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With the increase in contamination by microbial agents (bacteria, viruses, etc.) in the fields of agri-food, healthcare, and environment, it is necessary to detect and quantify these biological elements present in complex fluids in a short time with high selectivity, high sensitivity, and, if possible, moderate cost. Acoustic wave biosensors, based on immuno-detection, appear to meet a certain number of these criteria. In this context, we are developing a generic antibody-based biointerface that can detect a wide range of pathogenic bacterial agents using a specific bioreceptor. Based on the silane–oxide chemistry, the process is transferable to any kind of surface that can be either oxidized in surface or activated with O2-plasma, for instance. For this proof of concept, we have chosen to develop our biointerface on titanium and lithium niobate surfaces. The development of the biointerface consists of grafting antibodies via a self-assembled monolayer (SAM) composed of an aminopropyltriethoxysilane (APTES) and a linker (phenylene diisothiocyanate, PDITC). Two functionalization routes were tested for grafting APTES: in anhydrous toluene followed by a heating step at 110 °C or in chloroform at room temperature. The results obtained on titanium show comparable grafting efficiency between these two routes, allowing us to consider the transposition of the route at room temperature on lithium niobate. The latest route was chosen for fragile materials that do not require the heating steps necessary when using toluene for grafting aminopropyltriethoxysilane. Different surface characterization techniques were used, such as IR spectroscopy (FTIR-ATR), X-ray photoelectron spectroscopy (XPS), and contact angle (WCA), to verify the successful grafting of each layer. Biodetection experiments in static conditions were also carried out to demonstrate the specificity of pathogenic detection, testing an ideal medium with solely bacteria, with no other food sampling nutrients. This paper demonstrates the successful elaboration of a biointerface using APTES as the first anchoring layer, with chloroform as a mild solvent. The process is easily transferable to any kind of fragile surface. Moreover, following anti-L. monocytogenes antibodies, our biointerface shows a specificity of capture in static mode (at a concentration of 107 CFU/mL for an incubation time of 4 h at 37 °C) of up to 98% compared to a species negative control (E. coli) and up to 85% in terms of strain specificity (L. innocua).

The use of antimicrobial peptides (AMPs) covalently grafted on surfaces has been recognized in recent years as a promising strategy to fight against biofilm formation. However, after grafting, the understanding of AMP–bacteria interactions is still debated in the literature. In this study, Nisin, a cyclic AMP, was grafted onto gold surfaces via an indirect grafting on acidic thiol self-assembled monolayers using succinimide linkers. The physical and chemical properties of these SAMs were then finely characterized by XPS and FT-IR to confirm the covalent grafting of Nisin. The antiadhesion and bactericidal effects were then studied for Escherichia coli ATCC25922, Staphylococcus aureus ATCC 25923, and Listeria ivanovii Li4(pVS2) by a posteriori analysis of the culture supernatants (i.e., indirect technique) and ex situ by optical microscopy following crystal violet staining (i.e., direct technique). Statistical analysis reveals that the Nisin coating has bactericidal and antiadhesive properties towards Gram-positive bacteria, while no significant results were obtained for Gram-negative bacteria.

We present a mode localized mass sensor prototype based on a hybrid system excited at a fixed frequency slightly below the resonances. Indeed, we show, both theoretically and experimentally, that this condition yields higher sensitivities and similar sensitivity ranges than that of resonance peak tracking while being less time consuming than a classical open-loop configuration due to the absence of frequency sweep. The system is made of a quartz resonator and a hardware that includes a resonator and the coupling. The digital aspect allows maximum sensitivity to be achieved with a fine tuning of the different parameters and the implementation of a coupling, regardless of the physical resonator geometry. This allows the generation of mode localization on shear waves resonant structures such as the quartz cristal microbalance widely used in biosensing. This solution has been successfully implemented using resin micro balls depositions. The sensitivities reach almost their maximum theoretical values which means this fixed frequency method has the potential to reach lower limit of detection than the open loop frequency tracking method.

Microfluidics integration of acoustic biosensors is an actively developing field. Despite significant progress in “passive” microfluidic technology, integration with microacoustic devices is still in its research state. The major challenge is bonding polymers with monocrystalline piezoelectrics to seal microfluidic biosensors. In this contribution, we specifically address the challenge of microfluidics integration on gallium arsenide (GaAs) acoustic biosensors. We have developed a robust plasma-assisted bonding technology, allowing strong connections between PDMS microfluidic chip and GaAs/SiO2 at low temperatures (70 °C). Mechanical and fluidic performances of fabricated device were studied. The bonding surfaces were characterized by water contact angle measurement and ATR-FTIR, AFM, and SEM analysis. The bonding strength was characterized using a tensile machine and pressure/leakage tests. The study showed that the sealed chips were able to achieve a limit of high bonding strength of 2.01 MPa. The adhesion of PDMS to GaAs was significantly improved by use of SiO2 intermediate layer, permitting the bonded chip to withstand at least 8.5 bar of burst pressure. The developed bonding approach can be a valuable solution for microfluidics integration in several types of MEMS devices.

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.

A regenerable bulk acoustic wave (BAW) biosensor is developed for the rapid, label-free and selective detection of Escherichia coli in liquid media. The geometry of the biosensor consists of a GaAs membrane coated with a thin film of piezoelectric ZnO on its top surface. A pair of electrodes deposited on the ZnO film allows the generation of BAWs by lateral field excitation. The back surface of the membrane is functionalized with alkanethiol self-assembled monolayers and antibodies against E. coli. The antibody immobilization was investigated as a function of the concentration of antibody suspensions, their pH and incubation time, designed to optimize the immunocapture of bacteria. The performance of the biosensor was evaluated by detection tests in different environments for bacterial suspensions ranging between 103 and 108 CFU/mL. A linear dependence between the frequency response and the logarithm of E. coli concentration was observed for suspensions ranging between 103 and 107 CFU/mL, with the limit of detection of the biosensor estimated at 103 CFU/mL. The 5-fold regeneration and excellent selectivity towards E. coli detected at 104 CFU/mL in a suspension tinted with Bacillus subtilis at 106 CFU/mL illustrate the biosensor potential for the attractive operation in complex biological media.

Shear bulk acoustic type of resonant biosensors, such as the quartz crystal microbalance (QCM), give access to label-free in-liquid analysis of surface interactions. The general understanding of the sensing principles was inherited from past developments in biofilms measurements and applied to cells while keeping the same basic assumptions. Thus, the biosensor readouts are still quite often described using ‘mass’ related terminology. This contribution aims to show that assessment of cell deposits with acoustic biosensors requires a deep understanding of the sensor transduction mechanism. More specifically, the cell deposits should be considered as a structured viscoelastic load and the sensor response depends on both material and topological parameters of the deposits. This shifts the paradigm of acoustic biosensor away from the classical mass loading perspective. As a proof of the concept, we recorded QCM frequency shifts caused by blood platelet deposits on a collagen surface under different rheological conditions and observed the final deposit shape with atomic force microscopy (AFM). The results vividly demonstrate that the frequency shift is highly impacted by the platelet topology on the bio-interface. We support our findings with numerical simulations of viscoelastic unstructured and structured loads in liquid. Both experimental and theoretical studies underline the complexity behind the frequency shift interpretation when acoustic biosensing is used with cell deposits.

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.

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.

A regenerable bulk acoustic wave (BAW) biosensor is developed for the rapid, label-free and selective detection of

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