Thérèse Leblois

RECONSTITUTION OF A PROTEIN MONOLAYER ON THIOLATES FUNCTIONALIZED GaAs SURFACE

In the aim to realize an efficient resonant biosensor, gallium arsenide (GaAs) presents many advantages. In addition to its properties of transduction, GaAs is a crystal for which microfabrication processes were developed, conferring the possibility to miniaturize the device and integrate electronic circuit. Moreover, the biofunctionalization could be realized on the crystalline surface without layer deposition, constituting a real advantage to perform reusable sensor. The functionalization of GaAs surface was engaged in order to immobilize a protein monolayer on this substrate. Functionalization was done using a mixed self assembled monolayer of thiolate molecules. Characterizations at micro and nanoscale were performed to control the surface state, the establishment of thiolates self-assembled monolayer, the surface atomic composition and the topography of the GaAs substrate at the different steps of the process. Protein immobilization on thiolates modified GaAs was revealed through a detailed AFM study and in situ MALDI-TOF MS and MS/MS modified surface interrogations.

Influence of a Thiolate Chemical Layer on GaAs (100) Biofunctionalization: An Original Approach Coupling Atomic Force Microscopy and Mass Spectrometry Methods

Widely used in microelectronics and optoelectronics; Gallium Arsenide (GaAs) is a III-V crystal with several interesting properties for microsystem and biosensor applications. Among these; its piezoelectric properties and the ability to directly biofunctionalize the bare surface, offer an opportunity to combine a highly sensitive transducer with a specific bio-interface; which are the two essential parts of a biosensor. To optimize the biorecognition part; it is necessary to control protein coverage and the binding affinity of the protein layer on the GaAs surface. In this paper; we investigate the potential of a specific chemical interface composed of thiolate molecules with different chain lengths; possessing hydroxyl (MUDO; for 11-mercapto-1-undecanol (HS(CH2)11OH)) or carboxyl (MHDA; for mercaptohexadecanoic acid (HS(CH2)15CO2H)) end groups; to reconstitute a dense and homogeneous albumin (Rat Serum Albumin; RSA) protein layer on the GaAs (100) surface. The protein monolayer formation and the covalent binding existing between RSA proteins and carboxyl end groups were characterized by atomic force microscopy (AFM) analysis. Characterization in terms of topography; protein layer thickness and stability lead us to propose the 10% MHDA/MUDO interface as the optimal chemical layer to efficiently graft proteins. This analysis was coupled with in situ MALDI-TOF mass spectrometry measurements; which proved the presence of a dense and uniform grafted protein layer on the 10% MHDA/MUDO interface. We show in this study that a critical number of carboxylic docking sites (10%) is required to obtain homogeneous and dense protein coverage on GaAs. Such a protein bio-interface is of fundamental importance to ensure a highly specific and sensitive biosensor.

Formation Kinetics of Mixed Self-Assembled Monolayers of Alkanethiols on GaAs(100)

We report on the formation kinetics of mixed self-assembled monolayers (SAMs) comprising 16-mercaptohexadecanoic acid (MHDA) and 11-mercapto-1-undecanol (MUDO) thiols on GaAs(100) substrates. These compounds were selected for their potential in constructing highly selective and efficient architectures for biosensing applications. The molecular composition and quality of one-compound and mixed SAMs were determined by the Fourier transform infrared absorption spectroscopy measurements. The formation of enhanced-quality mixed SAMs was investigated as a function of the molecular composition of the thiol mixture and the proportion of ethanol/water solvent used during their arrangement. Furthermore, the formation of mixed SAMs has been carried out by successive immersion of MHDA SAMs in MUDO thiol solutions and MUDO SAMs in MHDA thiol solution through the process involving thiol-thiol substitution. Our results, in addition to confirming that water-ethanol-based solvents improve the packing density of single thiol monolayers, demonstrate the attractive role of water-ethanol solvents in forming superior quality mixed SAMs.

Regeneration of a thiolated and antibody functionalized GaAs (001) surface using wet chemical processes

Wet chemical processes were investigated to remove alkanethiol self-assembled monolayers (SAMs) and regenerate GaAs (001) samples studied in the context of the development of reusable devices for biosensing applications. The authors focused on 16-mercaptohexadecanoic acid (MHDA) SAMs that are commonly used to produce an interface between antibodies or others proteins and metallic or semiconductor substrates. As determined by Fourier transform infrared absorption spectroscopy, among the investigated solutions of HCl, H2O2, and NH4OH, the highest efficiency in removing alkanethiol SAM from GaAs was shown by NH4OH:H2O2 (3:1 volume ratio) diluted in H2O. The authors observed that this result was related to chemical etching of GaAs that even in a weak solution of NH4OH:H2O2:H2O (3:1:100) proceeded at a rate of 130 nm/min. The surface revealed by a 2-min etching under these conditions allowed depositing successfully a new MHDA SAM with comparable quality and density to the initial coating. This work provides an important view on the perspective of the development of a family of cost-effective GaAs-based biosensors designed for repetitive detection of a variety of biomolecules immobilized with dedicated antibody architectures.

A Fluidic Interface with High Flow Uniformity for Reusable Large Area Resonant Biosensors

Resonant biosensors are known for their high accuracy and high level of miniaturization. However, their fabrication costs prevent them from being used as disposable sensors and their effective commercial success will depend on their ability to be reused repeatedly. Accordingly, all the parts of the sensor in contact with the fluid need to tolerate the regenerative process which uses different chemicals (H3PO4, H2SO4 based baths) without degrading the characteristics of the sensor. In this paper, we propose a fluidic interface that can meet these requirements, and control the liquid flow uniformity at the surface of the vibrating area. We study different inlet and outlet channel configurations, estimating their performance using numerical simulations based on finite element method (FEM). The interfaces were fabricated using wet chemical etching on Si, which has all the desirable characteristics for a reusable biosensor circuit. Using a glass cover, we could observe the circulation of liquid near the active surface, and by using micro-particle image velocimetry (μPIV) on large surface area we could verify experimentally the effectiveness of the different designs and compare with simulation results.

Design and experimental studies of Gallium Arsenide bulk acoustic wave transducer under lateral field excitation

We have decided to develop high sensitive piezoelectric sensors in Gallium Arsenide (GaAs) for biological detection in liquid environment. The lateral field excitation is used to generate bulk acoustic waves through GaAs(001) membranes. This crystallographic plane is used to obtain the highest coupling coefficient. Gallium Arsenide presents interesting alternative to quartz crystal concerning resonant biosensors thanks to its piezoelectric, acoustic properties, and its common micro-fabrication processes. This study opens up new opportunities to fabricate high sensitive sensors using low cost microfabrication processes.

GaAs Coupled Micro Resonators with Enhanced Sensitive Mass Detection

This work demonstrates the improvement of mass detection sensitivity and time response using a simple sensor structure. Indeed, complicated technological processes leading to very brittle sensing structures are often required to reach high sensitivity when we want to detect specific molecules in biological fields. These developments constitute an obstacle to the early diagnosis of diseases. An alternative is the design of coupled structures. In this study, the device is based on the piezoelectric excitation and detection of two GaAs microstructures vibrating in antisymmetric modes. GaAs is a crystal which has the advantage to be micromachined easily using typical clean room processes. Moreover, we showed its high potential in direct biofunctionalisation for use in the biological field. A specific design of the device was performed to improve the detection at low mass and an original detection method has been developed. The principle is to exploit the variation in amplitude at the initial resonance frequency which has in the vicinity of weak added mass the greatest slope. Therefore, we get a very good resolution for an infinitely weak mass: relative voltage variation of 8%/1 fg. The analysis is based on results obtained by finite element simulation.

Langasite resonant structure: Micromachining and characterisation

This paper focuses on the micromachining of 3D microstructures on LGS plates by anisotropic wet etching. Several etching conditions are investigated to determine the best experimental way to fabricate 3D microstructures. The study covers the composition, the concentration and the temperature of the etching bath. Several types of experimental results are used to determine the conditions that provide consistent and reproducible results: the anisotropic factor, the etch rate and the degree of surface quality, the undercutting, the solution chemical aggressiveness toward masks. Etching profiles, SEM images are discussed on several orientations of etched plates. According to the experimental data base, an etchant is selected and the experimental conditions of etching are determined. 3D resonant structures are fabricated on the X cut using photolithographic process before the etching step. The final shapes of the structures are then characterized in view of inertial sensor design.