Murali Krishna Ghatkesar

Quantitative time-resolved measurement of membrane protein- ligand interactions using microcantilever array sensors

Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor--the FhuA receptor of Escherichia coli--is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein-ligand interactions in a micro-array format.

Graphene based piezoresistive pressure sensor

We present a pressure sensor based on the piezoresistive effect of graphene. The sensor is a 100 nm thick, 280 μm wide square silicon nitride membrane with graphene meander patterns located on the maximum strain area. The multilayer, polycrystalline graphene was obtained by chemical vapor deposition. Strain in graphene was generated by applying differential pressure across the membrane. Finite element simulation was used to analyze the strain distribution. By performing electromechanical measurements, we obtained a gauge factor of ∼1.6 for graphene and a dynamic range from 0 mbar to 700 mbar for the pressure sensor.

Higher modes of vibration increase mass sensitivity in nanomechanical microcantilevers

We evaluated the potential and limitations of resonating nanomechanical microcantilevers for the detection of mass adsorption. As a test system we used mass addition of gold layers of varying thickness. Our main findings are: (1) A linear increase in mass sensitivity with the square of the mode number—a sensitivity increase of two orders of magnitude is obtained from mode 1 to mode 7 with a minimum sensitivity of 8.6 ag Hz −1 μm −2 and mass resolution of 0.43 pg at mode 7 for a 1 μm thick cantilever. (2) The quality factor increases with the mode number, thus helping to achieve a higher sensitivity. (3) The effective spring constant of the cantilever remains constant for deposition of gold layers up to at least 4% of the cantilever thickness. (Some figures in this article are in colour only in the electronic version)

Resonating modes of vibrating microcantilevers in liquid

A study of nanomechanical cantilevers vibrating at various resonating modes in liquid is presented. Resonant frequency spectrum with 16 well resolved flexural modes is obtained. The quality factor increased from 1 at mode 1 to 30 at mode 16. The theoretical estimate of eigenfrequency using the Elmer–Dreier model [F.-J. Elmer and M. Dreier, J. Appl. Phys. 81, 12 (1997)] and Sader’s extended viscous model [C. A. Van Eysden and J. E. Sader, J. Appl. Phys. 101, 044908 (2007)] matched well with the experimental data. The apparent mass of the liquid comoved by the oscillating cantilevers decreased asymptotically with mode number.

Comprehensive Characterization of Molecular Interactions Based on Nanomechanics

Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (∼748*106 Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions.

Real-time mass sensing by nanomechanical resonators in fluid

We have developed a sensitive method for real-time mass sensing in a fluid using a microfabricated array of nanomechanical cantilevers actuated at their resonance frequencies. The sensor platform consists of a streptavidin layer immobilised onto gold-coated cantilevers and interacts with biotin-labeled latex beads. The addition of mass involved with this process decreased the resonance frequency. By monitoring frequency spectra of higher harmonics we measured a mass sensitivity of 3.3 Hz/pg. Our data demonstrate that the sensitivity can be increased by operating the cantilever at higher harmonics.

Digital processing of multi-mode nano-mechanical cantilever data

Nanomechanical sensors based on cantilever technology allow the measurement of various physical properties. Here we present a software for the comprehensive analysis of such data. An example for the combined measurement of mass and surface stress is presented.

In situ Stiffness Adjustment of AFM Probes by Two Orders of Magnitude

The choice on which type of cantilever to use for Atomic Force Microscopy (AFM) depends on the type of the experiment being done. Typically, the cantilever has to be exchanged when a different stiffness is required and the entire alignment has to be repeated. In the present work, a method to adjust the stiffness in situ of a commercial AFM cantilever is developed. The adjustment is achieved by changing the effective length of the cantilever by electrostatic pull-in. By applying a voltage between the cantilever and an electrode (with an insulating layer at the point of contact), the cantilever snaps to the electrode, reducing the cantilever’s effective length. An analytical model was developed to find the pull-in voltage of the system. Subsequently, a finite element model was developed to study the pull-in behavior. The working principle of this concept is demonstrated with a proof-of-concept experiment. The electrode was positioned close to the cantilever by using a robotic nanomanipulator. To confirm the change in stiffness, the fundamental resonance frequency of the cantilever was measured for varying electrode positions. The results match with the theoretical expectations. The stiffness was adjusted in situ in the range of 0.2 N/m to 27 N/m, covering two orders of magnitude in one single cantilever. This proof-of-concept is the first step towards a micro fabricated prototype, that integrates the electrode positioning system and cantilever that can be used for actual AFM experiments.

Technologies at Hand: Measuring the intrinsic nanomechanics of molecular interactions with microcantilever sensors

�icroarray techniques are of indisputable importance for today’s research in medicine and biology. classical microarray sensors depend on the labeling of molecules and do not provide real-time information. In recent years, diff erent micro�array technologies evolved not only measuring in a label�free manner but also providing kinetic information (kinetic microarrays, �raun, Huber et al., 2�����). �uch techniques involve surface plasmon imaging (Jordan �� corn, 199�), ellipsometry (Wang �� Jin, 2003), surface acoustic wave sensors (Gronewold, Baumgartner, Quandt, �� famulok, 2006), nano-wire based sensors (cui, Wei, park, �� Lieber, 2001) and cantilever based sensors (Hansen �� Thundat, 2005; Lang, Hegner, �� Gerber, 2006). note, that all these techniques depend on the activation of a sensor surface with cognitive molecules. This article aims to provide an overview of cantilever based microarray sensors. cantilevers are extremely thin springboards anchored at one end to a base (fig 1a). These micro-fabricated structures are traditionally used for imaging (Binnig, Quate, �� Gerber, 1986) but also enabled a new type of sensor (Gimzewski, Gerber, meyer, �� Schlittler, 1994; Thundat, Warmack, chen, �� allison, 1994; Waggoner �� Craighead, 2�����): the nano�mechanical measurement principles upon which this technique is based work label�free and provide quantitative real�time information in a micro�array format. During recent years a variety of applications for chemical, physical and biological sensing with cantilever technology was presented. in biology, cantilever sensors are proven to robustly measure data for “classical” genomic experiments (“gene fi shing”) (Zhang et al., 2����6), for proteomic research (e. g. antibody�antigen interaction) (Backmann et al., 2005), macrobiotic growth (Gfeller, nugaeva, �� Hegner, 2005; nugaeva et al., 2�����) and structural changes of membrane proteins (���lint et al., 2�����; �raun et al., 2����6). Here we discuss the principles of cantilever sensors and the unique potential of these devices to characterize molecular interactions. Braun, T Eur J nanomed 2009; 2:13-15