Microfluidic device for rapid (<15 min) automated microarray hybridization.

BACKGROUND: Current hybridization protocols on microarrays are slow and need skilled personnel. Microfluidics is an emerging science that enables the processing of minute volumes of liquids to perform chemical, biochemical, or enzymatic analyzes. The merging of microfluidics and microarray technologies constitutes an elegant solution that will automate and speed up microarray hybridization. METHODS: We developed a microfluidic flow cell consisting of a network of chambers and channels molded into a polydimethylsiloxane substrate. The substrate was aligned and reversibly bound to the microarray printed on a standard glass slide to form a functional microfluidic unit. The microfluidic units were placed on an engraved, disc-shaped support fixed on a rotational device. Centrifugal forces drove the sample and buffers directly onto the microarray surface. RESULTS: This microfluidic system increased the hybridization signal by approximately 10fold compared with a passive system that made use of 10 times more sample. By means of a 15-min automated hybridization process, performed at room temperature, we demonstrated the discrimination of 4 clinically relevant Staphylococcus species that differ by as little as a single-nucleotide polymorphism. This process included hybridization, washing, rinsing, and drying steps and did not require any purification of target nucleic acids. This platform was sensitive enough to detect 10 PCR-amplified bacterial genomes. CONCLUSION: This removable microfluidic system for performing microarray hybridization on glass slides is promising for molecular diagnostics and gene profiling.

Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid.

The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.

Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support.

BACKGROUND: Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization. RESULTS: Using surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray. CONCLUSION: This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.