Innovative Integrated System for Real-Time Measurement of Hybridization and Melting on Standard Format Microarrays

Abstract

Despite the great popularity and potential of microarrays, their use for research and clinical applications is still hampered by lengthy and costly design and optimization processes, mainly because the technology relies on the end point measurement of hybridization. Thus, the ability to monitor many hybridization events on a standard microarray slide in real time would greatly expand the use and benefit of this technology, as it would give access to better prediction of probe performance and improved optimization of hybridization parameters. Although real-time hybridization and thermal denaturation measurements have been reported, a complete walk-away system compatible with the standard format of microarrays is still unavailable. To address this issue, we have designed a biochip tool that combines a hybridization station with active mixing capability and temperature control together with a fluorescence reader in a single compact benchtop instrument. This integrated live hybridization machine (LHM) allows measuring in real time the hybridization of target DNA to thousands of probes simultaneously and provides excellent levels of detection and superior sequence discrimination. Here we show on an environmental single nucleotide polymorphism (SNP) model system that the LHM enables a variety of experiments unachievable with conventional biochip tools.

Protein–surface interactions: challenging experiments and computations

“…In addition to SPR, high-throughput fluorescent-based detection (Marcy et al, 2008) can be adapted to study protein adsorption (Claude Weisbuch, Ecole Polytechnique, France and UCSB, USA). Biochips with spatially selective adsorption of different proteins (Weisbuch; François Rossi, IHCP Joint Research Centre, EU) or surface spots with variable chemistry/surface morphology can be used in this format (Rossi; Paolo Milani, Univ.…”

Highly sensitive and reusable fluorescence microarrays based on hydrogenated amorphous silicon–carbon alloys

“…It is especially dramatic if the fluorophore is located at the surface of a metallic reflector (case |n| 1), i.e., at a node of the light electric field. On the opposite, a fluorophore located at a distance of /4 from the reflector (where is light wavelength in the spacer medium) lies at an anti-node of the light electric field, which may lead to an enhancement factor F as high as an order of magnitude (F 1) (Marcy et al, 2008). This has led us to explore the design schematized in Fig.…”

“…Several cycles of hybridization/dehybridization were monitored in situ (Marcy et al, 2008), using a Hyblive system (a real-time microarray platform from Genewave). The dehybridization was performed by in situ heating of the Hyblive chamber to 50 • C under a flowing mixture of formamide and 2.5X SSC (saline sodium citrate) (50:50 by vol.).…”

“…Improvements in the optical design, by using a back reflector located at a quarter wavelength behind the active surface, have pushed their sensitivity up to a level close to single-molecule detection capability (Choumane et al, 2005;Fouqué et al, 2007;Marcy et al, 2008). The immobilization of the biomolecule probes is generally performed on glass slides via hydrophobic or electrostatic interactions (Schena et al, 1995;Angenendt et al, 2003) or via iono-covalent bonding with a Si-O-Si-C link (Lindroos et al, 2001;Saldarriaga Fernández et al, 2007).…”

A mixed film composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) brushes to enhance selectivity for detection of single nucleotide polymorphisms

“…Therefore, rapid, selective, and sensitive tools are needed for the identification and detection of SNPs. Since established molecular biology techniques which screen for SNPs involve amplification and separation steps that are time-consuming and laborious [8,9], a number of sensor and microarray detection strategies have been reported based on colourimetric [10,11], electrochemical [12][13][14][15], and optical [8,[16][17][18][19][20] detection. Some of these involve large scale parallel analysis of samples for SNPs, example of sharpening of melting transitions has previously been demonstrated by Taton et al by implementation of gold nanoparticles [10].…”