less composition tail observed immobilization. means within UV-window study, no significant UV-damage found. standard procedure runs follows: incubated sample, followed washing step remove non-specific binding, then dried scanned. flow implies only measurement (end) used evaluate interactions. Since interactions depend factors, including preceding steps mixture itself, complete would additional reliable data. means, measurements should be done background signals requiring deployment methods suppression. Chapter 4 describes real melting curves repeated solution through porous substrate. During each cycle, suppressed pumping liquid method, Human Papilloma Virus (HPV) model assay, evaluated samples benchmarked against commonly (Reverse Line Blot). Both show comparable results, while flow-through enabled cross-hybridizations kinetics. contribute positive signals. Polymerase Chain Reaction (PCR) amplify concentration often performed prior Sample preparation variations amplification lower array PCR concept (qPCR) integrated, thereby combining advantages methods: multiplexing capabilities quantitative PCR. formed amplicons monitored, annealing Consequently curve obtained input derived compared qPCR. After amplification, further assess specificity. levels relatively short times greatest challenge concept. confocal fluorescence scanner significantly reduced levels. proof prototype instrument designed built components known storage technology. minimize costs goods, (reagents disposables) were possible. Amplification closed chamber, minimizing risks cross-contamination reducing manual steps, making suited testing. As 10 method. 6 mathematical insights taking place. Models qPCR Langmuir adsorption combined new describing protocol. Reasonably good agreement Using result array-based identified tested increase overall efficiency resulted similar performance bulk