The combination of microarray technologies with microfluidic sample delivery and real-time detection methods has the capability to simultaneously monitor 10-1000 s of biomolecular interactions in a single experiment. Despite the benefits that microfluidic systems provide, they typically operate in the laminar flow regime under mass transfer limitations, where large analyte depletion layers act as a resistance to analyte capture. By locally stirring the fluid and delivering fresh analyte to the capture spot, the use of passive mixing structures in a microarray environment can reduce the negative effects of these depletion layers and enhance the sensor performance. Despite their large potential, little attention has been given to the integration of these mixing structures in microarray sensing environments. In this study, we use passive mixing structures to enhance the mass transfer of analyte to a capture spot within a microfluidic flow cell. Using numerical methods, different structure shapes and heights were evaluated as means to increase local fluid velocities, and in turn, rates of mass transfer to a capture spot. These results were verified experimentally via the real-time detection of 20-mer ssDNA for an array of microspots. Both numerical and experimental results showed that a passive mixing structure situated directly over the capture spot can significantly enhance the binding rate of analyte to the sensing surface. Moreover, we show that these structures can be used to enhance mass transfer in experiments regarding an array of capture spots. The results of this study can be applied to any experimental system using microfluidic sample delivery methods for microarray detection techniques.
- MeSH
- Biomedical Research MeSH
- Databases, Genetic MeSH
- Research Support as Topic MeSH
- Financing, Organized MeSH
- Clinical Medicine MeSH
- Humans MeSH
- Oligonucleotide Array Sequence Analysis MeSH
- Software MeSH
- Gene Expression Profiling MeSH
- Systems Integration MeSH
- Database Management Systems MeSH
- Information Storage and Retrieval MeSH
- Computational Biology MeSH
- Check Tag
- Humans MeSH
Klinická onkologie, ISSN 0862-495X Ročník 19, supplement 2006, prosinec 2006
330-422 stran : ilustrace, tabulky ; 30 cm
- MeSH
- Molecular Targeted Therapy MeSH
- Molecular Diagnostic Techniques MeSH
- Precision Medicine MeSH
- Medical Oncology MeSH
- Microarray Analysis MeSH
- Research MeSH
- Publication type
- Collected Work MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- onkologie
Wilsonova choroba (WCH) je závažné, autozomálně recesivní onemocnění, jehož podstatou jsou mutace v ATP7B genu, který kóduje měď-specifickou ATPázu. U postižených jedinců dochází k poruše vylučování toxické mědi z organismu a k jejímu hromadění v tělesných orgánech. Molekulární diagnostika Wilsonovy choroby je důležitou součástí stanovení správné diagnózy. Cílem práce bylo navrhnout a zvalidovat genotypovací DNA čip, který umožňuje současně analyzovat 87 mutací a 17 polymorfismů v ATP7B genu. Metody a výsledky. V první fázi validace bylo testováno 97 WCH pacientů se známým genotypem a 46 vzorků uměle připravených mutagenezí. Všechny analyzované sekvenční varianty byly detekovány se 100% správností. Ve druhé fázi validace byly testovány reálné vzorky WCH suspektních pacientů. Dosud jsme zanalyzovali 58 nepříbuzných pacientů, z nichž u 10 byla čipovou analýzou potvrzena diagnóza WCH, u 13 byla nalezena jedna mutace a u 35 žádná. U pacientů s jednou nebo žádnou detekovanou mutací následovalo přímé sekvenování kódující oblasti genu ATP7B, přičemž nebyla nalezena žádná další kauzální mutace. Závěry. Wilsonův čip se jeví jako rychlá a spolehlivá vyhledávací metoda mutací v ATP7B genu.
Wilson disease (WD) is a serious autosomal recessive disorder caused by mutations in ATP7B-gene which encodes a copper-specific ATPase. WD patients suffer from impaired biliary excretion of copper from organism and its' accumulation in body organs. Molecular diagnostics of WD is an important part of correct diagnosis statement. The aim of the study was to design and validate a genotyping DNA microarray which enables to analyze 87 mutations and 17 polymorphisms in ATP7B gene, simultaneously. Methods and Results. 97 WD patients with known genotypes and 46 samples prepared by mutagenesis were tested in the first phase of chip validation. All analyzed sequence variants were detected with 100% accuracy. Samples from WD suspected patients were tested in the second phase of validation. We have analyzed 58 unrelated patients, yet. The diagnosis of WD was confirmed in 10 patients, 13 patients were heterozygous for some mutation and 35 had no mutation in ATP7B gene. Samples with one or no mutation found by microarray analysis were sequenced directly and no further causal mutation was revealed. Conclusions. Wilson chip seems to be a fast and reliable method for screening of mutations in ATP7B gene.
xiii, 647 stran : ilustrace
- MeSH
- Chromosome Aberrations * MeSH
- Cytogenetic Analysis MeSH
- In Situ Hybridization, Fluorescence MeSH
- Microarray Analysis MeSH
- Neoplasms pathology MeSH
- Terminology as Topic MeSH
- Publication type
- Monograph MeSH
- Conspectus
- Biochemie. Molekulární biologie. Biofyzika
- NML Fields
- molekulární biologie, molekulární medicína
Array srovnávací genomická hybridizace detekuje signifikantně větší množství i menších abnormalit než standardní chromosomová analýza. V určitých případech může tento test nahradit karyotypizaci v prenatální diagnostice genetických poruch.
- MeSH
- Chromosome Disorders diagnosis genetics MeSH
- DNA analysis MeSH
- In Situ Hybridization, Fluorescence methods utilization MeSH
- Karyotyping methods MeSH
- Clinical Laboratory Techniques MeSH
- Humans MeSH
- Prenatal Diagnosis methods MeSH
- Sequence Analysis, DNA methods trends utilization MeSH
- Pregnancy MeSH
- Check Tag
- Humans MeSH
- Pregnancy MeSH
- Female MeSH
