1 online zdroj
- MeSH
- Informatics MeSH
- Microarray Analysis instrumentation MeSH
- Sequence Analysis * instrumentation MeSH
- Publication type
- Periodical MeSH
- Conspectus
- Teorie systémů. Automatické systémy. Informační systémy. Kybernetika
- NML Fields
- přírodní vědy
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.
Multiplexní analýza umožňuje současné stanovení více analytů v tomtéž vzorku. Tyto postupy, původně rozšířené především v genomice, nyní významně pronikají i do dalších oblastí bioanalytiky. V analýze proteinů hrají zásadní roli imunochemické metody a jejich multiplexace a miniaturizace je široce využitelná jak v základním, tak v aplikovaném výzkumu. Také možnosti uplatnění těchto vysokokapacitních metod v laboratorní diagnostice jsou značné. Text popisuje dva hlavní přístupy k multiplexní imunoanalýze – provedení v planárním a suspenzním uspořádání. Krátce diskutuje principy, detekční metody a přednosti obou těchto strategií. V závěru jsou zmíněna některá úskalí spojená s integrací multiplexní analýzy do diagnostiky.
Multiplex analysis enables a simultaneous determination of multiple targets in one sample. This approach has been largely adopted in genomics and progressively expands to various domains of bioanalytics. In protein analysis, immunoassays play a fundamental role and their multiplexing and miniaturization is of great applicability to both basic and applied research. Furthermore, these high-throughput methodologies have a considerable potential in the field of laboratory diagnostics. The following text describes planar and bead-based arrays, two main strategies of immunoassay multiplexing. Principles, detection methods and strengths of each are shortly discussed. Finally, we mention several challenges linked with the integration of these methods to diagnostics.
Sdělení podává informace o novém přístupu k biochemickým analýzám. Dále shrnuje principy, použití, možné výhody či nevýhody nových multiplexních technologií. Většina prezentovaných metod je v současné době buď ve fázi vývoje, nebo je již používána v rámci výzkumných projektů. Pouze některé technologie postupně pronikají do laboratoří klinické biochemie.
The publication reports recent approach to biochemical analyses. There are summarized data about principles, usages, advantages or disadvantages of new multiplex technologies. Although, in the meantime, the most of presented methods are in development or they are used in research projects some technologies already expand to clinical biochemistry laboratories.
Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism that is caused by mutations in the ATP7B gene. To date, more than 300 mutations have been described in this gene. Molecular diagnostics of WD utilizes restriction enzyme digestion, multiplex ligation-dependent probe amplification or a direct sequencing of the whole gene. To simplify and speed up the screening of ATP7B mutations, we have developed a genotyping microarray for the simultaneous detection of 87 mutations and 17 polymorphisms in the ATP7B gene based on the arrayed primer extension reaction. The patient's DNA is amplified in four multiplex polymerase chain reactions, fragmented products are annealed to arrayed primers spotted on a chip, which enables DNA polymerase extension reactions with fluorescently labeled dideoxynucleotides. The Wilson microarray was validated by screening 97 previously genetically confirmed WD patients. In total, we detected 43 mutations and 15 polymorphisms that represent a majority of the common mutations occurring in the Czech and Slovak populations. All screened sequence variants were detected with 100% accuracy. The Wilson chip appears to be a rapid, sensitive and cost-effective tool, representing the prototype of a disease chip that facilitates and speeds up the screening of potential WD patients.
- MeSH
- Adenosine Triphosphatases genetics MeSH
- Point Mutation MeSH
- Genetic Carrier Screening methods MeSH
- Financing, Organized MeSH
- Genotype MeSH
- Hepatolenticular Degeneration diagnosis genetics MeSH
- Heterozygote MeSH
- Humans MeSH
- Microarray Analysis methods instrumentation MeSH
- Mutation MeSH
- DNA Mutational Analysis MeSH
- Cation Transport Proteins genetics MeSH
- Check Tag
- Humans MeSH
- MeSH
- Biocompatible Materials chemistry radiation effects MeSH
- Biological Assay methods instrumentation MeSH
- Cell Adhesion physiology MeSH
- Cell Line MeSH
- Endothelial Cells cytology physiology MeSH
- Factor VIII metabolism MeSH
- Research Support as Topic MeSH
- Photochemistry methods MeSH
- Kidney cytology embryology physiology MeSH
- Humans MeSH
- Microarray Analysis methods instrumentation MeSH
- Polytetrafluoroethylene analysis chemistry radiation effects MeSH
- Surface Properties MeSH
- Cell Proliferation MeSH
- Recombinant Proteins metabolism MeSH
- Gene Expression Profiling methods MeSH
- Light MeSH
- Cell Survival MeSH
- Check Tag
- Humans MeSH
- Publication type
- Comparative Study MeSH
- MeSH
- Gene Expression * MeSH
- Factor Xa MeSH
- Data Interpretation, Statistical MeSH
- Colorectal Neoplasms * genetics MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Humans MeSH
- Neoplasm Metastasis MeSH
- Microarray Analysis instrumentation MeSH
- Biomarkers, Tumor MeSH
- Nicotinamide N-Methyltransferase MeSH
- Image Processing, Computer-Assisted MeSH
- Oligonucleotide Array Sequence Analysis * MeSH
- Software MeSH
- Statistics as Topic MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
