Childérick Séverac

Detection of label-free cancer biomarkers using nickel nanoislands and quartz crystal microbalance

We present a technique for the label-free detection and recognition of cancer biomarkers using metal nanoislands intended to be integrated in a novel type of nanobiosensor. His-tagged (scFv)-F7N1N2 is the antibody fragment which is directly immobilized, by coordinative bonds, onto ~5 nm nickel islands, then deposited on the surface of a quartz crystal of a quartz crystal microbalance (QCM) to validate the technique. Biomarker GTPase RhoA was investigated because it has been found to be overexpressed in various tumors and because we have recently isolated and characterized a new conformational scFv which selectively recognizes the active form of RhoA. We implemented a surface chemistry involving an antibiofouling coating of polyethylene glycol silane (PEG-silane) (<2 nm thick) and Ni nanoislands to reach a label-free detection of the active antigen conformation of RhoA, at various concentrations. The methodology proposed here proves the viability of the concept by using Ni nanoislands as an anchoring surface layer enabling the detection of a specific conformation of a protein, identified as a potential cancer biomarker. Hence, this novel methodology can be transferred to a nanobiosensor to detect, at lower time consumption and with high sensitivity, specific biomolecules.

Automated and Multiplexed Soft Lithography for the Production of Low-Density DNA Microarrays.

Microarrays are established research tools for genotyping, expression profiling, or molecular diagnostics in which DNA molecules are precisely addressed to the surface of a solid support. This study assesses the fabrication of low-density oligonucleotide arrays using an automated microcontact printing device, the InnoStamp 40®. This automate allows a multiplexed deposition of oligoprobes on a functionalized surface by the use of a MacroStampTM bearing 64 individual pillars each mounted with 50 circular micropatterns (spots) of 160 µm diameter at 320 µm pitch. Reliability and reuse of the MacroStampTM were shown to be fast and robust by a simple washing step in 96% ethanol. The low-density microarrays printed on either epoxysilane or dendrimer-functionalized slides (DendriSlides) showed excellent hybridization response with complementary sequences at unusual low probe and target concentrations, since the actual probe density immobilized by this technology was at least 10-fold lower than with the conventional mechanical spotting. In addition, we found a comparable hybridization response in terms of fluorescence intensity between spotted and printed oligoarrays with a 1 nM complementary target by using a 50-fold lower probe concentration to produce the oligoarrays by the microcontact printing method. Taken together, our results lend support to the potential development of this multiplexed microcontact printing technology employing soft lithography as an alternative, cost-competitive tool for fabrication of low-density DNA microarrays.

Methods of Micropatterning and Manipulation of Cells for Biomedical Applications

Micropatterning and manipulation of mammalian and bacterial cells are important in biomedical studies to perform in vitro assays and to evaluate biochemical processes accurately, establishing the basis for implementing biomedical microelectromechanical systems (bioMEMS), point-of-care (POC) devices, or organs-on-chips (OOC), which impact on neurological, oncological, dermatologic, or tissue engineering issues as part of personalized medicine. Cell patterning represents a crucial step in fundamental and applied biological studies in vitro, hence today there are a myriad of materials and techniques that allow one to immobilize and manipulate cells, imitating the 3D in vivo milieu. This review focuses on current physical cell patterning, plus chemical and a combination of them both that utilizes different materials and cutting-edge micro-nanofabrication methodologies.