Emmanuel Maillart

One hundred spots parallel monitoring of DNA interactions by SPR imaging of polymer-functionalized surfaces applied to the detection of cystic fibrosis mutations

Abstract In the present paper, we report the detection of mutations implicated in human cystic fibrosis (CF). Nine different oligonucleotides are studied, including three possible mutations related to this specific genetic disease: a deletion of three bases, ΔF508, and two single-nucleotide polymorphisms 1540A/G and 1716G/A. We monitor, in real time and in parallel, hybridizations of a solution of unlabeled oligonucleotide targets to a matrix of 100 spots of oligonucleotide probes using surface plasmon resonance (SPR) imaging of a bio-functionalized gold slide. In order to functionalize our gold slide with the DNA probes, we have developed a self-assembled multilayer (SAM) based on electrostatic interactions and formed with 11-mercaptoundecanoic acid (MUA), poly(ethylenimine) (PEI) and ExtrAvidin layers. Probes are then linked to this SAM by the usual strong binding affinity of the avidin–biotin duplex. The 100 spots array deposited by a robot can be addressed either several times, sequentially, with the various oligonucleotide targets, or once, in parallel, with a mixture of some oligonucleotides. The specific response of our system is established along with the possibility of discriminating between a totally complementary sequence and its mutant form, even for a single base mismatch thus demonstrating the capacity of parallel diagnostic using patient like material.

Polypyrrole oligosaccharide array and surface plasmon resonance imaging for the measurement of glycosaminoglycan binding interactions.

In order to construct tools able to screen oligosaccharide-protein interactions, we have developed a polypyrrole-based oligosaccharide chip constructed via a copolymerization process of pyrrole and pyrrole-modified oligosaccharide. For our study, GAG (glycosaminoglycans) or GAG fragments, which are involved in many fundamental biological processes, were modified by the pyrrole moiety on their reducing end and then immobilized on the chip. The parallel binding events on the upperside of the surface can be simultaneously monitored and quantified in real time and without labeling by surface plasmon resonance imaging (SPRi). We show that electrocopolymerization of the oligosaccharide-pyrrole above a gold surface enables the covalent immobilization of multiple probes and the subsequent monitoring of their binding capacities using surface plasmon resonance imaging. Moreover, a biological application was made involving different GAG fragments and different proteins, including stromal cell-derived factor-1alpha (SDF-1alpha), interferon-gamma (IFN-gamma), and monoclonal antibody showing different affinity pattern.

Polypyrrole based DNA hybridization assays: study of label free detection processes versus fluorescence on microchips.

In this paper, we present different ways to detect DNA hybridization on a solid support. The grafting chemistry is based on the electro-controlled copolymerization of a pyrrole-modified oligonucleotide and pyrrole. This process allows an easy functionalization of conducting materials. Three kind of devices were studied: silicon chips bearing an array of addressable 50 or 4 microm microelectrodes, quartz crystal microbalance (QCM) and a non patterned gold/glass slide bearing 500 microm spots. Each device is compatible with a specific detection process: a classical indirect fluorescence detection for the microchips, a microgravimetric measurement for the QCM and a surface plasmon resonance imaging process (SPRi) for the gold slides. Both QCM and SPRi are a label-free real time detection process whereas the fluorescence methodology gives end-point data but only the fluorescence and the SPRi give multiparametric results. Although the hybridization experiments show that the detection limit for an oligonucleotide is better for the fluorescence (1-10 pM) than that found for SPRi (10 nM) and QCM (250 nM), the information content of real time measurement techniques such as SPRi is of interest for many biological studies.

Generalization of the Rouard method to an absorbing thin-film stack and application to surface plasmon resonance

In the context of surface plasmon resonance (SPR) kinetic biochips, it is important to model the SPR phenomenon (i.e., extinction of reflectivity) toward biochip design and optimization. The Rouard approach that models reflectivity off a thin-film stack is shown to be extendable to any number of absorbing layers with no added complexity. Using the generalized Rouard method, the effect of SPR is simulated as a function of the wavelength for various metal thicknesses. Given an optimal metal thickness, the dependence of SPR on the angle of incidence and wavelength is also demonstrated. Such a model constitutes a potential basis for the efficient design and optimization of multidimensional sensors.

Versatile analysis of multiple macromolecular interactions by SPR imaging: application to p53 and DNA interaction

The greatest challenge in the postgenomic era is the description of proteome interactions, such as protein–protein or protein–DNA interactions. Surface plasmon resonance (SPR) is an optical technique in which binding of an analyte to the surface changes the refractive index at the surface/solution interface. Molecular interactions are analysed in real time without a labeling step. Currently, the limit to SPR imaging is the small number of reactions that can be simultaneously analysed. Using a novel grafting technology and a new imaging system, we increased the throughput of SPR imaging. The interaction between p53 and DNA was chosen as a paradigm for validation of this assay. Using a tagged DNA methodology, we simultaneously targeted multiple DNA sequences on a single chip. The interaction between p53 and these DNA sequences was monitored by SPR imaging. Qualitative and quantitative analysis provides results similar to those obtained with conventional technologies.

Optimization of angularly resolved Bloch surface wave biosensors.

Bloch surface wave (BSW) sensors to be used in biochemical analytics are discussed in angularly resolved detection mode and are compared to surface plasmon resonance (SPR) sensors. BSW supported at the surface of a dielectric thin film stack feature many degrees of design freedom that enable tuning of resonance properties. In order to obtain a figure of merit for such optimization, the measurement uncertainty depending on resonance width and depth is deduced from different numerical models. This yields a limit of detection which depends on the sensors free measurement range and which is compared to a figure of merit derived previously. Stack design is illustrated for a BSW supporting thin film stack and is compared to the performance of a gold thin film for SPR sensing. Maximum sensitivity is obtained for a variety of stacks with the resonance positioned slightly above the TIR critical angle. Very narrow resonance widths of BSW sensors require sufficient sampling but are also associated with long surface wave propagation lengths as the limiting parameter for the performance of this kind of sensors.

Bloch surface wave enhanced biosensor for the direct detection of Angiopoietin-2 tumor biomarker in human plasma

Quantitative detection of angiogenic biomarkers provides a powerful tool to diagnose cancers in early stages and to follow its progression during therapy. Conventional tests require trained personnel, dedicated laboratory equipment and are generally time-consuming. Herein, we propose our developed biosensing platform as a useful tool for a rapid determination of Angiopoietin-2 biomarker directly from patient plasma within 30 minutes, without any sample preparation or dilution. Bloch surface waves supported by one dimensional photonic crystal are exploited to enhance and redirect the fluorescence arising from a sandwich immunoassay that involves Angiopoietin-2. The sensing units consist of disposable and low-cost plastic biochips coated with the photonic crystal. The biosensing platform is demonstrated to detect Angiopoietin-2 in plasma samples at the clinically relevant concentration of 6 ng/mL, with an estimated limit of detection of approximately 1 ng/mL. This is the first Bloch surface wave based assay capable of detecting relevant concentrations of an angiogenic factor in plasma samples. The results obtained by the developed biosensing platform are in close agreement with enzyme-linked immunosorbent assays, demonstrating a good accuracy, and their repeatability showed acceptable relative variations.

Surface plasmon resonance imaging and versatile surface functionalization for real time comparisons of biochemical interactions

Surface plasmon resonance imaging is an optical method that allows the real time detection of small changes in the physical properties of a dielectric medium near a metallic surface. Using proper surface functionalization and structuration, this technique can be applied to the realization of optical biochips where multiple unlabeled interactions can be monitored. More precisely, thanks to the use of an adequate optical set-up built around a gold surface realized by self assembled monolayers or electrocopolymerization, we studied DNA:DNA interactions with potential application to genetic diagnostic and DNA:protein interactions to demonstrate the ability of the system to determine simultaneously different affinity constants.

Label-free and fluorescence biosensing platform using one dimensional photonic crystal chips

The increasing demand for early detection of diseases drives the efforts to develop more and more sensitive techniques to detect biomarkers in extremely low concentrations. Electromagnetic modes at the surface of one dimensional photonic crystals, usually called Bloch surface waves, were demonstrated to enhance the resolution and constitute an attractive alternative to surface plasmon polariton optical biosensors. We report on the development of Bloch surface wave biochips operating in both label-free and fluorescence modes and demonstrate their use in ovalbumin recognition assays.

Multidimension potential of surface plasmon resonance imaging for dynamic surface characterization: application to optical biochip systems

We have realized a surface plasmon resonance imaging system allowing accurate characterization of biochips. In this paper, the Rouard approach is extended to absorbing layers to model the reflectivity information contained in the multidimensional data. The multidimension potential is also expressed to demonstrate the power of the SPR imaging system. To conclude, towards the development of a biosensor based on SPR, a theoretical study is also performed on the sensitivity to changes in reflectivity of such multidimension optical biosensor. The sensitivity of the sytem shows the power of this biophotonic technology.