Nicolas Lafitte

Improvement of Silicon Nanotweezers Sensitivity for Mechanical Characterization of Biomolecules Using Closed-Loop Control

In this paper, we show that closed-loop control can be advantageously used for the characterization of mechanical properties of biomolecules using silicon nanotweezers (SNT). SNT have already been used in open-loop mode for the characterization of mechanical properties of DNA molecules. Up to now, such an approach allows the detection of stiffness variations equivalent to about 15 DNA molecules. Here, it is shown that this resolution is inversely proportional to the resonance frequency of the whole system and that real-time feedback control with state observer can drastically improve the performances of the tweezers used as biosensors. Such improvement is experimentally validated in the case of the manipulation of fibronectin molecules. The results are promising for the accurate characterization of biopolymers such as DNA molecules.

Real-time mechanical characterization of DNA degradation under therapeutic X-rays and its theoretical modeling

The killing of tumor cells by ionizing radiation beams in cancer radiotherapy is currently based on a rather empirical understanding of the basic mechanisms and effectiveness of DNA damage by radiation. By contrast, the mechanical behaviour of DNA encompassing sequence sensitivity and elastic transitions to plastic responses is much better understood. A novel approach is proposed here based on a micromechanical Silicon Nanotweezers device. This instrument allows the detailed biomechanical characterization of a DNA bundle exposed to an ionizing radiation beam delivered here by a therapeutic linear particle accelerator (LINAC). The micromechanical device endures the harsh environment of radiation beams and still retains molecular-level detection accuracy. In this study, the first real-time observation of DNA damage by ionizing radiation is demonstrated. The DNA bundle degradation is detected by the micromechanical device as a reduction of the bundle stiffness, and a theoretical model provides an interpretation of the results. These first real-time observations pave the way for both fundamental and clinical studies of DNA degradation mechanisms under ionizing radiation for improved tumor treatment.

A rapid and practical technique for real-time monitoring of biomolecular interactions using mechanical responses of macromolecules

Monitoring biological reactions using the mechanical response of macromolecules is an alternative approach to immunoassays for providing real-time information about the underlying molecular mechanisms. Although force spectroscopy techniques, e.g. AFM and optical tweezers, perform precise molecular measurements at the single molecule level, sophisticated operation prevent their intensive use for systematic biosensing. Exploiting the biomechanical assay concept, we used micro-electro mechanical systems (MEMS) to develop a rapid platform for monitoring bio/chemical interactions of bio macromolecules, e.g. DNA, using their mechanical properties. The MEMS device provided real-time monitoring of reaction dynamics without any surface or molecular modifications. A microfluidic device with a side opening was fabricated for the optimal performance of the MEMS device to operate at the air-liquid interface for performing bioassays in liquid while actuating/sensing in air. The minimal immersion of the MEMS device in the channel provided long-term measurement stability (>10 h). Importantly, the method allowed monitoring effects of multiple solutions on the same macromolecule bundle (demonstrated with DNA bundles) without compromising the reproducibility. We monitored two different types of effects on the mechanical responses of DNA bundles (stiffness and viscous losses) exposed to pH changes (2.1 to 4.8) and different Ag+ concentrations (1 μM to 0.1 M).

Direct bio-mechanical sensing of enzymatic reaction On DNA by silicon nanotweezers

This paper demonstrates the real-time sensing of enzymatic reactions on a DNA bundle trapped by silicon nanotweezers. The digestion of DNA in protein solution (Hind III) is monitored by tracking the change in frequency response of the immersed tweezers. This new direct biomechanical detection clears the way for systematic bio tests at the molecular level by an integrated MEMS device.

Integrated MEMS platform with silicon nanotweezers and open microfluidic device for molecular and cellular biomechanical assays

This paper describes an integrated bio-reaction platform composed of silicon nanotweezers and open microfluidics for real time biomechanical assays. The silicon nanotweezers can sense slight biological modification of the trapped sample due to stable frequency response with high Q factor in liquid. The microfluidic device integrates active valves for controlling the biological medium. Biomolecular assay (DNA/Hind III enzymatic reaction kinetics), and cell contraction/relaxation dynamic assay illustrate the capabilities of this integrated platform that is a breakthrough capability for real time and routine biological tests.

Silicon Nanotweezers with a microfluidic cavity for the real time characterization of DNA damage under therapeutic radiation beams

We report the biomechanical characterization of λ-DNA bundle exposed to a therapeutic radiation beam by silicon Nanotweezers. The micromechanical device endures the harsh environment of radiation beams, and still retains molecular-level detection accuracy. The real-time DNA bundle degradation is observed in terms of biomechanical stiffness and viscosity reduction, both in air and in solution. These results pave the way for both fundamental and clinical studies of DNA degradation mechanisms under ionizing radiation for improved tumor treatment.

Elucidating the mechanism of the considerable mechanical stiffening of DNA induced by the couple Zn 2+ /Calix[4]arene-1,3-O-diphosphorous acid

The couple Calix[4]arene-1,3-O-diphosphorous acid (C4diP) and zinc ions (Zn2+) acts as a synergistic DNA binder. Silicon NanoTweezer (SNT) measurements show an increase in the mechanical stiffness of DNA bundles by a factor of >150, at Zn2+ to C4diP ratios above 8, as compared to Zinc alone whereas C4diP alone decreases the stiffness of DNA. Electroanalytical measurements using 3D printed devices demonstrate a progression of events in the assembly of C4diP on DNA promoted by zinc ions. A mechanism at the molecular level can be deduced in which C4diP initially coordinates to DNA by phosphate-phosphate hydrogen bonds or in the presence of Zn2+ by Zn2+ bridging coordination of the phosphate groups. Then, at high ratios of Zn2+ to C4diP, interdigitated dimerization of C4diP is followed by cross coordination of DNA strands through Zn2+/C4diP inter-strand interaction. The sum of these interactions leads to strong stiffening of the DNA bundles and increased inter-strand binding.

Closed-loop control of silicon nanotweezers for improvement of sensitivity to mechanical stiffness measurement and bio-sensing on DNA molecules

In this work we show that implementation of closed-loop control to silicon nanotweezers improves the sensitivity of the tool for mechanical characterization of biological molecules. Micromachined tweezers have already been used for the characterization of mechanical properties of DNA molecules [1] as well as for the sensing of enzymatic reactions on DNA bundle [2], [3]. However the resolution of the experiments does not allow the sensing on single molecules. Hereafter we show theoretically and experimentally that, reducing the resonance frequency of the system by the implementation of a state feedback, the sensitivity to stiffness variation is enhanced. Such improvement leads to better resolution for detection of enzymatic reactions on DNA [4].

Integrated MEMS platform with silicon nanotweezers and open microfluidic device for real-time and routine biomechanical probing on molecules and cells

This paper describes an integrated biomechanical platform for real-time molecular or cellular assays. This platform is composed of silicon nanotweezers to manipulate the biological samples and an open microfluidic to handle solution and reactive agents. The tweezers are fabricated by standard Silicon-On-Insulator based micromachining processes (2 masks +1 additional mask for special tips) and integrate actuator, trapping tips and sensor. The microfluidic device is produced from common polydimethylsiloxane (PDMS) micromolding and integrates active valves for controlling the biological medium. Combining both technologies, a versatile experimental setup, built up in an enclosed space (<; 10 cm3), enables direct interrogation of molecules or cells in solution. The silicon nanotweezers sense slight biological modifications of the trapped molecules or cell by monitoring the mechanical resonance response, which keeps a high Q factor (over 20) in liquid. Biomolecular assays (molecule trapping and enzymatic reaction kinetics) as well as characterizations of cells are reported here. The system provide molecular level resolution and is sensitive enough to capture cell biomechano-transduction activities. Moreover as the system is handy, it may be an easy, fast and quantitative alternative to existing methods.